Protocol for analyzing TRAIL- and Fas-induced signaling complexes by immunoprecipitation from human cells.

Engagement of TRAIL or Fas death receptors can trigger the assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) signaling complexes that promote nuclear factor κB (NF-κB) activation. Here, we present a protocol for immunoprecipitation of TRAIL- or Fas-induced FADDosomes from human cell lines. We describe steps for stimulating human cells with TRAIL or Fas ligand, followed by preparation of membrane death receptor-associated, as well as cytoplasmic FADDosome, signaling complexes. This protocol has application in the analysis of death receptor-induced signaling complex formation. For complete details on the use and execution of this protocol, please refer to Davidovich et al.1.

Corosolic acid attenuates cardiac ischemia/reperfusion injury through the PHB2/PINK1/parkin/mitophagy pathway.

Despite advances in treatment, myocardial infarction remains the leading cause of heart failure and death worldwide, and the restoration of coronary blood flow can also cause heart damage. In this study, we found that corosolic acid (CA), also known as plant insulin, significantly protects the heart from ischemia-reperfusion (I/R) injury. In addition, CA can inhibit oxidative stress and improve mitochondrial structure and function in cardiomyocytes. Subsequently, our study demonstrated that CA improved the expression of the mitophagy-related proteins Prohibitin 2 (PHB2), PTEN-induced putative kinase protein-1 (PINK1), and Parkin. Meanwhile, through molecular docking, we found an excellent binding between CA and PHB2 protein. Finally, the knockdown of PHB2 eliminated the protective effect of CA on hypoxia-reoxygenation in cardiomyocytes. Taken together, our study reveals that CA increases mitophagy in cardiomyocytes via the PHB2/PINK1/Parkin signaling pathway, inhibits oxidative stress response, and maintains mitochondrial function, thereby improving cardiac function after I/R.

Review of AlphaFold 3: Transformative Advances in Drug Design and Therapeutics.

Google DeepMind Technologies Limited (London, United Kingdom) recently released its new version of the biomolecular structure predictor artificial intelligence (AI) model named AlphaFold 3. Superior in accuracy and more powerful than its predecessor AlphaFold 2, this innovation has astonished the world with its capacity and speed. It takes humans years to determine the structure of various proteins and how the shape works with the receptors but AlphaFold 3 predicts the same structure in seconds. The version's utility is unimaginable in the field of drug discoveries, vaccines, enzymatic processes, and determining the rate and effect of different biological processes. AlphaFold 3 uses similar machine learning and deep learning models such as Gemini (Google DeepMind Technologies Limited). AlphaFold 3 has already established itself as a turning point in the field of computational biochemistry and drug development along with receptor modulation and biomolecular development. With the help of AlphaFold 3 and models similar to this, researchers will gain unparalleled insights into the structural dynamics of proteins and their interactions, opening up new avenues for scientists and doctors to exploit for the benefit of the patient. The integration of AI models like AlphaFold 3, bolstered by rigorous validation against high-standard research publications, is set to catalyze further innovations and offer a glimpse into the future of biomedicine.

Deciphering the actin structure-dependent preferential cooperative binding of cofilin.

The mechanism underlying the preferential and cooperative binding of cofilin and the expansion of clusters toward the pointed-end side of actin filaments remains poorly understood. To address this, we conducted a principal component analysis based on available filamentous actin (F-actin) and C-actin (cofilins were excluded from cofilactin) structures and compared to monomeric G-actin. The results strongly suggest that C-actin, rather than F-ADP-actin, represented the favourable structure for binding preference of cofilin. High-speed atomic force microscopy explored that the shortened bare half helix adjacent to the cofilin clusters on the pointed end side included fewer actin protomers than normal helices. The mean axial distance (MAD) between two adjacent actin protomers along the same long-pitch strand within shortened bare half helices was longer (5.0-6.3 nm) than the MAD within typical helices (4.3-5.6 nm). The inhibition of torsional motion during helical twisting, achieved through stronger attachment to the lipid membrane, led to more pronounced inhibition of cofilin binding and cluster formation than the presence of inorganic phosphate (Pi) in solution. F-ADP-actin exhibited more naturally supertwisted half helices than F-ADP.Pi-actin, explaining how Pi inhibits cofilin binding to F-actin with variable helical twists. We propose that protomers within the shorter bare helical twists, either influenced by thermal fluctuation or induced allosterically by cofilin clusters, exhibit characteristics of C-actin-like structures with an elongated MAD, leading to preferential and cooperative binding of cofilin.

