Milton assembles large mitochondrial clusters, mitoballs, to sustain spermatogenesis.

Mitochondria are dynamic organelles that undergo frequent remodeling to accommodate developmental needs. Here, we describe a striking organization of mitochondria into a large ball-like structure adjacent to the nucleus in premeiotic Drosophila melanogaster spermatocytes, which we term "mitoball". Mitoballs are transient structures that colocalize with the endoplasmic reticulum, Golgi bodies, and the fusome. We observed similar premeiotic mitochondrial clusters in a wide range of insect species, including mosquitos and cockroaches. Through a genetic screen, we identified that Milton, an adaptor protein that links mitochondria to microtubule-based motors, mediates mitoball formation. Flies lacking a 54 amino acid region in the C terminus of Milton completely lacked mitoballs, had swollen mitochondria in their spermatocytes, and showed reduced male fertility. We suggest that the premeiotic mitochondrial clustering is a conserved feature of insect spermatogenesis that supports sperm development.

The predictive utility of cytokines, procalcitonin and C-reactive protein among febrile pediatric hematology and oncology patients with severe sepsis or septic shock.

Severe sepsis and septic shock are life-threatening for pediatric hematology and oncology patient receiving chemotherapy. Th1/Th2 cytokines, C-reactive protein (CRP), and procalcitonin (PCT) are all thought to be associated with disease severity. The aim of this study was to prospectively verify the utility of Th1/Th2 cytokines and compare them with PCT and CRP in the prediction of adverse outcomes. Data on patients were collected from January 1, 2011, to December 31, 2020. Blood samples were taken for Th1/Th2 cytokine, CRP, and PCT measurements at the initial onset of infection. Severe infection (SI) was defined as severe sepsis or septic shock. Th1/Th2 cytokine levels were determined by using flow cytometric bead array technology. In total, 7,735 febrile episodes were included in this study. For SI prediction, the AUCs of IL-6, IL-10 and TNF-α were 0.814, 0.805 and 0.624, respectively, while IL-6 and IL-10 had high sensitivity and specificity. IL-6 > 220.85 pg/ml and IL-10 > 29.95 pg/ml had high odds ratio (OR) values of approximately 3.5 in the logistic regression. Within the subgroup analysis, for bloodstream infection (BSI) prediction, the AUCs of IL-10 and TNF-α were 0.757 and 0.694, respectively. For multiorgan dysfunction syndrome (MODS) prediction, the AUC of CRP was 0.606. The AUC of PCT for mortality prediction was 0.620. In conclusion, IL-6 and IL-10 provide good predictive value for the diagnosis of SI. For children with SI, IL-10 and TNF-α are associated with BSI, while CRP and PCT are associated with MODS and death, respectively.

Two new jatrophane diterpenoids from Euphorbia helioscopia with activity towards autophagic flux.

Nine jatrophane diterpenoids were isolated from the whole plant Euphorbia helioscopia, including two new ones, helioscopnins A (1) and B (2). Comprehensive spectroscopic data analysis and ECD calculations elucidated their structures, including absolute configurations. All compounds were evaluated for bioactivity towards autophagic flux by flow cytometry using HM mCherry-GFP-LC3 cells. Compounds 1, 3, 4, 5, 8, and 9 significantly increased autophagic flux.

Structural characterization of a novel GPVI-nanobody complex reveals a biologically active domain-swapped GPVI dimer.

Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterized their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nbs from 6 different binding classes showed the strongest binding to recombinant GPVI-Fc, suggesting that there was not a single dominant class. The most potent 3, Nb2, 21, and 35, inhibited collagen-induced platelet aggregation with nanomolar half maximal inhibitory concentration (IC50) values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the collagen-related peptide (CRP) binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C' loop, which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signaling was abolished in a cell-based NFAT reporter assay. This demonstrates that the C-C' loop region is important for GPVI signaling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.

Experimental validation of computerised models of clustering of platelet glycoprotein receptors that signal via tandem SH2 domain proteins.

The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects.

Measurement of tigecycline in dried blood spots by LC-MS/MS and comparison tigecycline concentrations between whole blood and plasma.

