Dual Role of IGF2BP2 in Osteoimmunomodulation during Periodontitis.

Periodontitis is a complex disease characterized by distinct inflammatory stages, with a peak of inflammation in the early phase and less prominent inflammation in the advanced phase. The insulin-like growth factor 2-binding proteins 2 (IGF2BP2) has recently been identified as a new m6A reader that protects m6A-modified messenger RNAs (mRNAs) from decay, thus participating in multiple biological processes. However, its role in periodontitis remains unexplored. Here, we investigated the role of IGF2BP2 in inflammation and osteoclast differentiation using a ligature-induced periodontitis model. Our findings revealed that IGF2BP2 responded to bacterial-induced inflammatory stimuli and exhibited differential expression patterns in early and advanced periodontitis stages, suggesting its dual role in regulating this disease. Depletion of Igf2bp2 contributed to increased release of inflammatory cytokines, thereby exacerbating periodontitis after 3 d of ligature while suppressing osteoclast differentiation and ameliorating periodontitis after 14 d of ligature. Mechanistically, we demonstrated that IGF2BP2 directly interacted with Cd5l and Cd36 mRNA via RNA immunoprecipitation assay. Overexpression of CD36 or recombinant CD5L rescued the osteoclast differentiation ability of Igf2bp2-null cells upon lipopolysaccharide stimulus, and thus the downregulation of Cd36 and Cd5l effectively reversed periodontitis in the advanced stage. Altogether, this study deepens our understanding of the potential mechanistic link among the dysregulated m6A reader IGF2BP2, immunomodulation, and osteoclastogenesis during different stages of periodontitis.

[Preliminary study on centrifugation time of liquid plasmatrix for soft tissue regeneration].

Objective: To provide reference for clinical application of liquid plasmatrix, and to investigate the optimal centrifugation time of liquid plasmatrix prepared by horizontal centrifugation for soft tissue regeneration from the aspects of mechanical properties, biological properties, and the effect of promoting soft tissue regeneration. Methods: Venous blood was collected from 6 healthy volunteers [3 males and 3 females, aged (26±2) years, with informed consent] who volunteered to donate blood at School of Stomatology, Wuhan University from September to November 2021. The collected venous blood was centrifuged at 500 ×g for 3, 5, 8 and 12 min to obtain liquid plasmatrix. The volume, weight, solidification time, and mechanical properties of liquid plasmatrix prepared at different centrifugation time were measured and recorded (the sample size at each time point was 3). The microstructure of different groups of liquid plasmatrix clot was observed by scanning electron microscope (SEM). The rheological properties of each group of liquid plasmatrix clot were measured by rheological test. The number and concentration of cells in the whole blood group and in each liquid plasmatrix group were measured using complete blood count test. The distribution of cells in the liquid plasmatrix clots was observed by hematoxylin-eosin staining. The effect of control group (Dulbecco's modified Eagle's medium containing 20% fetal bovine serum) and liquid plasmatrix clot exudates in 3, 5, 8, 12 min group (the sample size at each time point was 3) on gingival fibroblast migration was detected by cell migration method. Finally, the effects of control group and liquid plasmatrix clot exudates on the morphology of gingival fibroblasts were observed by fluorescence microscope. Results: The volume of liquid plasmatrix in 3, 5, 8 and 12 min group were approximately (2.47±0.12), (2.67±0.12), (3.53±0.12) and (3.73±0.12) ml, respectively. The weight of liquid plasmatrix in 3, 5, 8 and 12 min group were approximately (0.35±0.01), (0.46±0.02), (0.88±0.06) and (1.03±0.01) g, respectively. The maximum tensile force of liquid plasmatrix clots in 3, 5, 8 and 12 min group were (0.55±0.03), (0.56±0.03), (1.31±0.05) and (1.38±0.02) N, respectively. SEM results showed that the fibers inside the liquid plasmatrix clot became denser with increased centrifugation time. Compared with other groups, the concentrations of leukocytes, neutrophils, monocytes and lymphocytes in 8 min group were the highest, and the distribution of cell was more even. Compared with other groups, the efficiency of stimulating gingival fibroblast migration in 8 min group was the best (1.60±0.01). Fluorescence staining test showed that the liquid plasmatrix clot exudates could make gingival fibroblasts more stretched compared with control group. Conclusions: The present study shows that liquid plasmatrix prepared by centrifugation with 500 ×g centrifugal force for 8 min has higher concentration of viable cells and the ability to promote the migration of gingival fibroblasts.

Mitochondrial fission is necessary for mitophagy, development and virulence of the insect pathogenic fungus Beauveria bassiana.

