TRAP Assay Research Articles

Transduction of Human Fetal Liver Hematopoietic CD34+ Stem and Progenitor Cells into a Cell Line by Enhancing Telomerase Activity

Background: Human fetal liver hematopoietic stem cells have proven potential as therapeutics but lack extensive research due to their limited supply. Even in vitro expanded fetal liver hematopoietic stem cells enter senescence or lose their self-renewal capacity after a few days in culture. The present study aimed to obtain a homogeneous and persistent supply of hematopoietic stem cells from the fetal liver by establishing a cell line through immortalization of cells by enhancing telomerase activity. Materials and Methods: Human fetal liver hematopoietic CD34+ stem and progenitor cells were transformed and immortalized using retroviruses carrying the human telomerase (hTERT) gene. Following transduction, telomerase activity was assessed using the TRAP assay and telomere length was examined by Southern blotting in transduced cells. Their characterization was conducted using flowcytometry to analyze the CD34+ population of hematopoietic stem cells and their colony forming potential using colony forming unit (CFU) assay. Results: After transduction with hTERT, the life span of human fetal liver hematopoietic CD34+ stem and progenitor cells were extended to 80 population doublings, without any change in cell morphology or population doubling times. Constitutive hTERT expression enhanced the replicative capacity and prevented terminal differentiation of CD34+ fetal liver hematopoietic stem and progenitor cells (FLHSPCs). Moreover, hTERT-transduced stem cells maintained their telomere length and telomerase activity. Conclusion: By introducing telomerase activity into hematopoietic stem and progenitor cells, their lifespan can be extended while maintaining stemness. These modified cells hold promise for in vitro research focused on studying hematopoietic stem cells derived from fetal liver.

Small Molecules Inducing Autophagic Degradation of Expanded Polyglutamine Protein through Interaction with Both Mutant ATXN3 and LC3.

Polyglutamine (polyQ)-mediated spinocerebellar ataxia (SCA), including SCA1, 2, 3, 6, 7, and 17, are caused by mutant genes with expanded CAG repeats, leading to the intracellular accumulation of aggregated proteins, the production of reactive oxygen species, and cell death. Among SCA, SCA3 is caused by a mutation in the ATXN3 (ataxin-3) gene. In a circumstance of polyQ aggregation, the autophagic pathway is induced to degrade the aggregated proteins, thereby suppressing downstream deleterious effects and promoting neuronal survival. In this study, we tested the effects of synthetic indole (NC009-1, -2, -3, -6) and coumarin (LM-022, -031) derivatives as chemical chaperones to assist mutant ATXN3-Q75 folding, as well as autophagy inducers to clear aggregated protein. Among the tested compounds, NC009-1, -2, and -6 and LM-031 interfered with Escherichia coli-derived ATXN3-Q75 aggregation in thioflavin T binding and filter trap assays. In SH-SY5Y cells expressing GFP-fused ATXN3-Q75, these compounds displayed aggregation-inhibitory and neurite growth-promoting potentials compared to untreated cells. Furthermore, these compounds activated autophagy by increasing the phosphatidylethanolamine-conjugated LC3 (microtubule associated protein 1 light chain 3)-II:cytosolic LC3-I ratio in these cells. A biochemical co-immunoprecipitation assay by using a mixture of HEK 293T cell lysates containing recombinant ATXN3-Q75-Venus-C-terminus (VC) or Venus-N-terminus (VN)-LC3 protein indicated that NC009-1 and -2 and LM-031 served as an autophagosome-tethering compound (ATTEC) to interact with ATXN3-Q75 and LC3, and the interaction was further confirmed by bimolecular fluorescence complementation analysis in cells co-expressing both ATXN3-Q75-VC and VN-LC3 proteins. The study results suggest the potential of NC009-1 and -2 and LM-031 as an ATTEC in treating SCA3 and, probably, other polyQ diseases.

A ribonuclease T2 protein FocRnt2 contributes to the virulence of Fusarium oxysporum f. sp. cubense tropical race 4.

Banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a major disease of banana plants worldwide. Effector proteins play critical roles in banana-Foc TR4 interaction. Our previous studies highlighted a ribonuclease protein belonging to the T2 family (named as FocRnt2) in the Foc TR4 secretome, which was predicted to be an effector. However, its biological function in Foc TR4 infection is still unclear. Herein, we observed significant expression of FocRnt2 during the early stage of fungal infection in planta. A yeast signal sequence trap assay showed that FocRnt2 contained a functional signal peptide for secretion. FocRnt2 possessed ribonuclease activity that could degrade the banana total RNA invitro. Subcellular localization showed that FocRnt2 was localized in the nucleus and cytoplasm of Nicotiana benthamiana leaves. Transient expression of FocRnt2 suppressed the expression of salicylic acid- and jasmonic acid-signalling marker genes, reactive oxygen species accumulation, and BAX-mediated cell death in N. benthamiana. FocRnt2 deletion limited fungal penetration, reduced fusaric acid biosynthesis in Foc TR4, and attenuated fungal virulence against banana plants, but had little effect on Foc TR4 growth and sensitivity to various stresses. Furthermore, FocRnt2 deletion mutants induced higher expression of the defence-related genes in banana plants. These results suggest that FocRnt2 plays an important role in full virulence of Foc TR4, further improving our understanding of effector-mediated Foc TR4 pathogenesis.

Genotype-specificity in putative competitive endophytes modulated by root exudation of rice

The putative competitive endophytic bacteria with plant-growth-promoting attributes could be potential bio inputs to enhance agricultural sustainability. These special rhizosphere colonizing bacteria possess distinct strategies to enter and colonize the host plant asymptomatically and offer several plant growth-promoting benefits. However, the molecular interaction between the endophyte and host plant remains unclear in many crop spheres. In this study, we categorize putative endophytes in rice into two groups—common endophytes and genotype-specific endophytes—based on their ability to colonize alternative hosts. The putative competitive endophytes of rice landrace Norungan and high-yielding cultivar Co51 were cross-inoculated with each other under gnotobiotic conditions and attempted to re-isolate from the inoculated plants. Two Norungan endophytes (Priestia endophytica NE14 and NE21) and one Co51-endophyte (Peribacillus endoradicis CE10) could not colonize the internal tissues of the alternate hosts, and they were considered as genotype-specific endophytes. In contrast, the rest of the strains could colonize both genotypes (common endophytes). The bait trap assay with flow cytometry revealed that the Norungan root exudate significantly enhanced the chemotactic movement of NE14 and NE21 compared to Co51-endophyte (CE10) and common endophytes (NE09 and CE07). Likewise, the Co51-root exudate triggered high levels of chemotaxis of CE10 but not the others. The root exudates did not alter the growth and biofilm-producing capability of genotype-specific and common endophytes. The cell wall degrading enzyme, pectinase of genotype-specific endophytes (NE14 and NE21), had positively enhanced due to Norungan root exudate but not with Co51 exudate. When the genotype-specific endophytes were inoculated to alternate host, a strain-level difference was observed in the induction of rice defense enzymes. NE14 inoculation in Co51 rice had high levels of peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, and glutathione reductase; NE21 induced high levels of glutathione reductase and peroxidase alone in Co51. Likewise, CE10 triggered relatively high levels of catalase, glutathione reductase, peroxidase, and polyphenol oxidase in Norungan rice. No apparent difference between the two rice genotypes in defense enzymes’ levels was observed due to common endophytes (NE09 and CE07). These results authenticate the occurrence of genotype-specific putative competitive endophytes in rice, and exploring them for crop growth and yield would be a better choice for rice sustainability.

Trehalose prevents the formation of aggregates of mutant ataxin-3 and reduces soluble ataxin-3 protein levels in an SCA3 cell model

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by mutant ataxin-3 with an abnormally expanded polyQ tract and is the most common dominantly inherited ataxia worldwide. There are no suitable therapeutic options for this disease. Autophagy, a defense mechanism against the toxic effects of aggregation-prone misfolded proteins, has been shown to have beneficial effects on neurodegenerative diseases. Thus, trehalose, which is an autophagy inducer, may have beneficial effects on SCA3. In the present study, we examined the effects of trehalose on an SCA3 cell model. After trehalose treatment, aggregate formation, soluble ataxin-3 protein levels and cell viability were evaluated in HEK293T cells overexpressing ataxin-3-15Q or ataxin-3-77Q. We also explored the mechanism by which trehalose affects autophagy and stress pathways. A filter trap assay showed that trehalose decreased the number of aggregates formed by mutant ataxin-3 containing an expanded polyQ tract. Western blot and Cell Counting Kit-8 (CCK-8) results demonstrated that trehalose also reduced the ataxin-3 protein levels and was safe for ataxin-3-expressing cells, respectively. Western blot and total antioxidant capacity assays suggested that trehalose had great therapeutic potential for treating SCA3, likely through its antioxidant activity. Our data indicate that trehalose plays a neuroprotective role in SCA3 by inhibiting the aggregation and reducing the protein level of ataxin-3, which is also known to protect against oxidative stress. These findings provide a new insight into the possibility of treating SCA3 with trehalose and highlight the importance of inducing autophagy in SCA3.