RATIONALE: An LC-MS/MS method was established to measure tigecycline in dried blood spots (DBSs). METHODS: The DBS specimens obtained by applying 30 µl of blood to filter paper were extracted with hydrogen oxide and subsequently precipitated protein with perchloric acid, then the extract was directly analyzed by liquid chromatography tandem mass spectrometry. A Hypersil GOLD aQ column was utilized for separating the analytes, and detection was carried out in positive and selective reaction monitoring modes. The precursors to product ion transitions m/z 586.3 → 513.1 and m/z 586.3 → 569.2 were monitored for tigecycline, and m/z 473.2 → 456.0 and m/z 473.2 → 367.0 for 9-amino minocycline as internal standard. RESULTS: The validation parameters of specificity and selectivity, linearity (0.02-5 µg ml-1 ), sensitivity (limit of quantification 0.02 µg ml-1 ), intra- and interday precision (within 15%) and relative error (within ±15%) were acceptable. The recoveries were from 84.65% to 90.49% and from 85.41% to 95.72% for tigecycline and internal standard, respectively, and the matrix effect was not evident to influence accuracy. The impact of hematocrit on measurement of the analyte was negligible, and after preserving at ambient temperature for 24 h and at 4°C for 1 month it remained steady. CONCLUSIONS: The advantages of nonintrusive blood collection and micro-volume sample requirements make DBS a potent surrogate to conventional venepuncture for sampling.

Munronin V with 7/7/6 tricarbocyclic framework from Munronia henryi harms inhibits tau pathology by activating autophagy.

Munronin V (1), isolated from Munronia henryi Harms, is the first example, to the best of our knowledge, of an unprecedented 7/7/6 tricarbocyclic framework featuring an unusual A,B-seco-limonoid ring. The structures of munronin V were established from extensive spectroscopic and electronic circular dichroism (ECD) analyses. The novel A,B-seco with two seven-membered lactones was formed as a result of Baeyer-Villiger oxidation. Compound 1 activated autophagy and inhibited Tau pathology as revealed by flow cytometric analyses, confocal imaging analysis and western blotting, and this effect was mediated by transcription factor EB (TFEB). These findings suggested that 1 might have potential as a compound for combating Alzheimer's disease.

Terpenoids from Euphorbia helioscopia and Their Cytotoxic Activities against H1975 Cells.

Three previously undescribed diterpenoids, helioscopnoids A-C, and eight known compounds were isolated from the whole plants of Euphorbia helioscopia. Their structures were established by extensive analysis of spectra and data comparison with previous literatures. Among them, compound 4 was identified as 24,24-dimethoxy-25,26,27-trinoreuphan-3ß-ol with revised configurations of C-13, C-14, and C-17 (13R*, 14R*, 17R*). Cytotoxicity assays revealed that all compounds exhibited varying levels of cytotoxicity against H1975 cells, with compound 9 displaying the most potent activity, as indicated by cell viability rates of 18.13 % and 20.76 % at concentrations of 20 µM and 5 µM, respectively. This study expands the understanding of E. helioscopia terpenoids' structural diversity and biological activities, contributing to the exploration of potential therapeutic applications.

Quassinoids from Twigs of Harrisonia perforata (Blanco) Merr and Their Anti-Parkinson's Disease Effect.

Six new C-20 and one new C-19 quassinoids, named perforalactones F-L (1-7), were isolated from twigs of Harrisonia perforata. Spectroscopic and X-ray crystallographic experiments were conducted to identify their structures. Through oxidative degradation of perforalactone B to perforaqussin A, the biogenetic process from C-25 quassinoid to C-20 via Baeyer-Villiger oxidation was proposed. Furthermore, the study evaluated the anti-Parkinson's disease potential of these C-20 quassinoids for the first time on 6-OHDA-induced PC12 cells and a Drosophila Parkinson's disease model of PINK1B9. Perforalactones G and I (2 and 4) showed a 10-15% increase in cell viability of the model cells at 50 µM, while compounds 2 and 4 (100 µM) significantly improved the climbing ability of PINK1B9 flies and increased the dopamine level in the brains and ATP content in the thoraces of the flies.

Phosphoproteomic Analysis of Platelets in Severe Obesity Uncovers Platelet Reactivity and Signaling Pathways Alterations.

OBJECTIVE: Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. Approach and Results: We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- and gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients. CONCLUSIONS: Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.

Diterpenoids with an unprecedented ring system from Euphorbia peplus and their activities in the lysosomal-autophagy pathway.

Three novel jatrophane diterpenes, cyclojatrophanes A-C (1-3), were isolated from the seeds of Euphorbia peplus. Compounds 1-3 featured an unprecedented 5/5/5/11 tetracyclic ring system incorporating ditetrahydropyran rings. Their structures including their absolute configurations were established by extensive spectroscopic analysis, X-ray crystallographic experiments and chemical transformations. In addition, these compounds could significantly activate the lysosomal-autophagy pathway.

Perforalactones D and E, two new C-20 quassinoids with potential activity to induce lysosomal biogenesis from the twigs of Harrisonia perforata (Blanco) Merr.

