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The three-dimensional organization of chromatin is influenced by chromatin-binding proteins through both specific and non-specific interactions. However, the roles of chromatin sequence and the interactions between binding proteins in shaping chromatin structure remain elusive. By employing a simple polymer-based model of chromatin that explicitly considers sequence-dependent protein binding and protein–protein interactions, we elucidate a mechanism for chromatin organization. We find that tuning protein–protein interactions and protein concentration is sufficient to either promote or inhibit chromatin compartmentalization. Moreover, chromatin sequence and protein–protein attraction strongly affect the structural and dynamic exponents that describe the spatiotemporal organization of chromatin. Strikingly, our model's predictions for the exponents governing chromatin structure and dynamics successfully capture experimental observations, in sharp contrast to previous chromatin models. Overall, our findings have the potential to reinterpret data obtained from various chromosome conformation capture technologies, laying the groundwork for advancing our understanding of chromatin organization. [ABSTRACT FROM AUTHOR]

Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in the biosynthesis of taurine from cysteine and in the downstream metabolism of secondary taurine metabolites4,5. One taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by stimuli that alter taurine or acetate flux, including endurance exercise7, dietary taurine supplementation8 and alcohol consumption6,9. So far, the identities of the enzymes involved in N-acetyltaurine metabolism, and the potential functions of N-acetyltaurine itself, have remained unknown. Here we show that the body mass index associated orphan enzyme phosphotriesterase-related (PTER)10 is a physiological N-acetyltaurine hydrolase. In vitro, PTER catalyses the hydrolysis of N-acetyltaurine to taurine and acetate. In mice, PTER is expressed in the kidney, liver and brainstem. Genetic ablation of Pter in mice results in complete loss of tissue N-acetyltaurine hydrolysis activity and a systemic increase in N-acetyltaurine levels. After stimuli that increase taurine levels, Pter knockout mice exhibit reduced food intake, resistance to diet-induced obesity and improved glucose homeostasis. Administration of N-acetyltaurine to obese wild-type mice also reduces food intake and body weight in a GFRAL-dependent manner. These data place PTER into a central enzymatic node of secondary taurine metabolism and uncover a role for PTER and N-acetyltaurine in body weight control and energy balance.

BACKGROUND AND AIMS: Extracellular vesicles (EVs) secreted by cardiosphere-derived cells exert immunomodulatory effects through the transmission of small non-coding RNAs. METHODS: The mechanism and role of yREX3, a small Y RNA abundant in EVs in myocardial injury, was investigated. RESULTS: yREX3 attenuates cardiac ischaemic injury by selective DNA methylation. Synthetic yREX3 encapsulated in lipid nanoparticles triggers broad transcriptomic changes in macrophages, localizes to the nucleus, and mediates epigenetic silencing of protein interacting with C kinase-1 (Pick1) through methylation of upstream CpG sites. Moreover, yREX3 interacts with polypyrimidine tract binding protein 3 (PTBP3) to methylate the Pick1 gene locus in a DNA methyltransferase-dependent manner. Suppression of Pick1 in macrophages potentiates Smad3 signalling and enhances efferocytosis, minimizing heart necrosis in rats with myocardial infarction. Adoptive transfer of Pick1-deficient macrophages recapitulates the cardioprotective effects of yREX3 in vivo. CONCLUSIONS: These findings highlight the role of a small Y RNA mined from EVs with a novel gene-methylating mechanism.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Acute poisoning with organophosphorus cholinesterase inhibitors (OPs), such as OP nerve agents and pesticides, can cause life threatening cholinergic crisis and status epilepticus (SE). Survivors often experience significant morbidity, including brain injury, acquired epilepsy, and cognitive deficits. Current medical countermeasures for acute OP poisoning include a benzodiazepine to mitigate seizures. Diazepam was long the benzodiazepine included in autoinjectors used to treat OP-induced seizures, but it is now being replaced in many guidelines by midazolam, which terminates seizures more quickly, particularly when administered intramuscularly. While a direct correlation between seizure duration and the extent of brain injury has been widely reported, there are limited data comparing the neuroprotective efficacy of diazepam versus midazolam following acute OP intoxication. To address this data gap, we used non-invasive imaging techniques to longitudinally quantify neuropathology in a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP) with and without post-exposure intervention with diazepam or midazolam. Magnetic resonance imaging (MRI) was used to monitor neuropathology and brain atrophy, while positron emission tomography (PET) with a radiotracer targeting translocator protein (TSPO) was utilized to assess neuroinflammation. Animals were scanned at 3, 7, 28, 65, 91, and 168 days post-DFP and imaging metrics were quantitated for the hippocampus, amygdala, piriform cortex, thalamus, cerebral cortex and lateral ventricles. In the DFP-intoxicated rat, neuroinflammation persisted for the duration of the study coincident with progressive atrophy and ongoing tissue remodeling. Benzodiazepines attenuated neuropathology in a region-dependent manner, but neither benzodiazepine was effective in attenuating long-term neuroinflammation as detected by TSPO PET. Diffusion MRI and TSPO PET metrics were highly correlated with seizure severity, and early MRI and PET metrics were positively correlated with long-term brain atrophy. Collectively, these results suggest that anti-seizure therapy alone is insufficient to prevent long-lasting neuroinflammation and tissue remodeling.

Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.

Macroautophagy/autophagy receptors target their substrates to phagophores for subsequent sequestration within autophagosomes. During phagophore membrane expansion in mammalian cells, autophagy receptors simultaneously interact with the ubiquitinated substrates and the LC3/GABARAP proteins on the expanding membrane. In this punctum, we summarize and discuss our recent research progress on synthetic autophagy receptors (AceTACs). The series of AceTACs were designed by engineering the essential interacting domains and motifs of SQSTM1/p62 (sequestosome 1), a major mammalian autophagy receptor. Particularly, we replaced the ubiquitin-associated domain of SQSTM1 with a target-specific antibody, redirecting the bifunctional interactions of wild-type SQSTM1 and directing the degradation target into the autophagy process. We successfully demonstrated the targeted degradation of aggregation-prone proteins using the AceTAC degraders. Moreover, we presented a model system with a guideline to induce targeted degradation of organelles through the autophagy machinery.

Metal-reducing bacteria have adapted the ability to respire extracellular solid surfaces instead of soluble oxidants. This process requires an electron transport pathway that spans from the inner membrane, across the periplasm, through the outer membrane, and to an external surface. Multiheme cytochromes are the primary machinery for moving electrons through this pathway. Recent studies show that the chiral-induced spin selectivity (CISS) effect is observable in some of these proteins extracted from the model metal-reducing bacteria, Shewanella oneidensis MR-1. It was hypothesized that the CISS effect facilitates efficient electron transport in these proteins by coupling electron velocity to spin, thus reducing the probability of backscattering. However, these studies focused exclusively on the cell surface electron conduits, and thus, CISS has not been investigated in upstream electron transfer components such as the membrane-associated MtrA, or periplasmic proteins such as small tetraheme cytochrome (STC). By using conductive probe atomic force microscopy measurements of protein monolayers adsorbed onto ferromagnetic substrates, we show that electron transport is spin selective in both MtrA and STC. Moreover, we have determined the spin polarization of MtrA to be ∼77% and STC to be ∼35%. This disparity in spin polarizations could indicate that spin selectivity is length dependent in heme proteins, given that MtrA is approximately two times longer than STC. Most significantly, our study indicates that spin-dependent interactions affect the entire extracellular electron transport pathway. [ABSTRACT FROM AUTHOR]

BACKGROUND: Diabetes is a spectrum of metabolic diseases affecting millions of people worldwide. The loss of pancreatic β-cell mass by either autoimmune destruction or apoptosis, in type 1-diabetes (T1D) and type 2-diabetes (T2D), respectively, represents a pathophysiological process leading to insulin deficiency. Therefore, therapeutic strategies focusing on restoring β-cell mass and β-cell insulin secretory capacity may impact disease management. This study took advantage of powerful integrative bioinformatic tools to scrutinize publicly available diabetes-associated gene expression data to unveil novel potential molecular targets associated with β-cell dysfunction. METHODS: A comprehensive literature search for human studies on gene expression alterations in the pancreas associated with T1D and T2D was performed. A total of 6 studies were selected for data extraction and for bioinformatic analysis. Pathway enrichment analyses of differentially expressed genes (DEGs) were conducted, together with protein-protein interaction networks and the identification of potential transcription factors (TFs). For noncoding differentially expressed RNAs, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), which exert regulatory activities associated with diabetes, identifying target genes and pathways regulated by these RNAs is fundamental for establishing a robust regulatory network. RESULTS: Comparisons of DEGs among the 6 studies showed 59 genes in common among 4 or more studies. Besides alterations in mRNA, it was possible to identify differentially expressed miRNA and lncRNA. Among the top transcription factors (TFs), HIPK2, KLF5, STAT1 and STAT3 emerged as potential regulators of the altered gene expression. Integrated analysis of protein-coding genes, miRNAs, and lncRNAs pointed out several pathways involved in metabolism, cell signaling, the immune system, cell adhesion, and interactions. Interestingly, the GABAergic synapse pathway emerged as the only common pathway to all datasets. CONCLUSIONS: This study demonstrated the power of bioinformatics tools in scrutinizing publicly available gene expression data, thereby revealing potential therapeutic targets like the GABAergic synapse pathway, which holds promise in modulating α-cells transdifferentiation into β-cells.