Effect of gelling agents on properties of coumestrol-loaded nanoemulsions – Formulation, skin retention/permeation, antioxidant and skin photoprotective activities

Coumestrol is an isoflavonoid that exhibits antioxidant activity. In this study, hydrogels containing coumestrol-loaded nanoemulsions were designed to protect the skin against UVA/UVB light. The systems investigated were coumestrol-loaded nanoemulsions obtained by spontaneous emulsification procedure formulated in semisolid hydrogels composed of ionic (Carbopol 940®), cationic (chitosan) or non-ionic (hydroxyethylcellulose) gelling materials. Transmission electron microscopy images of formulations showed oil droplets exhibiting 200–300 nm. Hydrogels displayed non-Newtonian shear-thinning behavior. Coumestrol content in formulations remained close to 100 % (1 mg/mL) over time (up to 90 days). Regardless of the gelling material used, a lowering of coumestrol release rate was observed, in comparison with the release from nanoemulsion. The permeation and retention of coumestrol in porcine ear skin was evaluated using Franz-type diffusion cells. A higher retention of coumestrol in the epidermis was noticed (20 μg/g) from Carbopol 940-based formulation after 8 h of kinetics. No histological evidence of tissue damage was observed. The nanoemulsion containing coumestrol thickened with Carbopol 940® was selected for further studies. The formulation exhibited antioxidant potential as estimated by TRAP and TAR assays. The skin photoprotective effect was evidenced by means of TBARS, protein carbonylation, and protein thiol content experiments. Overall, our research suggests that the formulation is promising to protect skin against oxidative damage from UVA/UVB light.

One must reconstitute the functions of interest from purified proteins.

I am often asked by students and younger colleagues and now by the editors of this issue to tell the history of the development of the in vitro motility assay and the dual-beam single-molecule laser trap assay for myosin-driven actin filament movement, used widely as key assays for understanding how both muscle and nonmuscle myosin molecular motors work. As for all discoveries, the history of the development of the myosin assays involves many people who are not authors of the final publications, but without whom the assays would not have been developed as they are. Also, early experiences shape how one develops ideas and experiments, and influence future discoveries in major ways. I am pleased here to trace my own path and acknowledge the many individuals involved and my early science experiences that led to the work I and my students, postdoctoral fellows, and sabbatical visitors did to develop these assays. Mentors are too often overlooked in historical descriptions of discoveries, and my story starts with those who mentored me.

Benzaldehyde acts as a behaviorally active component in brewer's yeast protein powder which attracts B. dorsalis through olfaction.

The Oriental fruit fly, Bactrocera dorsalis, is a significant pest that damages a variety of fruit crops. The effectiveness of chemical pesticides against such pests is limited, raising concerns about pesticide residues and resistance. Proteins naturally attract B. dorsalis and have led to the development of a management strategy known as protein bait attractant technology (BAT). Although the attraction of protein sources to B. dorsalis is well-documented, the biologically active components within these sources are not fully understood. This study employed analytical chemistry, behavioral tests, and electrophysiological techniques to investigate the behaviorally active components of beer yeast protein powder (BYPD), aiming to provide a basis for improving and developing protein baits. An olfactory trap assay confirmed the attractiveness of BYPD, and five components with high abundance were identified from its headspace volatiles using GC-MS. These components included ethanol, isoamyl alcohol, ethyl decanoate, benzaldehyde, and phenylethyl alcohol. Mixtures of these five components demonstrated significant attraction to B. dorsalis adults, with benzaldehyde identified as a potential key component. The attractiveness of benzaldehyde required a relatively large dose, and it was most attractive to adults that had been starved from dusk until the following morning. Attraction of adult flies to benzaldehyde appeared mainly mediated by inputs from olfactory receptors. While EAG data supports that ionotropic receptors could influence the detection of benzaldehyde in female adults, they did not affect female behavior towards benzaldehyde. These findings indicate that benzaldehyde is an important behaviorally active component in BYPD and offer insights for developing novel protein lures to control B. dorsalis in an environmentally friendly manner.