Impact of protein and small molecule interactions on kinase conformations.

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

The mouse epididymal amyloid matrix is a mammalian counterpart of a bacterial biofilm.

The mouse epididymis is a long tubule connecting the testis to the vas deferens. Its primary functions are to mature spermatozoa into motile and fertile cells and to protect them from pathogens that ascend the male tract. We previously demonstrated that a functional extracellular amyloid matrix surrounds spermatozoa in the epididymal lumen and has host defense functions, properties not unlike that of an extracellular biofilm that encloses and protects a bacterial community. Here we show the epididymal amyloid matrix also structurally resembles a biofilm by containing eDNA, eRNA, and mucin-like polysaccharides. Further these structural components exhibit comparable behaviors and perform functions such as their counterparts in bacterial biofilms. Our studies suggest that nature has used the ancient building blocks of bacterial biofilms to form an analogous structure that nurtures and protects the mammalian male germline.

Variation of retinal and choroidal microvascularization after an exercise stress test in patients with effort angina.

PURPOSE: To describe retinal and choroidal vascular changes following an exercise stress test (ET) in patients with effort angina and to determine whether optical coherence tomography angiography (OCT-A) could play a role in the prediction of ischemic cardiac events. METHODS: Prospective comparative study including patients with effort angina. All patients underwent OCT-A before and after an ET was performed. Blood flow, intercapillary spaces, and vessel density were analyzed in the superficial capillary plexus (SCP) and the deep capillary plexus (DCP). Vessel density in the choriocapillaris and the parameters of the central avascular zone (CAZ) were also analyzed. RESULTS: Of the 38 eyes included in the study, a decrease in blood flow was found in 39.5% in the large SCP vessels, in 50% in the capillaris of the SCP, and in 81.6% in the DCP. An increase in intercapillary spaces was observed in the SCP in 68.4% of eyes and in the DCP in 55.3% of eyes. A statistically significant decrease in the DCP density was observed after an ET (p = 0.03). There were no significant modifications in the CAZ parameters, the SCP density, nor the choriocapillaris density. Patients with a positive ET had a decreased DCP density in 83.3%. Among patients with an increased DCP density, 92.85% had a negatif ET. CONCLUSION: This pilot study suggests that DCP density significantly decreases after an ET. The DCP appears to be most affected in patients with effort angina. A larger trial is needed to further investigate these hypotheses.

Impacts of conventional and biodegradable microplastics in maize-soil ecosystems: Above and below ground.

The increasing accumulation of microplastics (MPs) in agroecosystems has raised significant environmental and public health concerns, facilitating the application of biodegradable plastics. However, the comparative effects of conventional and biodegradable MPs in agroecosystem are still far from fully understood. Here we developed microcosm experiments to reveal the ecological effects of conventional (polyethylene [PE] and polypropylene [PP]) and biodegradable (polyadipate/butylene terephthalate [PBAT] and polycaprolactone [PCL]) MPs (0, 1%, 5%; w/w) in the maize-soil ecosystem. We found that PCL MPs reduced plant production by 73.6-75.2%, while PE, PP and PBAT MPs elicited almost negligible change. The addition of PCL MPs decreased specific enzyme activities critical for soil nutrients cycling by 71.5-95.3%. Biodegradable MPs tended to reduce bacterial α-diversity. The 1% treatments of PE and PBAT, and PCL enhanced bacterial networks complexity, whereas 5% of PE and PBAT, and PP had adverse effect. Moreover, biodegradable MPs appeared to reduce the α-diversity and networks complexity of fungal community. Overall, PCL reduced the ecosystem multifunctionality, mainly by inhibiting the microbial metabolic activity. This study offers evidence that biodegradable MPs can impair agroecosystem multifunctionality, and highlights the potential risks to replace the conventional plastics by biodegradable ones in agricultural practices.

HTRF-based assay for detection of mono-ADP-ribosyl hydrolyzing macrodomains and inhibitor screening.

The COVID-19 pandemic has highlighted the lack of effective, ready-to-use antivirals for the treatment of viruses with pandemic potential. The development of a diverse drug portfolio is therefore crucial for pandemic preparedness. Viral macrodomains are attractive therapeutic targets as they are suggested to play an important role in evading the innate host immune response, making them critical for viral pathogenesis. Macrodomains function as erasers of mono-ADP-ribosylation (deMARylation), a post-translational modification that is involved in interferon signaling. Herein, we report the development of a modular HTRF-based assay, that can be used to screen for inhibitors of various viral and human macrodomains. We characterized the five most promising small molecule SARS-CoV-2 Mac1 inhibitors recently reported in the literature for potency and selectivity and conducted a pilot screen demonstrating HTS suitability. The ability to directly detect enzymatic activity makes the DeMAR assay a valuable addition to the existing tools for macrodomain drug discovery.