AIMS: Mitochondrial fission is an essential dynamics that maintains mitochondrial morphology and function. This study seeks to determine the roles of mitochondrial fission in the filamentous entomopathogenic fungus Beauveria bassiana. MATERIAL AND METHODS: Three fission-related genes (BbFis1, BbMdv1 and BbDnm1) were functionally characterized via protein intracellular localization and construction of gene disruption mutants. RESULTS: Mitochondrial localization was only observed for BbFis1 which interacts with BbMdv1, but BbMdv1 did not have interaction with BbDnm1. Single disruption mutant of three genes generated the elongated and enlarged mitochondria which could not be eliminated via the mitophagy. Three mutant strains displayed the reduced ATP synthesis and vegetative growth compared with the wild type. Three genes were involved in the early stage of conidiation and unnecessary for the late stage. However, all three genes significantly contribute to blastospore development under submerged condition, and the loss of BbMdv1 had the greatest effects compared with the losses of BbFis1 or BbDnm1. Finally, disruption of three genes significantly attenuated fungal virulence, but their mutations had different influences. CONCLUSIONS: In addition to their consistent roles in mitochondrial division and mitophagy, three fission-related genes perform divergent roles in the development and virulence of the entomopathogenic fungus B. bassiana. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that mitochondrial fission is associated with lifecycle of B. bassiana. These findings provide information for the manipulation of fungal physiology and facilitate the application of entomopathogenic fungi.

High resistance of Isaria fumosorosea to carbendazim arises from the overexpression of an ATP-binding cassette transporter (ifT1) rather than tubulin mutation.

AIMS: Probing possible mechanisms involved in the resistance of entomopathogenic fungus Isaria fumosorosea to carbendazim fungicide. METHODS AND RESULTS: A carbendazim-sensitive strain (If116) selected from 15 wild-type strains was subjected to NaNO(2) -induced mutagenesis, yielding nine mutants with carbendazim resistance increased by 82- to 830-fold and thermotolerance decreased by 15-51%. Comparing the protein sequences deduced from the α- and ß-tubulin genes of If116 and its mutants revealed no traceable site mutation relating to the enhanced resistance although the transcripts levels of ß-tubulin gene in all mutants were 0·87- to 7·16-fold of that in If116. Three examined mutants showed multidrug resistance because they were significantly more resistant to glufosinate, imidacloprid and other six fungicides than If116 during growth. Further examination of rhodamine-stained blastospores revealed existence of drug efflux pump protein(s) in all carbendazim-resistant mutants. Thus, the sequences of an ATP-binding cassette (ABC) transporter gene (ifT1) and its promoter region cloned from the wild-type and mutant strains were analysed. Three common point mutations were located, respectively, at the binding sites of Gal4, Abf1 and Raf, which are crucial transcription factors in the regulative network of numerous protein loci. Such point mutations elevated the ifT1 expression by 17 to 137-fold in all the mutants. CONCLUSIONS: The overexpression of the ABC transporter caused by the point mutations at the binding sites was responsible for the fungal resistance to various pesticides including carbendazim. SIGNIFICANCE AND IMPACT OF STUDY: The transporter-mediated multidrug resistance found for the first time in entomopathogenic fungi is potential for use in improving mycoinsecticide compatibility with chemical pesticides.

Sporulation, storage and infectivity of obligate aphid pathogen Pandora nouryi grown on novel granules of broomcorn millet and polymer gel.

AIMS: Producing granular cultures of obligate aphid pathogen Pandora nouryi for improved sporulation and storage. METHODS AND RESULTS: Small millet-gel granules were made of the mixtures of 80-95% millet powder with 5-20% polymer gel (polyacrylamide, polyacrylate or acrylate-acrylamide copolymer) and inoculated with mycelia at 30 mg biomass g(-1) dry granules plus 87.5% water, followed by static incubation at 20 degrees C for 4-12 days. The fungus grew well on 12 preparations but best on that including 10% copolymer. An 8-day culture of this preparation discharged maximally 58.5 x 10(4) conidia mg(-1) granule at 100% RH and was capable of ejecting conidia at the nonsaturated regimes of 86-97% RH. During storage at 6 degrees C, granular cultures with >85% water content had twofold longevity (120 days) and half-decline period (34-36 days) of those stored at room temperature. The steadily high water content preserved the cultures better than that decreasing at 6 degrees C. However, conidia from 70-day-stored granules were less infective to Myzus persicae nymphs than those from fresh ones based on their LC(50)s. CONCLUSIONS: The millet-gel granules had higher sporulation capacity than reported Pandora cultures and a capability of spore discharge at nonsaturated humidity. SIGNIFICANCE AND IMPACT OF THE STUDY: The granular cultures are more useful for aphid control.