Synthesis of cNDI‐Peptide‐Dimer as a G4 Cluster‐Selective Binder and its Interaction with G4 Dimer

AbstractThe amino acid unit carrying cyclic naphthalene diimide (cNDI) was synthesized by linking the amino moiety of cNDI as a G4‐specific ligand with the γ‐carboxylic acid moiety of Fmoc‐Glu. cNDI‐peptide‐dimer, 1 was synthesized by peptide synthesizer using this unit. 1 exhibited an extremely strong binding constant of the order of 108 M−1 with telomeric G4 dimer, probably due to cooperative stacking of the its two G4 planes. It also showed an IC50 at the nM level by the TRAP assay. The results obtained here are expected to enable the design of peptide ligands with multiple cNDI sites targeting the G4 cluster by using the amino acid units of cNDI.

Abstract 4485: Identification of novel lead compounds and repurposing FDA approved drugs as telomerase inhibitors using structure-based drug designing approach and their evaluation using in-vitro and ex-vivo assays

Abstract Introduction: Upregulated telomerase activity is an established hallmark in about 85-90% of cancer malignancies making it an attractive therapeutic target. However, the development of approved small-molecule telomerase inhibitors has been impeded by a lack of structural ambiguity for decades. In our study, we have employed a molecular dynamics approach to validate the binding site of BIBR1532, an approved telomerase inhibitor in the hTERT thumb domain followed by a structure-based drug designing approach to repurpose FDA-approved drugs and identify novel lead compounds targeting the enzyme. Material and Methods: Due to the unavailability of a co-crystalized structure of BIBR1532 with the catalytic hTERT thumb domain, we employed a Molecular dynamics-based method to identify and validate the exact binding site of the inhibitor. Two pharmacophore models were generated for the putative and newly identified binding pockets which were screened virtually through an anti-cancer library and Drug Bank database. The models were validated on the basis of the fit value of the BIBR1532 and selectivity value indicating the favorable feature set required. The top hits obtained were filtered using drug-likeliness parameters like Lipinski, ADME, and TOPKAT followed by redocking into their binding site. Finally, lead molecules that were able to dock in the new pocket were validated using Molecular Dynamics (MD)simulation studies and their binding free energy was calculated using MM-PBSA calculations followed by their evaluation using in-vitro TRAP assay. Results: Structural investigation, molecular docking studies, and confirmatory molecular dynamics revealed that the exact binding site of BIBR1532 is 8.4 Å away from the reported FVYL pocket strikingly with clinically relevant and characteristic interactions conserved. We have identified five lead compounds and four lead compounds from DrugBank and the anti-cancer library respectively, that were stable in the new binding pocket based on their MD trajectory analysis, and parameters like RMSD, RMSF, H-bonds and Radius of Gyration as well as their MM-PBSA scores. The lead compounds are being evaluated in vitro using TRAP assay and ex-vivo assays. Conclusion: The study identified five existing FDA-approved drugs in the market for other diseases as potential telomerase inhibitors alongside four novel compounds that can be used for the treatment of cancer in the future after further validation studies. Citation Format: Divpreet Kaur, Madhu Chopra, Daman Saluja. Identification of novel lead compounds and repurposing FDA approved drugs as telomerase inhibitors using structure-based drug designing approach and their evaluation using in-vitro and ex-vivo assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4485.

A root-knot nematode effector targets the Arabidopsis cysteine protease RD21A for degradation to suppress plant defense and promote parasitism.