Characterizing replisome disassembly in human cells.

To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for "backup" mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes.

The role of m6A in angiogenesis and vascular diseases.

Angiogenesis, whether physiological or pathological, plays a pivotal role in various physiological and disease conditions. This intricate process relies on a complex and meticulously orchestrated signal transduction network that connects endothelial cells, their associated parietal cells (VSMCs and pericytes), and various other cell types, including immune cells. Given the significance of m6A and its connection to angiogenesis and vascular disease, researchers must adopt a comprehensive and ongoing approach to their investigations. This study aims to ascertain whether a common key mechanism of m6A exists in angiogenesis and vascular diseases and to elucidate the potential application of m6A in treating vascular diseases.

Allosteric regulation of kinase activity.

The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.

ADAMTS18-fibronectin interaction regulates the morphology of liver sinusoidal endothelial cells.

Liver sinusoidal endothelial cells (LSECs) have a unique morphological structure known as "fenestra" that plays a crucial role in liver substance exchange and homeostasis maintenance. In this study, we demonstrate that ADAMTS18 protease is primarily secreted by fetal liver endothelial cells. ADAMTS18 deficiency leads to enlarged fenestrae and increased porosity of LSECs, microthrombus formation in liver vessels, and an imbalance of liver oxidative stress. These defects worsen carbon tetrachloride (CCl4)-induced liver fibrosis and diethylnitrosamine (DEN)/high-fat-induced hepatocellular carcinoma (HCC) in adult Adamts18-deficient mice. Mechanically, ADAMTS18 functions as a modifier of fibronectin (FN) to regulate the morphological acquisition of LSECs via the vascular endothelial growth factor A (VEGFA) signaling pathways. Collectively, a mechanism is proposed for LSEC morphogenesis and liver homeostasis maintenance via ADAMTS18-FN-VEGFA niches.

Biomolecular alterations temporally anticipate microarchitectural modifications of collagen in oral tongue squamous cell carcinoma.

High resolution analysis of collagen bundles could provide information on tumor onset and evolution. This study was focused on the microarchitecture and biomolecular organization of collagen bundles in oral tongue squamous cell carcinoma (OTSCC). Thirty-five OTSCC biopsy samples were analyzed by synchrotron-based phase-contrast microcomputed tomography and Fourier transform infrared imaging (FTIRI) spectroscopy. PhC-microCT evidenced the presence of reduced and disorganized collagen in the tumor area compared to the extratumoral (ExtraT) one. FTIRI also revealed a reduction of folded secondary structures in the tumor area, and highlighted differences in the peritumoral (PeriT) areas in relation with the OTSCC stage, whereby a significantly lower amount of collagen with less organized fibers was found in the PeriT stroma of advanced-OTSCC stages. Interestingly, no significant morphometrical mismatches were detected in the same region by PhC-microCT analysis. These results suggest that biomolecular alterations in the OTSCC stroma temporally anticipate structural modifications of collagen bundle microarchitecture.

The protective role of GATA6+ pericardial macrophages in pericardial inflammation.

Prior research has suggested that GATA6+ pericardial macrophages may traffic to the myocardium to prevent interstitial fibrosis after myocardial infarction (MI), while subsequent literature claims that they do not. We demonstrate that GATA6+ pericardial macrophages are critical for preventing IL-33 induced pericarditis and attenuate trafficking of inflammatory monocytes and granulocytes to the pericardial cavity after MI. However, absence of GATA6+ macrophages did not affect myocardial inflammation due to MI or coxsackievirus-B3 induced myocarditis, or late-stage cardiac fibrosis and cardiac function post MI. GATA6+ macrophages are significantly less transcriptionally active following stimulation in vitro compared to bone marrow-derived macrophages and do not induce upregulation of inflammatory markers in fibroblasts. This suggests that GATA6+ pericardial macrophages attenuate inflammation through their interactions with surrounding cells. We therefore conclude that GATA6+ pericardial macrophages are critical in modulating pericardial inflammation, but do not play a significant role in controlling myocardial inflammation or fibrosis.

The toxicity effects of imidacloprid and chlorpyrifos on oxidative stress and blood biochemistry in Cyprinus carpio.