Medium components and culture conditions affect the thermotolerance of aerial conidia of fungal biocontrol agent Beauveria bassiana.

AIMS: To produce more thermotolerable conidia of Beauveria bassiana, a well-known fungal biocontrol agent, by optimizing the medium components and culture conditions. METHODS AND RESULTS: The conidia produced on media including 0.5-6% glucose, sucrose or starch as carbon source and 50-300-microg ml(-1) Cu2+, Zn2+, Mn2+ or Fe3+ as additive to Sabouraud dextrose medium at 15-30 degrees C, pH 4-8 or KCl-adjusted water availabilities were exposed to 30-min wet heat stress at 48 degrees C. The medium components for conidial production with greatly enhanced thermotolerance included 4% glucose as optimum or 1% starch as alternative for the carbon source and < or =50-microg ml(-1) Mn2+ for the metal additive. The culture conditions were optimized as 25 degrees C and pH 5-6. Conidial thermotolerance decreased remarkably when sucrose and Fe3+ or Cu2+ were used in the cultures, but altered slightly when 50-200-microg ml(-1) Zn2+ were included. CONCLUSIONS: The tolerance of B. bassiana conidia to the thermal stress was significantly affected by the medium composition and culture conditions under which the conidia were produced. SIGNIFICANCE AND IMPACT OF THE STUDY: Proper treatment of small grains as mass production substrates for more glucose release and supplement of glucose or 50-microg ml(-1) Mn2+ are possible means to enhancing conidial thermotolerance and field persistence for improved insect control.

New solid-state fermentation chamber for bulk production of aerial conidia of fungal biocontrol agents on rice.

A novel solid-state fermentation apparatus, namely an upright multi-tray conidiation chamber, was developed to facilitate the production of aerial conidia of fungal biocontrol agents, such as Beauveria bassiana. The chamber with 25 bottom-meshed metal trays had a capacity of > or =50 kg rice with each tray holding > or =2 kg. In repeated trials, a mean yield of 2.4 (1.8-2.7) x 10(12) conidia kg(-1) rice was harvested from the 7-day cultures of B. bassiana in a fully loaded chamber. The new apparatus has a high potential for bulk production of fungal conidia.

Relationship between thermotolerance and hydrophobin-like proteins in aerial conidia of Beauveria bassiana and Paecilomyces fumosoroseus as fungal biocontrol agents.

AIMS: This study was to illustrate the relationship between the thermotolerance and the contents of hydrophobin-like or formic-acid-extractable (FAE) proteins in aerial conidia of Beauveria bassiana and Paecilomyces fumosoroseus produced on rice-based substrate. METHODS AND RESULTS: Survival indices of 11 isolates were separately assessed as a ratio of the viability of conidia after 3-150 min thermal stress at 48 degrees C over that of unstressed conidia and fitted well to a survival model (r(2) >/= 0.97). For a given isolate, the fitted model generated an LT(50), the time for 50% viability loss under the stress. The LT(50)s of six B. bassiana isolates (10.1-61.9 min) and five P. fumosoroseus isolates (2.8-6.2 min) were correlated (r(2) = 0.81) with FAE protein contents (6.9-23.4 microg mg(-1)). The survival indices of a fixed B. bassiana isolate after 45-min thermal stress at 48 degrees C were also correlated to the FAE protein contents from conidia produced on glucose-, sucrose-, or starch-based substrate (0.79

[Optional growth medium and conditions for mass production of Pandora delphacis mycelia in submerged culture].

The entomophthoraceous fungi are important microbial control agents for insect control but their mass production is usually difficult and expensive. To produce in large quantity the mycelia of the entomophthoraceous fungus, Pandora delphacis (isolate F95129), this study was aimed at replacing expensive components of liquid medium that is usually used in laboratory only with cheap materials easily available. Based on comparative experiments with primary components of several media designed and optional conditions for submerged culture, an appropriate medium was recognized that included(per liter) 10 g of homemade yeast extract, 10 g industrial fish meal, 10 g wheat bran, 15 g corn meal, 1.0 g KH2PO4, 3.0 g NH4NO3, and 0.25 g MgSO(4).7H2O. The optional conditions for submerged culture was 25-30 degrees C, initial pH 6.5, and 40% (V/V) of flask filled with the medium and 10% of initial inoculum (liquid culture containing mycelia) for inoculation. Using the selected medium and the conditions considered, 48 h culture resulted in a considerably high yield of dry mycelia(> 25 mg/mL) with desirable capacity of spore production.

Selective effect of tempol on renal medullary hemodynamics in spontaneously hypertensive rats.

