Thymosin β4 Is an Endogenous Iron Chelator and Molecular Switcher of Ferroptosis

Thymosin β4 (Tβ4) was extracted forty years agofrom calf thymus. Since then, it has been identified as a G-actin binding protein involved in blood clotting, tissue regeneration, angiogenesis, and anti-inflammatory processes. Tβ4 has also been implicated in tumor metastasis and neurodegeneration. However, the precise roles and mechanism(s) of action of Tβ4 in these processes remain largely unknown, with the binding of the G-actin protein being insufficient to explain these multi-actions. Here we identify for the first time the important role of Tβ4 mechanism in ferroptosis, an iron-dependent form of cell death, which leads to neurodegeneration and somehow protects cancer cells against cell death. Specifically, we demonstrate four iron2+ and iron3+ binding regions along the peptide and show that the presence of Tβ4 in cell growing medium inhibits erastin and glutamate-induced ferroptosis in the macrophage cell line. Moreover, Tβ4 increases the expression of oxidative stress-related genes, namely BAX, hem oxygenase-1, heat shock protein 70 and thioredoxin reductase 1, which are downregulated during ferroptosis. We state the hypothesis that Tβ4 is an endogenous iron chelator and take part in iron homeostasis in the ferroptosis process. We discuss the literature data of parallel involvement of Tβ4 and ferroptosis in different human pathologies, mainly cancer and neurodegeneration. Our findings confronted with literature data show that controlled Tβ4 release could command on/off switching of ferroptosis and may provide novel therapeutic opportunities in cancer and tissue degeneration pathologies.

Toxicity of Legiayu incense as Insecticide and Larvicide Against Aedes aegypti Mosquitoes Mortality

Community-based DHF vector control has been implemented in Indonesia but has not yet obtained optimal results. Thus, in the community choosing synthetic insecticides to control disease vectors. However, irregular and excessive use of insecticides has a toxic effect and resistance to mosquitoes. Burning mosquito coils and incense containing synthetic dyes and fragrances have the potential to reduce environmental quality. Therefore, this research was conducted to analyze the toxicity of Legiayu incense as an insecticide and larvicide against Aedes aegypti mosquito mortality. The research design is experimental with a completely randomized design. Testing was conducted by providing exposure to smoke and ash of Legiayu incense five times on twenty-five Aedes aegypti mosquitoes. Statistical analysis used one-way ANOVA, LSD, and probit test. The test result as insecticide value (p=0.000) effective exposure for 20 minutes with a durability of 6 hours. The test result as larvicide value (p=0.000) effective exposure for 24 hours. Thus, exposure toismoke and ash of iLegiayuiincense has a very noticeable effect on the mortality of Aedes aegypti mosquitoes. Exposureito Legiayu incense smoke obtained an LT50 value of 0,9012 ≤ 5 (super toxic category) with a time of 15 minutes 39 seconds, coefficient determination of 99.24%, and correlation coefficient of 99.62% while exposure to the ash of Legiayu incense obtained LT50 value of 0,05896 ≤ 5 (super toxic category) with time 19 hours 15 minutes 34 seconds, coefficient determination and correlation coefficient of 100%. Histopathological test results showed that Legiayu incense smoke did not cause tissue degeneration, necrosis, hyperplasia, and metaplasia in the lung tissue of mice (mus musculus) within a period of 12 weeks. Thus, Legiayu incense is effective as insecticides and larvicides against Aedes aegypti mosquitoes. Legiayu incense has potential substitute for mosquito repellent coils, temephos, and synthetic incense circulating in the market.