PURPOSE: Germline pathogenic variants in checkpoint kinase 2 (CHEK2) are associated with a moderately increased risk of breast cancer (BC). The spectrum of clinicopathologic features and genetics of these tumors has not been fully established. METHODS: We characterized the histopathologic and clinicopathologic features of 44 CHEK2-associated BCs from 35 women, and assessed responses to neoadjuvant chemotherapy. A subset of cases (n = 23) was additionally analyzed using targeted next-generation DNA sequencing (NGS). RESULTS: Most (94%, 33/35) patients were heterozygous carriers for germline CHEK2 variants, and 40% had the c.1100delC allele. Two patients were homozygous, and five had additional germline pathogenic variants in ATM (2), PALB2 (1), RAD50 (1), or MUTYH (1). CHEK2-associated BCs occurred in younger women (median age 45 years, range 25-75) and were often multifocal (20%) or bilateral (11%). Most (86%, 38/44) were invasive ductal carcinomas of no special type (IDC-NST). Almost all (95%, 41/43) BCs were ER + (79% ER + HER2-, 16% ER + HER2 + , 5% ER-HER2 +), and most (69%) were luminal B. Nottingham grade, proliferation index, and results of multiparametric molecular testing were heterogeneous. Biallelic CHEK2 alteration with loss of heterozygosity was identified in most BCs (57%, 13/23) by NGS. Additional recurrent alterations included GATA3 (26%), PIK3CA (226%), CCND1 (22%), FGFR1 (22%), ERBB2 (17%), ZNF703 (17%), TP53 (9%), and PPM1D (9%), among others. Responses to neoadjuvant chemotherapy were variable, but few patients (21%, 3/14) achieved pathologic complete response. Most patients (85%) were without evidence of disease at time of study (n = 34). Five patients (15%) developed distant metastasis, and one (3%) died (mean follow-up 50 months). CONCLUSION: Almost all CHEK2-associated BCs were ER + IDC-NST, with most classified as luminal B with or without HER2 overexpression. NGS supported the luminal-like phenotype and confirmed CHEK2 as an oncogenic driver in the majority of cases. Responses to neoadjuvant chemotherapy were variable but mostly incomplete.

Membrane protein biogenesis poses acute challenges to protein homeostasis, and how they are selectively escorted to the target membrane is not well understood. Here we address this question in the guided-entry-of-tail-anchored protein (GET) pathway, in which tail-anchored membrane proteins (TAs) are relayed through an Hsp70-Sgt2-Get3 chaperone triad for targeting to the endoplasmic reticulum. We show that the Hsp70 ATPase cycle and TA substrate drive dimeric Sgt2 from a wide-open conformation to a closed state, in which TAs are protected by both substrate binding domains of Sgt2. Get3 is privileged to receive TA from closed Sgt2, whereas off-pathway chaperones remove TAs from open Sgt2. Sgt2 closing is less favorable with suboptimal GET substrates, which are rejected during or after the Hsp70-to-Sgt2 handover. Our results demonstrate how fine-tuned conformational dynamics in Sgt2 enable hydrophobic TAs to be effectively funneled onto their dedicated targeting factor while also providing a mechanism for substrate selection.

We synthesized a WNT5A β-hairpin peptide responsible for the protein's binding to its receptor, Frizzled. Full affinity to Frizzled requires all three disulfides but not an invariant tryptophan that significantly contributes to the buried surface area. Our work opens the avenue to target the Wnt–Fzd interface using synthetic peptides. [ABSTRACT FROM AUTHOR]

Melatonin is produced in the pineal gland under very tight control through the influences of light and the suprachiasmatic nucleus. As such, melatonin circulates in the blood at levels <3 pg/ml during the day and is only actively secreted at night reaching levels of approximately 100 pg/ml. As a consequence of binding to plasma proteins, free melatonin appears in saliva at approximately one third the plasma concentration. Measurement of melatonin is technically challenging because of these very low concentrations and while a number of commercial immunoassay kits are available and mass spectrometry assay methods have been published, not all are fit for purpose and can lead to unreliable conclusions. In this review I discuss the aspects of pineal melatonin production that saliva melatonin reflects, the factors influencing melatonin production or metabolism, saliva collection and analysis methods. Examples are provided of the appropriate use of saliva melatonin measurements; Dim Light Melatonin Onset (DLMO) assessment, impact of light on melatonin and the monitoring of rhythms prior to specific treatments. Examples of inappropriate use of saliva melatonin measurements are also provided including the use of poorly validated assays, morning saliva collections, attempts to stimulate melatonin, and linking specific illnesses to saliva melatonin levels. [ABSTRACT FROM AUTHOR]

Organs obtained from brain dead donors can have suboptimal outcomes. Activation of the innate immune system and translocation of intestinal bacteria could be causative. Thirty two pigs were assigned to control, brain death (BD), BD + luminal intestinal polyethylene glycol (PEG), and BD + luminal intestinal University of Wisconsin solution (UW) groups. Animals were observed for 360 min after BD before organ retrieval. 2,000 mL luminal intestinal preservation solution was instilled into the duodenum at the start of organ procurement. Repeated measurements of plasma C3a, Terminal Complement Complex (TCC), IL-8, TNF, and lipopolysaccharide binding protein were analysed by immunoassays. C3a was significantly higher in the BD groups compared to controls at 480 min after brain death. TCC was significantly higher in BD and BD + UW, but not BD + PEG, compared to controls at 480 min. TNF was significantly higher in the BD group compared to all other groups at 480 min. LPS binding protein increased following BD in all groups except BD + PEG, which at 480 min was significantly lower compared with all other groups. Brain death induced innate immune system activation was decreased by luminal preservation using PEG during organ procurement, possibly due to reduced bacterial translocation. [ABSTRACT FROM AUTHOR]

Background: Renal involvement resulting from hyperuricemia, known as hyperuricemia nephropathy (HN), is characterized by chronic tubulointerstitial inflammation caused by extensive urate crystal deposition. Managing this condition requires straightforward preventive or therapeutic interventions, primarily through dietary measures. Methods: In this study, the mouse model of HN was established using yeast extract combined with potassium oxonate. The effect and potential mechanism of β‐sitosterol in treating HN were investigated through biochemical indexes, pathological changes, untargeted metabolomics, and network pharmacology. Results: β‐Sitosterol reduced the levels of four biomarkers of HN: uric acid (UA), creatinine (CRE), blood urea nitrogen (BUN), and xanthine oxidase (XOD). It also mitigated inflammatory injury in renal tissues and reversed the abnormal expression of four key urate transporter proteins: glucose transporter protein 9 (GLUT9), organic anion transporter 1 (OAT1), ATP‐binding cassette transporter G2 (ABCG2), and urate transporter 1 (URAT1). To explore the mechanism of β‐sitosterol in treating HN, this study employed network pharmacology and metabolomics to analyze 27 intersecting gene targets and 14 differential metabolites. The findings indicated that glutathione (GSH) metabolism might be a crucial pathway. Treatment with β‐sitosterol increased the levels of reduced GSH as well as the activity and expression of 6‐phosphogluconate dehydrogenase (G6PDH) in mice, thereby effectively modulating GSH metabolism. This study proposes a novel strategy using β‐sitosterol for treating HN, providing a promising approach for addressing this condition. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic, caused by SARS-CoV-2, demonstrated that zoonotic transmission of animal sarbecoviruses threatens human health but the determinants of transmission are incompletely understood. Here, we show that most spike (S) proteins of horseshoe bat and Malayan pangolin sarbecoviruses employ ACE2 for entry, with human and raccoon dog ACE2 exhibiting broad receptor activity. The insertion of a multibasic cleavage site into the S proteins increased entry into human lung cells driven by most S proteins tested, suggesting that acquisition of a multibasic cleavage site might increase infectivity of diverse animal sarbecoviruses for the human respiratory tract. In contrast, two bat sarbecovirus S proteins drove cell entry in an ACE2-independent, trypsin-dependent fashion and several ACE2-dependent S proteins could switch to the ACE2-independent entry pathway when exposed to trypsin. Several TMPRSS2-related cellular proteases but not the insertion of a multibasic cleavage site into the S protein allowed for ACE2-independent entry in the absence of trypsin and may support viral spread in the respiratory tract. Finally, the pan-sarbecovirus antibody S2H97 enhanced cell entry driven by two S proteins and this effect was reversed by trypsin while trypsin protected entry driven by a third S protein from neutralization by S2H97. Similarly, plasma from quadruple vaccinated individuals neutralized entry driven by all S proteins studied, and availability of the ACE2-independent, trypsin-dependent pathway reduced neutralization sensitivity. In sum, our study reports a pathway for entry into human cells that is ACE2-independent, can be supported by TMPRSS2-related proteases and may be associated with antibody evasion. Author summary: Bats host a wide range of coronaviruses, including those related to SARS-CoV-1 and SARS-CoV-2 (subgenus Sarbecovirus), but their ability to infect human cells remains poorly understood. Identifying sarbecoviruses with zoonotic potential and understanding the factors controlling their entry into human cells are crucial for risk assessment and antiviral development. In this study, we examined how bat sarbecovirus spike proteins facilitate entry into human cells and identified key host factors involved. Using pseudovirus particles, we show that several bat sarbecovirus spike proteins use human and animal ACE2 receptors for entry. Additionally, we confirm that some spike proteins utilize an ACE2-independent entry pathway requiring trypsin treatment and demonstrate that this process is controlled by the spike protein receptor binding domain. Furthermore, we reveal that a subset of ACE2-using bat sarbecovirus spike proteins can switch to ACE2-independent entry following trypsin exposure and show that certain human type II transmembrane serine proteases can substitute for trypsin, enabling ACE2-independent entry. Finally, we demonstrate that repeated COVID-19 vaccination generates cross-neutralizing activity against bat sarbecoviruses, though ACE2-independent entry reduces neutralization sensitivity. [ABSTRACT FROM AUTHOR]