This study aimed to assess the toxicity effects of chlorpyrifos and imidacloprid, alone and in combination, on oxidative biomarkers and blood biochemistry of Cyprinus carpio. A total of 324 common carp (Cyprinus carpio) were distributed among 27 tanks and exposed to concentrations of 0.0, 100, and 200 µg L-1 of chlorpyrifos and 0.0, 10.0, and 20.0 µg L-1 of imidacloprid for 28 days. Changes in enzyme activities in the plasma of fish exposed to chlorpyrifos depended on the dose. In contrast, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatine phosphokinase (CPK), gamma-glutamyl transferase (GGT) activities were significantly increased in fish exposed to imidacloprid, alone and in combination with chlorpyrifos. However, the activity of butyrylcholinesterase (BChE) was significantly decreased. Exposure to imidacloprid and chlorpyrifos, alone and in combination, increased glucose, urea, cholesterol, triglycerides, and creatinine levels, whereas total protein and albumin levels were significantly decreased. The activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), and catalase (CAT) was significantly increased, while glutathione reductase (GR) was significantly decreased. Additionally, although the total antioxidant capacity (TAN) was significantly decreased, malondialdehyde (MDA) levels increased after exposure to imidacloprid and chlorpyrifos, alone and in combination. In conclusion, exposure to imidacloprid and chlorpyrifos, alone and in combination, induced oxidative stress and altered blood biochemistry in carp fish. Moreover, imidacloprid and chlorpyrifos had synergistic effects on some oxidative and biochemical biomarkers.

Swept-source optical coherence tomography early findings in patients with carotid artery disease.

PURPOSE: Ocular ischemic syndrome can be the first and only hint of life-threatening carotid artery disease. The early recognition of carotid stenosis-related retinal signs, as well as the comprehension of the pathophysiology behind retinal changes could become relevant for physicians to predict the risk of stroke. The aim of this study is to assess the carotid artery disease-induced early structural retinochoroidal changes by means of swept-source optical coherence tomography (SS-OCT). METHODS: A prospective observational study was conducted in 72 eyes with carotid stenosis. According to the degree of stenosis, the participants were divided into a normal group (34 eyes), a mild-moderate stenosis group (22 eyes), a severe stenosis group (16 eyes). SS-OCT and OCTA were performed to scan macular fovea. Central macular thickness (CMT), subfoveal choroidal thickness (SCT) and foveal avascular zona (FAZ) area were the major measurements for our study. RESULTS: CMT was significantly thicker in group 3 when compared to group 2 and 1. SCT was significantly thinner in group 3 vs group 1, being thicker in group 2 when compared to group 1. No significant differences were obtained when comparing FAZ in the superficial and middle capillary plexus although it was significant when comparing the FAZ in the deep capillary plexus between group 1 and 3. CONCLUSION: internal carotid artery stenosis greater than 70% leads to a significant increase in CMT and a decrease in SCT prior the development of clinical findings of ocular ischemia syndrome.

Enrichment of rare codons at 5' ends of genes is a spandrel caused by evolutionary sequence turnover and does not improve translation.

Previously, Tuller et al. found that the first 30-50 codons of the genes of yeast and other eukaryotes are slightly enriched for rare codons. They argued that this slowed translation, and was adaptive because it queued ribosomes to prevent collisions. Today, the translational speeds of different codons are known, and indeed rare codons are translated slowly. We re-examined this 5' slow translation 'ramp.' We confirm that 5' regions are slightly enriched for rare codons; in addition, they are depleted for downstream Start codons (which are fast), with both effects contributing to slow 5' translation. However, we also find that the 5' (and 3') ends of yeast genes are poorly conserved in evolution, suggesting that they are unstable and turnover relatively rapidly. When a new 5' end forms de novo, it is likely to include codons that would otherwise be rare. Because evolution has had a relatively short time to select against these codons, 5' ends are typically slightly enriched for rare, slow codons. Opposite to the expectation of Tuller et al., we show by direct experiment that genes with slowly translated codons at the 5' end are expressed relatively poorly, and that substituting faster synonymous codons improves expression. Direct experiment shows that slow codons do not prevent downstream ribosome collisions. Further informatic studies suggest that for natural genes, slow 5' ends are correlated with poor gene expression, opposite to the expectation of Tuller et al. Thus, we conclude that slow 5' translation is a 'spandrel'--a non-adaptive consequence of something else, in this case, the turnover of 5' ends in evolution, and it does not improve translation.