Two new quassinoids (1 and 2) were isolated from the twigs of Harrisonia perforata (Blanco) Merr. Perforalactone E (2) possesses an uncommon hexacyclic 1α,12α:5α,13α-dicyclo-9ßH-picrasane skeleton. Its structure was determined based on spectroscopic data and X-ray crystallography. Compounds 1 and 2 could significantly induce lysosomal biogenesis through transcriptional activation of lysosomal genes.

The structure of CLEC-2: mechanisms of dimerization and higher-order clustering.

The platelet C-type lectin-like receptor CLEC-2 drives inflammation-driven venous thrombosis in mouse models of thrombo-inflammatory disease with a minimal effect on hemostasis identifying it as a target for a new class of antiplatelet agent. Here, we discuss how the protein structure and dynamic arrangement of CLEC-2 on the platelet membrane helps the receptor, which has a single YxxL motif (known as a hemITAM), to trigger intracellular signaling. CLEC-2 exists as a monomer and homo-dimer within resting platelets and forms higher-order oligomers following ligand activation, a process that is mediated by the multivalent nature of its ligands and the binding of the tandem SH2 domains of Syk to the phosphorylated hemITAM and concomitantly to PIP2 or PIP3 to localize it to the membrane. We propose that a low level of active Syk is present at the membrane in resting platelets due to phosphorylation by Src family kinases and that clustering of receptors disturbs the equilibrium between kinases and phosphatases, triggering phosphorylation of the CLEC-2 hemITAM and recruitment of Syk. Knowledge of the structure of CLEC-2 and the mechanism of platelet activation has important implications for development of therapeutics.

Lymphatic blood filling in CLEC-2-deficient mouse models.

C-type lectin-like receptor 2 (CLEC-2) is considered as a potential drug target in settings of wound healing, inflammation, and infection. A potential barrier to this is evidence that CLEC-2 and its ligand podoplanin play a critical role in preventing lymphatic vessel blood filling in mice throughout life. In this study, this aspect of CLEC-2/podoplanin function is investigated in more detail using new and established mouse models of CLEC-2 and podoplanin deficiency, and models of acute and chronic vascular remodeling. We report that CLEC-2 expression on platelets is not required to maintain a barrier between the blood and lymphatic systems in unchallenged mice, post-development. However, under certain conditions of chronic vascular remodeling, such as during tumorigenesis, deficiency in CLEC-2 can lead to lymphatic vessel blood filling. These data provide a new understanding of the function of CLEC-2 in adult mice and confirm the essential nature of CLEC-2-driven platelet activation in vascular developmental programs. This work expands our understanding of how lymphatic blood filling is prevented by CLEC-2-dependent platelet function and provides a context for the development of safe targeting strategies for CLEC-2 and podoplanin.

Novel role of LINC01013/miR-6795-5p/FMNL3 axis in the regulation of hepatocellular carcinoma stem cell features.

Cancer stem cells (CSCs) are major contributors to tumor initiation, recurrence, and metastasis of hepatocellular carcinoma (HCC). Some long non-coding RNAs have been reported as modulators of stem-like properties in cancer cells. However, the role of LINC01013 in liver CSCs has not yet been clarified. In this study, we aimed to elucidate the expression pattern and functions of LINC01013 in HCC. HCC tissues and normal controls were collected, and the expression pattern of LINC01013 and miR-6795-5p was identified by quick real-time polymerase chain reaction. Cell counting kit-8 assay, colony formation, and spheroid formation were performed to measure cell viability, proliferation, and self-renewal of HCC cell lines. The expression of stem markers was detected by western blot analysis. The effect of LINC01013 on viability, proliferation, and stem-like properties was detected through gain-of-function and loss-of-function experiments. The direct interaction among LINC01013, miR-6795-5p, and FMNL3 was testified by dual-luciferase reporter gene assay. Tumor-bearing mice were constructed to ascertain the functions of LINC01013 in vivo. HCC tissues showed increased LINC01013 and FMNL3 expression, while it showed a decreased miR-6795-5p expression as compared to the relative controls. Moreover, the high level of LINC01013 was closely related to the poor prognosis of HCC patients. LINC01013 directly binds to miR-6795-5p and subsequently relieves FMNL3. Silencing LINC01013, FMNL3, or overexpression of miR-6795-5p could suppress spheroid and colony formation, proliferation, as well as expression of stemness markers in HepG2 and SNU-182 cells. LINC01013 knockdown suppressed growth and stem-like traits of HCC cells in vivo by reducing FMNL3 expression. LINC01013/miR-6795-5p/FMNL3 axis may be a novel therapeutic target for HCC.