Psoriasis is chronic immune-mediated inflammatory disorder characterized by various comorbidities, erythematous plaques with silvery scale which can lead to psoriatic arthritis. The phosphodiesterase 4 (PDE4) protein is a potential drug target to control Psoriasis. In the current study, pharmacophore-based virtual screening of Diversity library of ChemDiv database was first performed, and then the screened hits were docked to the active site of PDE4 to choose the best binding modes. Forty-six hits generated during the virtual screening were prepared and docked to the PDE4 receptor by SP docking module of glide. The binding affinities of the selected hits were calculated by molecular docking and based on the affinities, ten hits were selected for the bioactivity scores prediction and ADMET analysis. Based on the ADMET profiling, four hits D356-2630, C700-2058, G842-0420 and F403-0203 were processed to MD simulations for stability analysis. The outcomes showed that these compounds showed strong binding with proteins with better binding free energies. Based on the results of our study, we proposed that these hits can function as lead in the biological assays and in vitro studies are required to develop the novel drug candidates. [ABSTRACT FROM AUTHOR]

Radiotherapy is a curative arsenal for prostate cancer (PCa), but radioresistance seriously compromises its effectiveness. Dysregulated RNA splicing factors are extensively involved in tumor progression. Nonetheless, the role of splicing factors in radioresistance remains largely unexplored in PCa. Here, 23 splicing factors that are differentially expressed between PCa and adjacent normal tissues across multiple public PCa databases are identified. Among those genes, polypyrimidine tract binding protein 1 (PTBP1) is significantly upregulated in PCa and is positively associated with advanced clinicopathological features and poor prognosis. Gain‐ and loss‐of‐function experiments demonstrate that PTBP1 markedly reinforces genomic DNA stability to desensitize PCa cells to irradiation in vitro and in vivo. Mechanistically, PTBP1 interacts with the heterogeneous nuclear ribonucleoproteins (hnRNP) associated with lethal yellow protein homolog (RALY) and regulates exon 5 splicing of DNA methyltransferase 3b (DNMT3B) from DNMT3B‐S to DNMT3B‐L. Furthermore, upregulation of DNMT3B‐L induces promoter methylation of dual‐specificity phosphatase‐2 (DUSP2) and subsequently inhibits DUSP2 expression, thereby increasing radioresistance in PCa. The findings highlight the role of splicing factors in inducing aberrant splicing events in response to radiotherapy and the potential role of PTBP1 and DNMT3B‐L in reversing radioresistance in PCa. [ABSTRACT FROM AUTHOR]

ABSTRACT Aluminum (Al) toxicity in acidic soils leads to a considerable reduction in crop yields. MicroRNAs play essential roles in abiotic stress responses, but little is known of their role in the response of peanut (Arachnis hypogea L.) to Al stress. In this study, a novel Ah‐miR2916 (miR2916)–AhERF13–AhSUC3 module was found to be involved in the Al‐stress response via ethylene‐mediated signaling in peanut. Overexpression of miR2916 in Arabidopsis resulted in reduced Al tolerance by downregulating ethylene biosynthesis, while knockdown miR2916 in peanut enhanced Al tolerance. Notably, the APETALA2/ethylene‐responsive factor (ERF), AhERF13, was identified as a potential target of miR2916. AhERF13 expression was increased in miR2916 knockdown peanut lines and displayed an opposing pattern to that of miR2916 under Al stress. Consistently, knockdown AhERF13 peanut lines indicated that AhERF13 positively regulates Al tolerance by upregulating ethylene biosynthesis. AhERF13 was shown capable of binding to an ERF motif in the promoter region of sucrose transport protein 3 (AhSUC3) and positively regulate its expression. Consequently, AhSUC3 improved Al tolerance by upregulating ethylene biosynthesis. These results provide further insights into the molecular mechanisms operating during peanut response to Al stress, and suggests targets for manipulation in breeding programs for improved Al tolerance. [ABSTRACT FROM AUTHOR]

Nanobodies (Nbs) are one of the most promising therapeutics for overcoming immune escape in various diseases, including SARS‐CoV‐2 infection and cancers. However, the small sizes of nanobodies make them prone to renal clearance, thus decreasing circulation half‐life and hindering therapeutic efficacy. Traditional modification technologies, i.e., biotinylation and Fc‐fusion, aim to enhance nanobody pharmacokinetics, but they may introduce heterogeneous products with impaired functions and potentially affect binding to the Fc receptor. Here, a versatile nanobody engineering strategy is presented via molecular modification mediated by an intrinsically disordered protein. The engineered nanobody nano‐formulations retain their high‐affinity binding to the spike protein receptor binding domain and possess submicromolar levels of half‐maximal inhibitory concentration (IC50) against the pseudotyped SARS‐CoV‐2 variants, comparable to the unmodified nanobodies. Notably, the nano‐formulations show elongated half‐lives that are up to ≈15 times higher than those of original nanobodies and superior to other reported modified nanobodies. Furthermore, the in vivo therapeutic efficacy of such nano‐formulation toward breast cancer is significantly enhanced. Therefore, this nanobody engineering strategy offers a convenient and broadly applicable solution to address the suboptimal in vivo performance of nanobodies, holding substantial promise for effectively combating treatment‐tolerant cancers and future pandemics. [ABSTRACT FROM AUTHOR]

A number of quinoidal molecules with symmetric end‐capping groups, particularly dicyanomethylene units, have been synthesized for organic optoelectronic materials. In comparison, dissymmetric quinoidal molecules, characterized by end‐capping with different groups, are less explored. In this paper, we present the unexpected formation of new formal quinoidal molecules, which are end‐capped with both dicyanomethylene and triphenylphosphonium moieties. The structures of these dissymmetric quinoidal molecules were firmly verified by single crystal structural analyses. On the basis of the control experiments and DFT calculations, we proposed the reaction mechanism for the formation of these dissymmetric quinoidal molecules. The respective zwitterionic forms should make contributions to the ground state structures of these quinoidal molecules based on the analysis of their bond lengths and aromaticity and Mayer Bond Orbital (MBO) calculation. This agrees well with the fact that negative solvatochromism was observed for these quinoidal molecules. Although these new quinoidal molecules are non‐emissive both in solutions and crystalline states, they become emissive with quantum yields up to 51.4 % after elevating the solvent viscosity or dispersing them in a PMMA matrix. Interestingly, their emissions can also be switched on upon binding with certain proteins, in particular with human serum albumin. [ABSTRACT FROM AUTHOR]