Meloidogyne incognita is one of the most widely distributed plant-parasitic nematodes and causes severe economic losses annually. The parasite produces effector proteins that play essential roles in successful parasitism. Here, we identified one such effector named MiCE108, which is exclusively expressed within the nematode subventral esophageal gland cells and is upregulated in the early parasitic stage of M. incognita. A yeast signal sequence trap assay showed that MiCE108 contains a functional signal peptide for secretion. Virus-induced gene silencing of MiCE108 impaired the parasitism of M. incognita in Nicotiana benthamiana. The ectopic expression of MiCE108 in Arabidopsis suppressed the deposition of callose, the generation of reactive oxygen species, and the expression of marker genes for bacterial flagellin epitope flg22-triggered immunity, resulting in increased susceptibility to M. incognita, Botrytis cinerea, and Pseudomonas syringae pv. tomato (Pst) DC3000. The MiCE108 protein physically associates with the plant defense protease RD21A and promotes its degradation via the endosomal-dependent pathway, or 26S proteasome. Consistent with this, knockout of RD21A compromises the innate immunity of Arabidopsis and increases its susceptibility to a broad range of pathogens, including M. incognita, strongly indicating a role in defense against this nematode. Together, our data suggest that M. incognita deploys the effector MiCE108 to target Arabidopsis cysteine protease RD21A and affect its stability, thereby suppressing plant innate immunity and facilitating parasitism.

The role of telomerase reverse transcriptase in the mitochondrial protective functions of Angiotensin-(1–7) in diabetic CD34+ cells

Angiotensin (Ang)-(1–7) stimulates vasoprotective functions of diabetic (DB) CD34+ hematopoietic stem/progenitor cells partly by decreasing reactive oxygen species (ROS), increasing nitric oxide (NO) levels and decreasing TGFβ1 secretion. Telomerase reverse transcriptase (TERT) translocates to mitochondria and regulates ROS generation. Alternative splicing of TERT results in variants α-, β- and α-β-TERT, which may oppose functions of full-length (FL) TERT. This study tested if the protective functions of Ang-(1–7) or TGFβ1-silencing are mediated by mitoTERT and that diabetes decreases FL-TERT expression by inducing splicing.CD34+ cells were isolated from the peripheral blood mononuclear cells of nondiabetic (ND, n = 68) or DB (n = 74) subjects. NO and mitoROS levels were evaluated by flow cytometry. TERT splice variants and mitoDNA-lesions were characterized by qPCR. TRAP assay was used for telomerase activity. Decoy peptide was used to block mitochondrial translocation (mitoXTERT).TERT inhibitor or mitoXTERT prevented the effects of Ang-(1–7) on NO or mitoROS levels in DB-CD34+ cells. FL-TERT expression and telomerase activity were lower and mitoDNA-lesions were higher in DB cells compared to ND and were reversed by Ang-(1–7) or TGFβ1-silencing. The prevalence of TERT splice variants, with predominant β-TERT expression, was higher and the expression of FL-TERT was lower in DB cells (n = 25) compared to ND (n = 30). Ang-(1–7) or TGFβ1-silencing decreased TERT-splicing and increased FL-TERT. Blocking of β-splicing increased FL-TERT and protected mitoDNA in DB-cells.The findings suggest that diabetes induces TERT-splicing in CD34+ cells and that β-TERT splice variant largely contributes to the mitoDNA oxidative damage.

Comparing Length and Telomere Expression at Oral Precancerous and Cancerous Stages.

Telomeres consist of repetitive G-rich nucleotides located at the end of each chromosome, acting as protein binding sites. The aim of this study was to examine the differences in telomere length in blood, saliva, and tissue samples at various stages of oral precancerous and cancerous lesions. Samples of blood, tissue, and saliva were collected from patients with oral precancerous and cancerous lesions. DNA extraction was performed. Then, a TRAP assay was conducted to assess and compare the telomere length and telomerase expression. The levels of telomerase activity (TA) in the DNA samples ranged from 0.19 to 6.91 (2.05+1.37) among oral squamous cell carcinoma (OSCC) patients and from 0.17 to 4.5 (0.28+4.25) among precancerous patients. A significant difference was observed in TA levels between OSCC and precancerous samples (t=3.9691, P= 0.0000). Assessing the telomerase activity is crucial for studying the behavior of carcinoma in the clinical setting. The augmented telomerase expression and the length of telomere contribute to OSCC progression. Hence, this study adds a diagnostic tool that can serve as a biomarker for the early detection and prognosis of OSCC.

Telomere Maintenance Is Associated with Type 2 Diabetes Remission in Response to a Long-Term Dietary Intervention without Non-Weight Loss in Patients with Coronary Heart Disease: From the CORDIOPREV Randomized Controlled Trial.