Macrocyclic diterpenoids from the seeds of Euphorbia peplus with potential activity in inducing lysosomal biogenesis.

The first phytochemical investigation of the seeds of Euphorbia peplus led to the isolation and characterization of five new (1-5), named euphopepluanones A-E, and five known diterpenoids (6-10). Their structures were established by extensive spectroscopic analysis and X-ray crystallographic experiments. Euphopepluanones A-E (1-3) feature a very rare 5/11/5-tricyclic skeleton, and euphopepluanones D-E (4-5) represent the first report of lathyrane type diterpenoids found in E. peplus. The new compounds 1-5 were assessed for their activities to induce lysosomal biogenesis through LysoTracker Red staining, in which compounds 1 and 3 could significantly induce lysosomal biogenesis. In addition, compounds 1 and 3 could promote the nuclear translocation of TFEB, a master transcriptional factor of lysosomal genes, indicating that compounds 1 and 3 induced lysosomal biogenesis through activation of TFEB.

Pretreatment biomarkers as prognostic predictors of survival in patients with Pancreatic Cancer treated with Gemcitabine-based Therapy and 5-Fluorouracil: Neutrophil-to-lymphocyte ratio vs Platelet-to-lymphocyte ratio.

Although elevated neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been reported to be inverse prognostic predictors of survival in patients with pancreatic cancer (PC), the comparison of their prognostic roles in patients with PC undergoing gemcitabine-based chemotherapy and 5-fluorouracil (5-FU) remains unclear. This study was designed and performed to determine the predictive roles of NLR and PLR in patients diagnosed with PC who underwent one of these two regimens. We retrospectively enrolled 95 patients diagnosed with PC undergoing supportive care, gemcitabine-based chemotherapy or 5-FU therapy from January 2015 to October 2018. Univariate and multivariate Cox regression analyses were done to identify clinicopathological predictors of time to treatment failure (TTF) and overall survival (OS), including pretreatment NLR and PLR. The statistical data showed that pretreatment NLR was significantly associated with metastasis. Among all analyzed variables, pretreatment NLR was an independent prognostic predictor of both TTF and OS of patients with PC, with NLR>4.0 predicting worse survival. PLR, however, didn't independently predict TTF or OS. There were no significant difference in the OS of patients undergoing gemcitabine-based regimens and 5-FU regimens when divided into two subgroups: NLR ≤4.0 and >4.0. In conclusion, pretreatment NLR is a promising independent outcome predictor for patients with PC, while NLR might not be a suitable factor in the selection of regimens for patients with PC.

Identification of Ingol and Rhamnofolane Diterpenoids from Euphorbia resinifera and Their Abilities to Induce Lysosomal Biosynthesis.

The phytochemical study of Euphorbia resinifera afforded 18 structurally diverse diterpenoids, including 14 new ingol-type diterpenoids, euphorblins A-N (1-14), a new rhamnofolane diterpenoid, euphorblin O (15), and three known analogues (16-18). The structures of these compounds were deduced using 2D NMR spectroscopy and NOE experiments. The structure of compound 1 was confirmed by single-crystal X-ray crystallography. The abilities of the compounds to enhance lysosomal biosynthesis were evaluated through LysoTracker Red staining. Among the 10 active compounds, compounds 2, 4, and 18 showed remarkable immunofluorescence strength, and their LysoTracker staining intensities were 155.9%, 143.5%, and 140.7%, respectively, greater than that of the control. A series of lysosomal genes were also found to be upregulated by these compounds, which further confirms their ability to induce lysosome biosynthesis and suggests that these diterpenoids have potential as lead compounds for the development of drugs for the treatment of lysosome-related diseases.

Mouse podoplanin supports adhesion and aggregation of platelets under arterial shear: A novel mechanism of haemostasis.

The podoplanin-CLEC-2 axis is critical in mice for prevention of hemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In this study, we demonstrate that mouse platelets form stable aggregates on mouse podoplanin at arterial shear through a CLEC-2 and Src kinase-dependent pathway. Adhesion and aggregation are also dependent on the platelet glycoprotein (GP) receptors, integrin αIIbß3 and GPIb, and the feedback agonists ADP and thromboxane A2 (TxA2). CLEC-2 does not bind to von Willebrand factor (VWF) suggesting that the interaction with podoplanin is sufficient to both tether and activate platelets. Consistent with this, the surface plasmon resonance measurements reveal that mouse CLEC-2 binds to mouse podoplanin with nanomolar affinity. The present findings demonstrate a novel pathway of hemostasis in which podoplanin supports platelet capture and activation at arteriolar rates of shear.