Background: Biofilm formation has a crucial role in the cariogenic virulence of Streptococcus mutans, which leads to resistance to common antibacterials. The antimicrobial resistance crisis has led to increased research about traditional natural products. Purpose: Quercus infectoria extract (QI extract) and nano hydrogels containing QI extract (QI-NH) and tannic acid (TA-NH) were evaluated against this pathogen. Methods: QI extract was analyzed by HPLC and the physiological characteristics of nanohydrogels were assessed by SEM, FTIR, zeta potential, DLS and determination of release kinetics and encapsulation efficiency. Determination of MIC and MBC of the material and their anti-biofilm effect was done by the microtiter method and on the extracted tooth surface. The properties of extracts and nano hydrogels in the expression of genes codifying glucosyltransferases (gtfB, gtfC and gtfD) and glucan binding protein B (gbpB) were quantified. Their toxicity was tested by the MTT method against the KB cell line. Results: According to HPLC, 55.18% of QI extract contained TA. The encapsulation efficiency of QI-NH and TA-NH was equal to 60% and 80%, respectively. SEM and FTIR exhibited that QI extract and TA were successfully entrapped in the networks resulting from the chemical bonding of chitosan and TPP. The average size of QI-NH and TA-NH was 70.45 and 58.43 nm, and their zeta potential was 6.17 ± 2.58 and 0.25 ± 0.03 mv, respectively. PDI < 0.3 of nano hydrogels indicated the favorable polydispersity of nanohydrogels. MIC of QI extract, QI-NH and TA-NH were 937.5, 30 and 10 µg/ml, respectively. Also their MBIC50 was 35.1, 2.1 and 0.95 µg/ml, respectively, and the extracts and nano hydrogels restrained the biofilm maturation on enamel. The pivotal genes of S. mutans in biofilm formation were significantly less expressed by treatment with QI-NH and TA-NH than others. Based on the MTT test, QI-NH had less acute toxicity than QI extract and TA-NH. IC50 of QI-NH was calculated as 775.4 µg/ml, while it was equal to 3.12 µg/ml for chlorhexidine as a common antibacterial agent. Conclusion: QI-NH, a new formulation derived from traditional anti-caries, can be a safe and efficient option to combat dental biofilm. [ABSTRACT FROM AUTHOR]

Anchoring more template proteins on the carrier surface is one of the most critical steps in the preparation process of protein-imprinted polymers. Inspired by the stable interactions between metal ions and proteins, high external surface iron-nickel double hydroxides (Fe–Ni LDH) derived from metal–organic framework were selected as carriers to develop high-performance surface bovine serum albumin (BSA) imprinted iron-nickel double hydroxides nanorods (Fe–Ni LDH@MIP). A hexagonal hollow structure Fe–Ni LDH was synthesized with nanosheets stacked on the surface by etching MIL-88A with Ni2+. The etching of Ni2+ increased the surface roughness of MIL-88A and the rough surface of the carrier was conducive to improving the anchorage amount of BSA, thus providing more effective imprinting sites. Controlled coating of the imprinted polymer layer on the surface of Fe–Ni LDH was obtained by aqueous phase precipitation polymerization. The protein adsorption amount reached 329.8 ± 7.8 mg/g in 60 min with an imprinting factor of 2.86. Fe–Ni LDH@MIP also demonstrated excellent and specific recognition ability in the separation of mixed proteins and fetal bovine serum (FBS) and had good adsorption selectivity, whose adsorption capacity only decreased by 11.5% after 7 adsorption–desorption cycles. In short, Fe–Ni LDH@MIP has great potential to apply in the separation and purification of biomolecules. [ABSTRACT FROM AUTHOR]

Plasmodium vivax malaria poses a major global health challenge, fueled by the parasite's ability to establish chronic infections via dormant liver hypnozoites that enable immune evasion and show transmission resilience. A key virulence determinant of P. vivax blood-stage infection is the ligand-receptor interaction of infected erythrocytes mediated by the Duffy Binding Protein (PvDBP) ligand. Gene duplication events leading to increased PvDBP copy numbers have been documented in parasite populations in Cambodia, Brazil, and Sudan, but whether such changes exist in Indian isolates is not known. India shoulders a disproportionately high P. vivax burden in the world. DNA extracted from malaria-positive samples from a multisite survey was subjected to diagnostic PCR to evaluate PvDBP duplication. We identified PvDBP duplication at 18.6% frequency across various regions in India via diagnostic PCR. Both 'Cambodian-type' and 'Malagasy-type' duplication patterns were detected. PvDBP copy number variations associated with specific Duffy antigen receptor genotypes were correlated in the patient cohort. While PvDBP duplication was widespread, its geographic distribution varied, occurring most prevalently in northeastern regions of India. The presence of Duffy binding protein gene duplication in nearly one-fifth of P. vivax isolates in India may have significant epidemiological and clinical implications. This genetic variation could potentially impact, parasite fitness and invasion efficiency, possibly leading to higher parasitemia levels and immune evasion capabilities, which may affect the efficacy of natural immunity and vaccine development efforts and / or transmission dynamics if the duplication confers any advantage in mosquito stages. To fully understand these implications, we propose longitudinal studies tracking patients with and without PvDBP duplications, comparing clinical outcomes, parasitemia levels, and transmission rates. Additionally, spatial and temporal analyses of duplication frequency across India could reveal patterns of spread and potential selective pressures driving this genetic change. Highlights: 18.6% frequency of PvDBP duplication found across various Indian regions. Both 'Cambodian-type' and 'Malagasy-type' duplication patterns were detected. PvDBP copy number variation was associated with specific ACKR1 antigen receptor genotypes in patients. Geographic distribution of PvDBP duplication varied, with highest prevalence in northeastern India. [ABSTRACT FROM AUTHOR]

The cAMP response element binding protein (CREB)‐binding protein (CBP) is a histone acetyltransferase that plays an indispensable role in regulating the acetylation of histone and non‐histone proteins. Recently, it has been discovered that chemical inhibitors A485 and C646 can bind to Bombyx mori's CBP (BmCBP) and inhibit its acetyltransferase activity. Notably, the binding ability of A485 with BmCBP showed a very low Kd value of 48 nM by surface plasmon resonance (SPR) test. Further identification showed that both A485 and C646 can decrease the acetylation level of known substrate H3K27 and only 1 μM of A485 can almost completely inhibit the acetylation of H3K27, suggesting that A485 is an effective inhibitor of BmCBP's acetyltransferase activity. Moreover, it was confirmed that A485 could downregulate the expression of acetylated Bm30K‐24 protein at a post‐translational level through acetylation modification by BmCBP. Additionally, it was found that A485 can downregulate the stability of Bm30K‐24 and improve its ubiquitination level, suggesting that the acetylation modification by BmCBP could compete with ubiquitination modification at the same lysine site on Bm30K‐24, thereby affecting its protein stability. Here, we predict that A485 may be a potent CBP acetyltransferase inhibitor which could be utilized to inhibit acetyltransferase activity in insects, including silkworms. [ABSTRACT FROM AUTHOR]

Salinity stress is a major concern in regions where irrigation relies on saline water. This study aimed to investigate the relative water content (RWC), electrolytic leakage (EL), total chlorophyll content, free amino acid content, and total soluble sugar content were analyzed in different groundnut species subjected to various salinity treatments. The results showed that salinity stress significantly reduced the RWC in groundnut leaves, with A. duranensis (wild type) exhibiting higher RWC values compared to the Arachis hypogaea species. RNA sequencing was performed to identify differentially expressed genes (DEGs) during salt stress. A total of 9079 DEGs were identified, with 1372 genes upregulated and 2509 genes downregulated. Genes belonging to transcription factor families, such as WRKY, MYB, bHLH, E2F, and Auxin efflux carrier proteins, were induced under salt stress in the tolerant genotype. Conversely, genes encoding NADH dehydrogenase, glutathione S-transferase, protein kinases, UDP-glycosyltransferase, and peroxidase were downregulated. Gene ontology and pathway analyses revealed several enriched categories and metabolic pathways associated with salt stress response, including catalytic activity, response to salt stress, ATP-dependent activity, and oxidative phosphorylation. The findings of this study provide insights into the physiological and molecular responses of groundnut to salinity stress. A. duranensis exhibited better salinity tolerance than Arachis hypogaea , as indicated by higher RWC values, lower electrolytic leakage, and differential gene expression patterns. These results contribute to our understanding of the mechanisms underlying salt stress tolerance in groundnut and may guide future efforts to develop salinity-tolerant groundnut species, ultimately improving crop yield in saline-affected regions. • Salinity stress decreases crop yield by affecting water content and leaf damage in groundnut plants. • A. duranensis variety shows higher tolerance to salinity stress compared to GG20. • Salinity stress reduces chlorophyll levels in groundnut leaves, with A. duranensis being more resilient. • Salinity stress triggers the accumulation of free amino acids and soluble sugars, especially in A. duranensis. • RNA-seq analysis reveals key genes involved in groundnut's response to salt stress, providing valuable insights. [ABSTRACT FROM AUTHOR]