In order to evaluate whether telomere maintenance is associated with type 2 diabetes remission, newly diagnosed type 2 diabetes patients without glucose-lowering treatment (183 out of 1002) from the CORDIOPREV study (NCT00924937) were randomized to consume a Mediterranean or low-fat diet. Patients were classified as Responders, those who reverted from type 2 diabetes during the 5 years of dietary intervention (n = 69), and Non-Responders, who did not achieve diabetes remission by the end of the follow-up period (n = 104). We found no differences in diabetes remission between the two diets, and we determined telomere length (TL) by measuring qPCR, telomerase activity using the TRAP assay, and direct redox balance based on the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSH) via colorimetric assay. Responders exhibited higher baseline TL in comparison with Non-Responders (p = 0.040), and a higher TL at baseline significantly predicted a higher probability of type 2 diabetes remission (OR 2.13; 95% CI, 1.03 to 4.41). After the dietary intervention, Non-Responders showed significant telomere shortening (-0.19, 95% CI -0.32 to 0.57; p = 0.005). Telomere shortening was significantly pronounced in type 2 diabetes patients with a worse profile of insulin resistance and/or beta-cell functionality: high hepatic insulin resistance fasting, a high disposition index (-0.35; 95% CI, -0.54 to -0.16; p < 0.001), and a low disposition index (-0.25; 95% CI, -0.47 to -0.01; p = 0.037). In addition, changes in TL were correlated to the GSH/GSSG ratio. Responders also showed increased telomerase activity compared with baseline (p = 0.048), from 0.16 (95% CI, 0.08 to 0.23) to 0.28 (95% CI, 0.15 to 0.40), with a more marked increase after the dietary intervention compared with Non-Responders (+0.07; 95% CI, -0.06-0.20; p = 0.049). To conclude, telomere maintenance may play a key role in the molecular mechanisms underlying type 2 diabetes remission in newly diagnosed patients. However, further larger-scale prospective studies are necessary to corroborate our findings.

An optimized filter trap assay for detecting recombinant authentic tau fibrils.

The development and optimization of the Filter Trap Assay (FTA) for the detection of authentic tau fibrils in vitro mark a pivotal advancement in the realm of tauopathy research, particularly by addressing the limitations of using polyanion-induced tau fibrils, which structurally differ from those isolated from tauopathy patients. Recently it has been shown that truncated tau fragment (297-391), also termed dGAE, can form authentic tau fibrils in the absence of polyanions. This study introduces a refined protocol that reliably detects authentic tau fibrils in a physiologically relevant framework, utilizing nitrocellulose membranes to achieve heightened sensitivity. Our investigation highlights the superior efficacy of sarkosyl, an anionic surfactant traditionally used to prepare protein lysates from brains and cultured neurons, in preserving the aggregated state of tau dGAE fibrils in vitro, underscoring its potential for further exploratory studies. By offering a user-friendly and economically feasible approach, this technique enables a broad range of laboratories to measure the presence of authentic tau fibrils. This methodological enhancement propels our understanding of tauopathies forward and bridges the gap between basic research and advanced structural analyses, enriching the scientific community's methodologies for studying neurodegenerative disorders.

Mechanical coupling coordinates microtubule growth

During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing in vitro are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubule pairs without any free fit parameters. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling k-fiber bundles with three or more microtubules, as found in many eukaryotes.

Mechanical coupling coordinates microtubule growth.

During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing in vitro are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubule pairs without any free fit parameters. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling k-fiber bundles with three or more microtubules, as found in many eukaryotes.

Secreted Effector Proteins of Poplar Leaf Spot and Stem Canker Pathogen Sphaerulina musiva Manipulate Plant Immunity and Contribute to Virulence in Diverse Ways.

Fungal effectors play critical roles in manipulating plant immune responses and promoting colonization. Sphaerulina musiva is a heterothallic ascomycete fungus that causes Septoria leaf spot and stem canker disease in poplar (Populus spp.) plantations. This disease can result in premature defoliation, branch and stem breakage, increased mortality, and plantation failure. However, little is known about the interaction between S. musiva and poplar. Previous work predicted 142 candidate secreted effector proteins in S. musiva (SmCSEPs), 19 of which were selected for further functional characterization in this study. SmCSEP3 induced plant cell death in Nicotiana benthamiana, while 8 out of 19 tested SmCSEPs suppressed cell death. The signal peptides of these eight SmCSEPs exhibited secretory activity in a yeast signal sequence trap assay. Confocal microscopy revealed that four of these eight SmCSEPs target both the cytoplasm and the nucleus, whereas four predominantly localize to discrete punctate structures. Pathogen challenge assays in N. benthamiana demonstrated that the transient expression of six SmCSEPs promoted Fusarium proliferatum infection. The expression of these six SmCSEP genes were induced during infection. SmCSEP2, SmCSEP13, and SmCSEP25 suppressed chitin-triggered reactive oxygen species burst and callose deposition in N. benthamiana. The candidate secreted effector proteins of S. musiva target multiple compartments in the plant cell and modulate different pattern-triggered immunity pathways. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Ulmus macrocarpa Hance trunk bark extracts inhibit RANKL-induced osteoclast differentiation and prevent ovariectomy-induced osteoporosis in mice