The present study assessed the short- and long-term effect of tempol, a membrane-permeable mimetic of superoxide dismutase, on renal medullary hemodynamics in spontaneously hypertensive rats (SHR). Tempol was given in the drinking water (1 mM) for 4 days or 7 wk (4-11 wk of age), and medullary blood flow (MBF) was measured over a wide range of renal arterial pressure by means of laser-Doppler flowmetry in anesthetized rats. In addition, the response of the medullary circulation to angiotensin II (5-50 ng x kg(-1) x min(-1) iv) was determined in SHR treated for 4 days with tempol. Compared with control SHR, short- and long-term treatment with tempol decreased arterial pressure by approximately 20 mmHg and increased MBF by 35-50% without altering total renal blood flow (RBF) or autoregulation of RBF. Angiotensin II decreased RBF and MBF dose dependently (approximately 30% at the highest dose) in control SHR. In SHR treated with tempol, angiotensin II decreased RBF (approximately 30% at the highest dose) but did not alter MBF significantly. These data indicate that the antihypertensive effect of short- and long-term administration of tempol in SHR is associated with a selective increase in MBF. Tempol also reduced the sensitivity of MBF to angiotensin II. Taken together, these data support the idea that tempol enhances vasodilator mechanisms of the medullary circulation, possibly by interacting with the nitric oxide system. Increased MBF and reduced sensitivity of MBF to angiotensin II may contribute to the antihypertensive action of tempol in SHR.

Abnormal renal medullary response to angiotensin II in SHR is corrected by long-term enalapril treatment.

This study tested the hypotheses that renal medullary blood flow (MBF) in spontaneously hypertensive rats (SHR) has enhanced responsiveness to angiotensin (ANG) II and that long-term treatment with enalapril can correct this. MBF, measured by laser Doppler flowmetry in anesthetized rats, was not altered significantly by ANG II in Wistar-Kyoto (WKY) rats, but was reduced dose dependently (25% at 50 ng. kg(-1). min(-1)) in SHR. Infusion of N(G)-nitro-L-arginine methyl ester (L-NAME) into the renal medulla unmasked ANG II sensitivity in WKY rats while L-arginine given into the renal medulla abolished the responses to ANG II in SHR. In 18- to 19-wk-old SHR treated with enalapril (25 mg. kg(-1). day(-1) when 4 to 14 wk old), ANG II did not alter MBF significantly, but sensitivity to ANG II was unmasked after L-NAME was infused into the renal medulla. Endothelium-dependent vasodilation (assessed with aortic rings) was significantly greater in treated SHR when compared with that in control SHR. These results indicate that MBF in SHR is sensitive to low-dose ANG II and suggest that this effect may be due to an impaired counterregulatory effect of nitric oxide. Long-term treatment with enalapril improves endothelium-dependent vascular relaxation and decreases the sensitivity of MBF to ANG II. These effects may be causally related to the persistent antihypertensive action of enalapril in SHR.

Midazolam produces vasodilation by mixed endothelium-dependent and -independent mechanisms.

Aortic rings were obtained from rat thoracic aorta and studied in vitro with and without functionally intact endothelium to determine whether "the mechanism" requires endothelium [or endothelium-derived relaxing factor (EDRF)]. In aortic rings precontracted with either phenylephrine (PE, 3 x 10(-8)-3 x 10(-7) mol/L) or KCl (40 mmol/L), midazolam produced concentration-dependent relaxation, with and without endothelium. Rings without endothelium demonstrated significantly less relaxation than those with endothelium regardless of whether they were precontracted with PE or KCl. With intact endothelium, midazolam produced greater relaxation in PE-contracted aortic rings than in KCl-contracted aortic rings; the midazolam concentration producing 50% relaxation from the contracted state (RC50) was 8.8 +/- 3.6 x 10(-7) mol/L for PE-contracted rings and 3.3 +/- 1.1 x 10(-6) mol/L for KCl-contracted rings (P < 0.05). In aortic rings with intact endothelium pretreated with NG-monomethyl-L-arginine (L-NMMA, 10(-4) mol/L), an inhibitor of nitric oxide (NO) synthesis, midazolam produced relaxation of similar magnitude to that seen in the denuded aortic rings except at the highest concentration (1 x 10(-5) mol/L). Pretreatment with the cyclooxygenase inhibitor, indomethacin (2.5 x 10(-5) mol/L), did not change the midazolam-induced relaxation in rings with intact endothelium as compared to untreated control aortic rings. In contrast to the intact endothelium state, when endothelium was removed, midazolam produced greater relaxation in the KCl-contracted aortic rings than in PE-contracted rings (RC50, 1.2 +/- 0.3 x 10(-5) mol/L vs 2.3 +/- 0.4 x 10(-5) mol/L, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)