The absorption and transport of selenium (Se) in rice depend on the shared transport proteins and channels with other elements. However, the interactions between Se and other elements within the soil–rice system and their relationship with Se-enriched rice are still not well understood. Hence, we conducted pot experiments to investigate the transformation of Se forms in soil and the absorption and enrichment of Se in rice, which varied with other elements influenced by straw and straw biochar returning in Se-rich red paddy soil. Partial least squares path modeling (PLS-PM) analysis was carried out to reveal the interaction between Se and other elements and the crucial processes in Se enrichment in rice grains. The results showed that the incorporation of straw and straw biochar into the fields increased the content of soil-soluble Se (SOL-Se) but significantly decreased the content of iron-manganese oxide-bound Se (FMO-Se) and organic matter-bound Se (OM-Se). Moreover, the rise in the soil-bioavailable Se was mainly attributed to the activation of FMO-Se and OM-Se. Compared with the NPK treatment, the contents of Se in rice grain were increased by 69.22% and 38.09%, under straw and biochar returning, respectively. However, the contents of Se in the leaves decreased. Variation partitioning analysis (VPA) indicated that the migration of Se in rice plants was significantly influenced by differences in rice tissues and their interactions with other nutrients [nitrogen (N), phosphorus (P), potassium (K), and Se], explaining 51.5% and 35.3% of the variations in Se content in different rice parts, respectively. The PLS-PM analysis demonstrated that the absorption of Se by rice roots and its transportation from the leaves to grains were crucial processes affecting Se enrichment in rice. However, these processes were modulated by the interaction between soil properties and root nutrients (N, P, and Se) induced by straw and straw biochar incorporation. The present study provides further understanding of the main factors and key processes in regulating Se absorption and transformation in the soil–rice system to more efficiently utilize Se-rich paddy fields through agricultural management measures. [ABSTRACT FROM AUTHOR]

Clathrin forms a triskelion, or three-legged, network that regulates cellular processes by facilitating cargo internalization and trafficking in eukaryotes. Its N-terminal domain is crucial for interacting with adaptor proteins, which link clathrin to the membrane and engage with specific cargo. The N-terminal domain contains up to four adaptor-binding sites, though their role in preferential occupancy by adaptor proteins remains unclear. In this study, we examine the binding hierarchy of adaptors for clathrin, using integrative biophysical and structural approaches, along with in vivo functional experiments. We find that yeast epsin Ent5 has the highest affinity for clathrin, highlighting its key role in cellular trafficking. Epsins Ent1 and Ent2, crucial for endocytosis but thought to have redundant functions, show distinct binding patterns. Ent1 exhibits stronger interactions with clathrin than Ent2, suggesting a functional divergence toward actin binding. These results offer molecular insights into adaptor protein selectivity, suggesting they competitively bind clathrin while also targeting three different clathrin sites. Clathrin helps cells internalize and transport cargo by interacting with membrane adaptor proteins. Here, the authors reveal that adaptor proteins bind selectively, hierarchically and simultaneously to three sites of clathrin's N-terminal domain. [ABSTRACT FROM AUTHOR]

Soybean protein is of plant origin and is commonly appropriate for improving the processing characteristics of foods. This study aimed to explore a novel functional ingredient that contained soybean protein isolate (SPI) and blueberry anthocyanins (BANs). The spatial conformation and secondary structure of SPI-BANs complexes were analyzed using circular dichroism and fluorescence spectroscopy, the processing properties were investigated as well as the retention of antioxidant activity during thermal treatments. Results showed that the contents of free sulfhydryl and free amino groups in complexes increased to 3.50 and 1.19 folds than those of SPI, respectively, while the surface hydrophobicity decreased by 74.23%. Compared with SPI, the BANs-modified SPI had a smaller particle size of 29.12 nm and a lower zeta-potential of -8.73 mV and on the other hand, the complexes possessed higher solubility (83.08%) and foaming and emulsifying properties (115.08% and 54.03 m2/g). After fortification with SPI-BANs, the baking loss rate and adhesiveness of chiffon cake were reduced by 10.82% and improved to 0.24 N.mm, respectively. The high antioxidant activities of SPI-BANs under heat led to the cake's bioactivities largely enhanced by 1.99 ~ 12.71 folds, being 345.19 µg Trolox/g for the DPPH radical scavenging activity. This study developed the functional food ingredients as antioxidants and a substitute for animal-based proteins in bakery products, which was safe and sustainable by using the dietary components from plant resources. [ABSTRACT FROM AUTHOR]

Junctions between the ER and plasma membrane (PM) are implicated in calcium homeostasis, non-vesicular lipid transfer, and other cellular functions. Two ER proteins that function both as tethers to the PM via a polybasic C-terminus motif and as phospholipid transporters are brain-enriched TMEM24 (C2CD2L) and its paralog C2CD2. We report that both proteins also form a complex with band 4.1 family members, which in turn bind PM proteins including cell adhesion molecules such as SynCAM 1. This complex enriches TMEM24 and C2CD2 containing ER/PM junctions at sites of cell contacts. Dynamic properties of TMEM24-dependent ER/PM junctions are impacted when band 4.1 is part of the junction, as TMEM24 at cell-adjacent ER/PM junctions is not shed from the PM by calcium rise, unlike TMEM24 at non-cell adjacent junctions. Lipid transport between the ER and the PM by TMEM24 and C2CD2 at sites where cells, including neurons, contact other cells may participate in adaptive responses to cell contact-dependent signaling. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases are often accompanied by neuroinflammation and impairment of the blood-brain barrier (BBB) mediated by activated glial cells through their release of proinflammatory molecules. To study the effects of glial cells on mouse brain endothelial cells (mBECs), we developed an in vitro BBB model with inflammation by preactivating mixed glial cells (MGCs) with lipopolysaccharide (LPS) before co-culturing with mBECs to study the influence of molecules released by activated MGCs. The response of the mBECs to activated MGCs was compared to direct stimulation with LPS. The cytokine profile of activated MGCs was analyzed together with their effects on the mBEC's integrity, expression of tight junction proteins, adhesion molecules, and BBB-specific transport proteins. Stimulation of MGCs significantly upregulated mRNA expression and secretion of several pro-inflammatory cytokines. Co-culturing mBECs with pre-stimulated MGCs significantly affected the barrier integrity of mBECs similar to direct stimulation with LPS. The gene expression levels of tight junction proteins were unaltered, but tight junction proteins revealed rearrangements with respect to subcellular distribution. Compared to direct stimulation with LPS, the expression of cell-adhesion molecules was significantly increased when mBECs were co-cultured with prestimulated MGCs and thus pre-activating MGCs transforms mBECs into a proinflammatory phenotype. [ABSTRACT FROM AUTHOR]

Fungal biosynthetic gene clusters often include genes encoding transmembrane proteins, which have been mostly thought to be transporters exporting the products. However, there is little knowledge about subcellular compartmentalization of transmembrane proteins essential for biosynthesis. Fungal mycotoxin cyclochlorotine is synthesized by non-ribosomal peptide synthetase, which is followed by modifications with three transmembrane UstYa-family proteins. Heterologous expression in Aspergillus oryzae revealed that total biosynthesis of cyclochlorotine requires additional two transporter proteins. Here, we investigated subcellular localizations of the five transmembrane proteins under heterologous expression in A. oryzae. Enhanced green fluorescent protein (EGFP) fusions to the transmembrane proteins, which were confirmed to normally function in cyclochlorotine production, were expressed together with organellar markers. All the transmembrane proteins exhibited localizations commonly in line of the trans -Golgi, endosomes, and vacuoles. This study suggests that subcellular compartmentalization of UstYa family proteins and transporters allows corporative functions of delivering intermediates and subsequent modifications, completing cyclochlorotine biosynthesis. [ABSTRACT FROM AUTHOR]

In yeast, control of sulfur amino acid metabolism relies upon Met4 , a transcription factor that activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by the SCF Met30 E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses sulfur status to tune its E3 ligase activity remains unresolved. Herein, we show that Met30 responds to flux through the trans-sulfuration pathway to regulate the MET gene transcriptional program. In particular, Met30 is responsive to the biological gas hydrogen sulfide, which is sufficient to induce ubiquitination of Met4 in vivo. Additionally, we identify important cysteine residues in Met30 's WD-40 repeat region that sense the availability of sulfur in the cell. Our findings reveal how SCF Met30 dynamically senses the flow of sulfur metabolites through the trans-sulfuration pathway to regulate the synthesis of these special amino acids. [ABSTRACT FROM AUTHOR]