Ethnopharmacological relevanceUlmus macrocarpa Hance (UmH) bark has been traditionally utilized for medicinal purposes. The bark extract of this plant has diverse health benefits, and its potential role in enhancing bone health is of distinct interest, particularly when considering the substantial health and economic implications of bone-related pathologies, such as osteoporosis. Despite the compelling theoretical implications of UmH bark in fortifying bone health, no definitive evidence at the in vivo level is currently available, thus highlighting the innovative and as-yet-unexplored potential of this field of study. Aim of the studyPrimarily, our study aims to conduct a meticulous analysis of the disparity in the concentration of active compounds in the UmH root bark (Umrb) and trunk bark (Umtb) extracts and confirm UmH bark's efficacy in enhancing bone health in vivo, illuminating the cellular mechanisms involved. Materials and methodsThe Umrb and Umtb extracts were subjected to component analysis using high-performance liquid chromatography and then assessed for their inhibitory effects on osteoclast differentiation through the TRAP assay. An ovariectomized (OVX) mouse model replicates postmenopausal conditions commonly associated with osteoporosis. Micro-CT was used to analyze bone structure parameters, and enzyme-linked immunosorbent assay and staining were used to assess bone formation markers and osteoclast activity. Furthermore, this study investigated the impact of the extract on the expression of pivotal proteins and genes involved in bone formation and resorption using mouse bone marrow-derived macrophages (BMMs). ResultsThe findings of our study reveal a significant discrepancy in the concentration of active constituents between Umrb and Umtb, establishing Umtb as a superior source for promoting bone health. I addition, a standardized pilot-scale procedure was conducted for credibility. The bone health benefits of Umtb were verified using an OVX model. This validation involved the assessment of various parameters, including BMD, BV/TV, and BS/TV, using micro-CT imaging. Additionally, the activation of osteoblasts was evaluated by Umtb by measuring specific factors such as ALP, OCN, OPG in blood samples and through IHC staining. In the same investigations, diminished levels of osteoclast differentiation factors, such as TRAP, NFATc1, were also observed. The observed patterns exhibited consistency in vitro BMM investigations. ConclusionsThrough verification at both in vitro levels using BMMs and in vivo levels using the OVX-induced mouse model, our research demonstrates that Umtb is a more effective means of improving bone health in comparison to Umrb. These findings pave the way for developing health-functional foods or botanical drugs targeting osteoporosis and other bone-related disorders and enhance the prospects for future research extensions, including clinical studies, in extract applications.

Temperature-dependent modulation of odor-dependent behavior in three drosophilid fly species of differing thermal preference

Rapid and ongoing climate change increases global temperature, impacts feeding, and reproduction in insects. The olfaction plays an important underlying role in these behaviors in most insect species. Here, we investigated how changing temperatures affect odor detection and ensuing behavior in three drosophilid flies: Drosophila novamexicana, D. virilis and D. ezoana, species adapted to life in desert, global, and subarctic climates, respectively. Using a series of thermal preference assays, we confirmed that the three species indeed exhibit distinct temperature preferences. Next, using single sensillum recording technique, we classified olfactory sensory neurons (OSNs) present in basiconic sensilla on the antenna of the three species and thereby identified ligands for each OSN type. In a series of trap assays we proceeded to establish the behavioral valence of the best ligands and chose guaiacol, methyl salicylate and isopropyl benzoate as representatives of a repellent, attractant and neutral odor. Next, we assessed the behavioral valence of these three odors in all three species across a thermal range (10-35 °C), with flies reared at 18 °C and 25 °C. We found that both developmental and experimental temperatures affected the behavioral performance of the flies. Our study thus reveals temperature-dependent changes in odor-guided behavior in drosophilid flies.