The synaptonemal complex (SC) is a meiotic interface that assembles between parental chromosomes and is essential for gamete formation. While the dimensions and ultrastructure of the SC are conserved across eukaryotes, its protein components are highly divergent. Recently, an unexpected component of the SC has been described in the nematode Caenorhabditis elegans : the Skp1-related proteins SKR-1/2, which are components of the Skp1, Cullin, F-box (SCF) ubiquitin ligase. Here, we find that the role of SKR-1 in the SC is conserved in Pristionchus pacificus. The P. pacificus Skp1 ortholog, Ppa-SKR-1 , colocalizes with other SC proteins throughout meiotic prophase, where it occupies the middle of the SC. Like in C. elegans , the dimerization interface of Ppa-SKR-1 is required for its SC function. A dimerization mutant, ppa-skr-1F105E , fails to assemble SC and produces almost no progeny. Interestingly, the evolutionary trajectory of SKR-1 contrasts with other SC proteins. Unlike most SC proteins, SKR-1 is highly conserved in nematodes. Our results suggest that the structural role of SKR-1 in the SC has been conserved since the common ancestor of C. elegans and P. pacificus , and that rapidly evolving SC proteins have maintained the ability to interact with SKR-1 for at least 100 million years. [ABSTRACT FROM AUTHOR]

Intracellular molecules are transported by motor proteins or move by diffusion resulting from random molecular motion. Cardiomyocytes are packed with structures that are crucial for function, but also confine the diffusional spaces, providing cells with a means to control diffusion. They form compartments in which local concentrations are different from the overall, average concentrations. For example, calcium and cyclic AMP are highly compartmentalized, allowing these versatile second messengers to send different signals depending on their location. In energetic compartmentalization, the ratios of AMP and ADP to ATP are different from the average ratios. This is important for the performance of ATPases fuelling cardiac excitation‐contraction coupling and mechanical work. A recent study suggested that compartmentalization modulates the activity of creatine kinase and adenylate kinase in situ. This could have implications for energetic signaling through, for example, AMP‐activated kinase. It highlights the importance of taking compartmentalization into account in our interpretation of cellular physiology and developing methods to assess local concentrations of AMP and ADP to enhance our understanding of compartmentalization in different cell types. [ABSTRACT FROM AUTHOR]

Plant aspartic proteinases (APs) from Cynara cardunculus feature unique plant-specific insert (PSI) domains, which serve as essential vacuolar sorting determinants, mediating the transport of proteins to the vacuole. Although their role in vacuolar trafficking is well established, the exact molecular mechanisms that regulate PSI interactions and functions remain largely unknown. This study explores the ability of PSI A and PSI B to form homo- and heterodimers using a combination of pull-down assays, the mating-based split-ubiquitin system (mbSUS), and FRET-FLIM analyses. Pull-down assays provided preliminary evidence of potential PSI homo- and heterodimer formation. This was conclusively validated by the more robust in vivo mbSUS and FRET-FLIM assays, which clearly demonstrated the formation of both homo- and heterodimers between PSI A and PSI B within cellular environments. These findings suggest that PSI dimerization is related to their broader functional role, particularly in protein trafficking. Results open new avenues for future research to explore the full extent of PSI dimerization and its implications in plant cellular processes. [ABSTRACT FROM AUTHOR]

RNA polymerase III (Pol III) transcribes tRNA genes using type II promoters. The internal control regions contain a Box A and a Box B, which are recognized by TFIIIC. The 5′-flanking regions of tRNA genes clearly play a role in the regulation of transcription, but consensus sequences in it have been found only in some plants and S. pombe; although, the TATA binding protein (TBP) is a component of the TFIIIB complex in all eukaryotes. Archaea utilize an ortholog of the TBP. The goal of this work is the detection of the positions of intragenic and extragenic promoters of Pol III, which regulate the transcription of tRNA genes in eukaryotes and archaea. For this purpose, we analyzed textual and some structural, mechanical, and physicochemical properties of the DNA in the 5′-flanking regions of tRNA genes, as well as in 30 bp at the beginning of genes and 60 bp at the end of genes in organisms possessing the TBP or its analog (eukaryotes, archaea) and organisms not possessing the TBP (bacteria). Representative tRNA gene sets of 11 organisms were taken from the GtRNAdb database. We found that the consensuses of A- and B-boxes in organisms from all three domains are identical; although, they differ in the conservativism of some positions. Their location relative to the ends of tRNA genes is also identical. In contrast, the structural and mechanical properties of DNA in the 5′-flanking regions of tRNA genes differ not only between organisms from different domains, but also between organisms from the same domain. Well-expressed TBP binding positions are found only in S. pombe and A. thaliana. We discuss possible reasons for the variability of the 5′-flanking regions of tRNA genes. [ABSTRACT FROM AUTHOR]

Alum is the most used vaccine adjuvant, due to its safety, low cost and adjuvanticity to various antigens. However, the mechanism of action of alum is complex and not yet fully understood, and the immune responses elicited can be weak and antigen-dependent. While several antigens rapidly desorb from alum upon exposure to serum, phosphorylated proteins remain tightly bound through a ligand-exchange reaction with surface hydroxyls on alum. Here, bacterial proteins and glycoconjugates have been modified with phosphoserines, aiming at enhancing the binding to alum and prolonging their bioavailability. Tetanus toxoid protein and Salmonella Typhi fragmented Vi-CRM conjugate were used. Both antigens rapidly and completely desorbed from alum after incubation with serum, verified via a competitive ELISA assay, and set up to rapidly evaluate in vitro antigen desorption from alum. After antigen modification with phosphoserines, desorption from alum was slowed down, and modified antigens demonstrated more prolonged retention at the injection sites through in vivo optical imaging in mice. Both modified antigens elicited stronger immune responses in mice, after a single injection only, compared to unmodified antigens. A stronger binding to alum could result in potent single-dose vaccine candidates and opens the possibility to design novel carrier proteins for glycoconjugates and improved versions of bacterial recombinant proteins. [ABSTRACT FROM AUTHOR]

Aptamers are short DNA or RNA sequences that adopt 3D structures and can bind to protein targets with high binding affinity and specificity. Aptamers exhibit excellent tissue penetration, are inexpensive to produce, and can be internalized by cells. Therefore, aptamers are attractive targeting ligands to direct the delivery of theranostic agents to the desired cells. Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that is aberrantly overexpressed on many epithelial-derived cancers, including on cholangiocarcinoma (CCA) cells. Its expression on treatment-resistant cancer stem cells, along with its abundance in the CCA tumor microenvironment, highlights the need to develop EpCAM-targeted therapies for CCA. Herein, an in silico approach was used to design and screen DNA aptamers capable of binding to the EpCAM monomer and homodimer. Two aptamers, PLD01 and PLD02, met the selection criteria and were validated in vitro. Both aptamers exhibited high affinity for EpCAM+ CCA cells, with negligible binding to EpCAM- leukemia cells. Modified versions of PLD01 and PLD02 were successfully incorporated into the membranes of milk-derived nanovesicles. PLD01-functionalized nanovesicles enabled EpCAM-targeted delivery of the therapeutic cargo to CCA cells. In summary, these EpCAM-targeting aptamers can be utilized to direct the delivery of theranostic agents to EpCAM-expressing cells. [ABSTRACT FROM AUTHOR]

Background/Objectives: Besides a multitude of consequences patients on chronic renal replacement therapy have, anemia is one of the most prominent factors making a significant number of patients dependent on erythropoiesis-stimulating agent (ESA) therapy. The aim of this study was to examine the relationship between the levels of a broad spectrum of thrombo-inflammatory and oxidative stress-related biomarkers and the presence and level of ESA hyporesponsiveness in patients undergoing regular chronic hemodialysis. Methods: This cross-sectional study included 96 patients treated with chronic hemodialysis. Levels of several thrombo-inflammatory and oxidative stress-related biomarkers, as well as demographic, clinical, and laboratory analyses, were collected and analyzed based on the calculated value of the ESA-hyporesponsiveness index (EHRI). Results: In the analyzed sample, 58 patients received ESAs. Of all the investigated parameters, only body mass index (BMI), level of plasminogen activator inhibitor-1, and level of L-type fatty acid binding protein (L-FABP) were observed as significant predictors of EHRI. A significant diagnostic potential for ESA resistance has been observed in BMI and L-FABP between ESA-resistant and ESA-non-resistant groups of patients (p = 0.004, area under the curve 0.763 and p = 0.014, area under the curve 0.712, respectively) with the cut-off values of 25.46 kg/m2 and 5355.24 ng/mL, respectively. Having a BMI of 25.46 kg/m2 or less and an L-FABP level higher than 5355.24 ng/mL were observed as significant predictors of ESA resistance (odds ratio 9.857 and 6.125, respectively). Conclusions: EHRI was positively predicted by low BMI and high levels of plasminogen activator inhibitor-1 and L-FABP. High levels of L-FABP and low BMI have been observed as strong predictors of ESA resistance. [ABSTRACT FROM AUTHOR]

The development of novel tools to tackle viral processes has become a central focus in global health, during the COVID-19 pandemic. The spike protein is currently one of the main SARS-CoV-2 targets, owing to its key roles in infectivity and virion formation. In this context, exploring innovative strategies to block the activity of essential factors of SARS-CoV-2, such as spike proteins, will strengthen the capacity to respond to current and future threats. In the present work, we developed and tested novel bispecific molecules that encompass: (i) oligonucleotide aptamers S901 and S702, which bind to the spike protein through its S1 domain, and (ii) hydrophobic tags, such as adamantane and tert-butyl-carbamate-based ligands. Hydrophobic tags have the capacity to trigger the degradation of targets recruited in the context of a proteolytic chimera by activating quality control pathways. We observed that S901-adamantyl conjugates promote the degradation of the S1 spike domain, stably expressed in human cells by genomic insertion. These results highlight the suitability of aptamers as target-recognition molecules and the robustness of protein quality control pathways triggered by hydrophobic signals, and place aptamer-Hytacs as promising tools for counteracting coronavirus progression in human cells. [ABSTRACT FROM AUTHOR]

Simple Summary: The Yes-associated protein (YAP) is part of a system that regulates cell proliferation in response to changes in cell–cell and cell–matrix interactions. In malignant tumors, that system is almost always disrupted in ways that promote the stability of YAP. Mutations in genes encoding Ras proteins are found in 30% of all human cancers, but the mechanisms by which these mutated proteins promote cancer are still incompletely understood. Here, we investigated the regulation of YAP stability by the Ras oncogenes and found that while Ras does not directly regulate YAP, the stability of YAP is directly controlled by one of Ras's own effectors, the Rac1 GTPase, a small regulatory protein involved in controlling cell shape and migration. This regulation of YAP by Rac1 could potentially play an important role in the development of therapeutic resistance to Ras inhibitors, which we know can be promoted by the overexpression of YAP. Background: Oncogenic mutations in the KRAS gene are detected in >90% of pancreatic cancers (PC). In genetically engineered mouse models of PC, oncogenic KRAS drives the formation of precursor lesions and their progression to invasive PC. The Yes-associated Protein (YAP) is a transcriptional coactivator required for transformation by the RAS oncogenes and the development of PC. In Ras-driven tumors, YAP can also substitute for oncogenic KRAS to drive tumor survival after the repression of the oncogene. Ras oncoproteins exert their transforming properties through their downstream effectors, including the PI3K kinase, Rac1 GTPase, and MAPK pathways. Methods: To identify Ras effectors that regulate YAP, YAP levels were measured in PC cells exposed to inhibitors of oncogenic K-Ras and its effectors. Results: In PC cells, the inhibition of Rac1 leads to a time-dependent decline in YAP protein, which could be blocked by proteosome inhibitor MG132. This YAP degradation after Rac1 inhibition was observed in a range of cell lines using different Rac1 inhibitors, Rac1 siRNA, or expression of dominant negative Rac1T17N mutant. Several E3 ubiquitin ligases, including SCFβTrCP, regulate YAP protein stability. To be recognized by this ligase, the βTrCP degron of YAP (amino acid 383–388) requires its phosphorylation by casein kinase 1 at Ser384 and Ser387, but these events must first be primed by the phosphorylation of Ser381 by LATS1/2. Using Flag-tagged mutants of YAP, we show that YAP degradation after Rac1 inhibition requires the integrity of this degron and is blocked by the silencing of βTrCP1/2 and by the inhibition of casein kinase 1. Unexpectedly, YAP degradation after Rac1 inhibition was still observed after the silencing of LATS1/2 or in cells carrying a LATS1/2 double knockout. Conclusions: These results reveal Rac1 as an oncogenic KRAS effector that contributes to YAP stabilization in PC cells. They also show that this regulation of YAP by Rac1 requires the SCFβTrCP ligase but occurs independently of the LATS1/2 kinases. [ABSTRACT FROM AUTHOR]

Peptide-based anticancer vaccines have shown some efficacy in generating cancer-specific immune responses in various cancer studies, but clinical success is limited, one of the reasons is due to its prone degradation and weak immunogenicity. So some tumor epitope peptide vaccines often require coupling or forming fusion proteins with corresponding protein carriers to enhance their stability and immunogenicity. Given the scarcity of validated carriers for clinical trials, there is an urgent requirement for the development of novel protein carrier. Our previous work has demonstrated that VEGF165b mutant could be used as an effective immunization adjunct to enhance anti-tumor immune response. By analyzing and evaluating the gene structure of VEGF, we speculated that mVEGF165b has the potential to be utilized as a tumor peptide vaccine carrier. An mVEGF165b-MUC1 chimeric tumor vaccine was produced by fusing the MUC1 peptide （(MUC1, a T-cell epitope dominant peptide from Mucin1） to the C-terminus of mVEGF165b, expressing the fusing protein in pichia yeast, followed by purification with a HiTrap heparin affinity chromatography column. We found that immunizing mice with mVEGF165b-MUC1 fusion protein induced high-titer antibodies against VEGF in a preventive context, which in turn reduced the proportion of Tregs and further stimulated mice to produce T-cell responses specific to mucin1. The high-titer VEGF antibody stimulated by mVEGF165b also promoted tumor blood vessel maturation and facilitated T-cell infiltration. In conclusion，immunized with mVEGF165b-MUC1 protein are beneficial for eliciting immune responses targeting Mucin1, mVEGF165b have the potential to be utilized as a peptide tumor vaccine carrier. • mVEGF165b can be used as a protein carrier for the MUC1 peptide. • High anti-VEGF antibody titers induced by mVEGF165b-MUC1 decreased immunosuppression by reducing the proportion of Tregs. • The decreased immunosuppression augmented the immune response induced by the peptide vaccine from Mucin-1. [ABSTRACT FROM AUTHOR]

This is the first case report of a non‐small‐cell lung cancer (NSCLC) patient harboring HIP1‐ALK (H28:A20) and CTNNB1 p.S45del treated with first‐line alectinib. Approximately 5% of NSCLC patients are reported to have anaplastic lymphoma kinase (ALK) rearrangements, and among these EML4‐ALK is the most frequent fusion variant. However, in recent years the use of next‐generation sequencing (NGS) in clinical laboratories has become increasingly widespread, identifying a lot of new ALK fusion partners as well as a large quantity of co‐occurring genomic alterations. Unfortunately, the growing number of genomic alterations detected by NGS does not always correspond to adequate knowledge of their clinical significance, often resulting in an empiric treatment of patients harboring uncommon mutations. [ABSTRACT FROM AUTHOR]

Hypothetical and experimental models of unbound fraction have been proposed to facilitate predicting the hepatic clearance (CL H) of drugs from values of intrinsic clearance for the unbound drug (CL int-in vitro-unbound) and the well-stirred model (WSM). The hypothetical model (fu -adjusted) is adjusting the unbound fractions determined in plasma in vitro to estimate the maximum unbound fractions at the hepatocytes if each drug-protein complex in plasma becomes fully dissociated at the membrane by any albumin (ALB)-facilitated hepatic uptake mechanism. The model of fu -adjusted is also adjusting the unbound fraction for a pH gradient effect across the membrane. Alternatively, the new experimental model (fu -dynamic) measures the unbound fractions resulting to the dynamic dissociation kinetics from proteins in the presence of plasma and a liver enzyme in an in vitro assay. The objective of this study was to conduct an in-depth analysis of previous CL H predictions made with these unbound fractions in a companion manuscript. Furthermore, a new dataset on transporter substrates was also included in this study. Finally, the physiological basis of fu -adjusted has been redefined to extend its applicability with more drugs. In this case, there are lower concentrations of binding proteins in liver versus plasma that could also explain the higher unbound fractions for that organ. The outcomes associated to additional analyses pointed out that fu -adjusted , again, generally provided the most accurate predictions of CL H because fu -dynamic has generated superior biases of underpredictions or overpredictions. For slowly metabolized drugs bound to ALB, fu -dynamic was definitively less accurate than fu -adjusted. For other drug properties, fu -dynamic fared better but it was still not generally more accurate than fu -adjusted. Furthermore, experimental values of fu -dynamic were sometimes incoherent. For example, drugs bound to alpha-acid glycoprotein (AGP) did not follow the principle of fu -dynamic (i.e. , values of fu -dynamic did not correlate with values of CL int-in vitro-unbound) by contrast to those drugs bound to ALB. Therefore, the current experimental setting for fu -dynamic might be unsuitable in some circumstances. Overall, this study confirmed that calculated values of fu -adjusted were as accurate as experimental values of fu -dynamic and can even be more accurate. A guidance on which unbound fraction to use in the WSM is also provided. [ABSTRACT FROM AUTHOR]

AbstractBackgroundMethodsResultsConclusionsGraves’ orbitopathy (GO) is an autoimmune condition that causes serious ocular symptoms; its treatment strategies are limited. Physalin A is a phytosterol that has shown various therapeutic properties, including anti-inflammatory and anti-fibrotic effects. In this study, we investigated whether physalin A could inhibit inflammation, fibrosis, hyaluronan (hyaluronic acid) production, and adipogenesis, which are crucial to the pathogenesis of GO.Orbital tissue explants were obtained from patients with GO during orbital decompression surgery and healthy controls. Orbital fibroblasts (OFs) were isolated and treated with different concentrations of physalin A. Using western blot and ELISA analyses, we determined the effects of physalin A on OFs.Physalin A treatment suppressed the production of interleukin (IL)-1β-induced prostaglandin E2 (PGE2) and pro-inflammatory molecules, including cyclooxygenase (COX)-2, IL-6, IL-8, and intercellular adhesion molecule (ICAM)-1. We discovered that physalin A attenuated hyaluronan production induced by IL-1β or insulin-like growth factor (IGF)-1. Moreover, physalin A reduced lipid droplet formation and production of peroxisome proliferator activator (PPAR) γ, CCAAT-enhancer-binding protein (C/EBP) α, C/EBP β, sterol regulatory element binding protein (SREBP)-1, leptin, and adiponectin proteins. Physalin A suppressed the phosphorylation of extracellular signal-related kinase (ERK), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and suppressor of mothers against decapentaplegic (SMAD) 2 signaling protein.Our study suggests that the major mechanisms by which physalin A suppresses GO include reducing inflammation, fibrosis, hyaluronan production, and adipogenesis in OFs. The findings of this study provide evidence of the therapeutic effect of physalin A in GO. [ABSTRACT FROM AUTHOR]

Amino acids are important for cellular metabolism. Their uptake across the plasma membrane is mediated by transport proteins. Despite the fact that the organic anion transporting polypeptide 4C1 (OATP4C1, Uniprot: Q6ZQN7) mediates transport of l‐arginine and l‐arginine derivatives, other members of the OATP family have not been characterized as amino acid transporters. The OATP family member OATP3A1 (gene symbol SLCO3A1, Uniprot: Q9UIG8) is ubiquitously expressed in human cells and highly expressed in many cancer tissues and cell lines. However, only a few substrates are known for OATP3A1. Accordingly, knowledge about its biological relevance is restricted. Our aim was to identify new substrates of OATP3A1 to gain insights into its (patho‐)physiological function. In an LC‐MS‐based untargeted metabolomics assay using untreated OATP3A1‐overexpressing HEK293 cells and control cells, we identified several amino acids as potential substrates of OATP3A1. Subsequent uptake experiments using exogenously added substrates revealed OATP3A1‐mediated transport of l‐tryptophan, l‐tyrosine, and l‐phenylalanine with 194.8 ± 28.7% (P < 0.05), 226.2 ± 18.7% (P < 0.001), and 235.2 ± 13.5% (P < 0.001), respectively, in OATP3A1‐overexpressing cells compared to control cells. Furthermore, kinetic transport parameters (Km values) were determined (Trp = 61.5 ± 14.2 μm, Tyr = 220.8 ± 54.5 μm, Phe = 234.7 ± 20.6 μm). In summary, we identified the amino acids l‐tryptophan, l‐tyrosine, and l‐phenylalanine as new substrates of OATP3A1. These findings could be used for a better understanding of (patho‐)physiological processes involving increased demand of amino acids, where OATP3A1 should be considered as an important uptake transporter of l‐tryptophan, l‐tyrosine, and l‐phenylalanine. [ABSTRACT FROM AUTHOR]

Identifying biomarkers can help in the early detection of muscle loss and drive the development of new therapies. Research suggests a potential link between retinol-binding protein 4 (RBP4) and muscle mass, particularly in postmenopausal women. This study aimed to examine the association between baseline RBP4 levels and changes in appendicular lean mass (ALM), an indicator of muscle mass, in postmenopausal women. A 12-month follow-up period (n = 153) included baseline and 12-month ALM assessments using DXA. ALM was normalized to squared height (ALMI). Baseline evaluations encompassed insulin resistance via HOMA-IR and immunoassay magnetic bead panel measurements of RPB4, IL-6, TNF-α, and IL-10. Postmenopausal women were categorized into higher (n = 77) and lower (n = 76) RPB4 groups based on baseline RPB4 values. Their changes in ALMI were compared using Mann–Whitney tests. General linear model was employed to evaluate the predictive power of baseline RBP4 for ALMI changes, adjusting for confounding variables: age, physical activity, smoking status, body fat, HOMA-IR, inflammatory markers (TNF-α and IL-6), and anti-inflammatory factor (IL-10). The higher RBP4 group exhibited a more pronounced reduction in ALMI compared to the lower RBP4 group (Higher RBP4 = −0.39 kg/m2, 95% CI: −0.48 to −0.31 kg/m2vs. Lower RBP4 = −0.24 kg/m2, 95% CI: −0.32 to −0.15 kg/m2, P = 0.011). After adjusting for confounding factors, the association between baseline RBP4 changes and ALMI remained (b = −0.008, SE = 0.002, P < 0.001), indicating higher baseline RBP4 values linked to greater ALMI reduction. Our findings support RBP4 as a potential biomarker for changes in muscle mass in postmenopausal women. [ABSTRACT FROM AUTHOR]

Background: Viral myocarditis (VMC) is a common cardiovascular disease, and circular RNAs (circRNAs) have been identified to play an important role in the pathophysiology of cardiovascular disease. However, the clinical signifi ance, biological functions, and regulatory mechanisms of circRNAs in VMC remain poorly understood. Therefore, this study explored the biological functions and regulatory mechanisms of circ-ACSL1 in VMC. Methods: The animal and cell models of VMC were established by infecting BABL/C mice and interleukin-2 cells with coxsackievirus B3 (CVB3). Pro-inflamma ory factors, markers of myocardial injury, apoptosis, and autophagy were detected to evaluate the degree of myocardial inflammation and myocardial injury after altering circ-ACSL1, microRNA- 7-5p (miR-7-5p), and X-box binding protein 1 (XBP1) expression alone or in combination. Results: Knocking down circ-ACSL1 could inhibit inflammation, autophagy, and apoptosis in VMC animals and cells. Mechanistically, circ-ACSL1 targeted miR-7-5p to regulate the downstream target XBP1. In addition, depleting miR-7-5p rescued the therapeutic effect of depleting circ-ACSL1. Overexpression of circ-ACSL1 aggravated VMC; however, this effect was saved by knocking down XBP1. Conclusion: By competitively absorbing miR-7-5p, circ-ACSL1 increases XBP1 expression and aggravates myocardial inflammation. Meaningfully, VMC treatment may benefit from circ-ACSL1 as a potential biomarker for precise diagnosis and as a potential therapeutic target. [ABSTRACT FROM AUTHOR]

SUMMARY: Genome stability is significantly influenced by the precise coordination of chromatin complexes that facilitate the loading and eviction of histones from chromatin during replication, transcription, and DNA repair processes. In this study, we investigate the role of the Arabidopsis H3 histone chaperones ANTI‐SILENCING FUNCTION 1 (ASF1) and HISTONE REGULATOR A (HIRA) in the maintenance of telomeres and 45S rDNA loci, genomic sites that are particularly susceptible to changes in the chromatin structure. We find that both ASF1 and HIRA are essential for telomere length regulation, as telomeres are significantly shorter in asf1a1b and hira mutants. However, these shorter telomeres remain localized around the nucleolus and exhibit a comparable relative H3 occupancy to the wild type. In addition to regulating telomere length, ASF1 and HIRA contribute to silencing 45S rRNA genes and affect their copy number. Besides, ASF1 supports global heterochromatin maintenance. Our findings also indicate that ASF1 transiently binds to the TELOMERE REPEAT BINDING 1 protein and the N terminus of telomerase in vivo, suggesting a physical link between the ASF1 histone chaperone and the telomere maintenance machinery. [ABSTRACT FROM AUTHOR]