Long-Term Maintenance and Meiotic Entry of Early Germ Cells in Murine Testicular Organoids Functionalized by 3D Printed Scaffolds and Air-Medium Interface Cultivation

Short-term germ cell survival and central tissue degeneration limit organoid cultures. Here, testicular organoids (TOs) were generated from two different mouse strains in 3D printed one-layer scaffolds (1LS) at the air-medium interface displaying tubule-like structures and Leydig cell functionality supporting long-term survival and differentiation of germ cells to the meiotic phase. Chimeric TOs, consisting of a mixture of primary testicular cells and EGFP+ germline stem (GS) cells, were cultured in two-layer scaffolds (2LSs) for better entrapment. They showed an improved spheroidal morphology consisting of one intact tubule-like structure and surrounding interstitium, representing the functional unit of a testis. However, GS cells did not survive long-term culture. Consequently, further optimization of the culture medium is required to enhance the maintenance and differentiation of germ cells. The opportunities TOs offer to manipulate somatic and germ cells are essential for the study of male infertility and the search for potential therapies.

A histopathological comparison of prophylactic effects of Rosmarinic Acid and Oleanolic Acid isolated from Salvia species (sage) in scopolamine induced dementia model

Objective: Alzheimer's disease (AD) is the most common form of dementia, can be created in experimental models by using toxins. Possible therapeutic effects of secondary metabolites Rosmarinic (RA) and Oleanolic acids (OA) obtained from Salvia species (sage) were investigated using histopathological and immunohistochemical tecniques in experimental Alzheimer's-like dementia model induced by scopolamine. Study Design: Male BALB/c mice (n: 48) of 3-4 weeks old, divided into 6 groups; control received only saline(i.p.) for 21 days, scopolamine group received 3 mg/kg of scopolamine (i.p.) between 8-21th days. Scopolamine + RA group received 5 mg/kg RA (i.p.) between days 0 and 21 and 3 mg/kg of scopolamine (i.p.) between 8-21th days. Scopolamine + OA group received 5 mg/kg of OA (i.p.) between days of 0-21 and 3 mg/kg of scopolamine (i.p.) between 8-21th days. RA group received 5 mg/kg RA (i.p.) between 0-21th days. OA group received 5 mg/kg of OA (i.p.) between 0-21th days. Animals were sacrificed under anesthesia, brain tissues were excised and placed in 10% formaldehyde, embedded in paraffin wax. 5 µm sections were cut, stained for histopathological examinations. Immunoreactivity for Beta-amyloid accumulation was observed. ImageJ 153 software was used to analyze IHC figures. Positive signaling for DAB density was calculated, ANOVA test with the post hoc dunnett's or tukey test were applied, p<0.05 was considered statistically significant. Results: In scopolamine+RA treated group, tissue degeneration was less compared to the scopolamine group. Scopolamine+OA group revealed signs of pyknosis in neurons. The amyloid beta immunoexpression was positive in scopolamine group partially positive in scopolamine+OA group but, negative in RA treated scopolamine group. Intensity of signal in scopolamine group was statistically increased compared to control (p=0.0102). Intensity of signal was reduced in Scopolamine+RA and Scopolamine+OA groups, was statistically significant (p=0.0281 and p=0.0362, respectively). Conclusion: We suggest that pretreatment of RA and OA decreased beta amyloid formation and ameliorated tissue structure but, further studies with different methods are needed to be commercially available. Keywords: Scopolamine, dementia, Rosmarinic acid, Oleanolic acid, histopathology, Amyloid beta

A molecular spectroscopy approach for the investigation of early phase ochronotic pigment development in Alkaptonuria

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs due to a deficiency in functional levels of the enzyme homogentisate 1,2-dioxygenase (HGD), required for the breakdown of HGA, because of mutations in the HGD gene. Over time, HGA accumulation causes the formation of the ochronotic pigment, a dark deposit that leads to tissue degeneration and organ malfunction. Such behaviour can be observed also in vitro for HGA solutions or HGA-containing biofluids (e.g. urine from AKU patients) upon alkalinisation, although a comparison at the molecular level between the laboratory and the physiological conditions is lacking. Indeed, independently from the conditions, such process is usually explained with the formation of 1,4-benzoquinone acetic acid (BQA) as the product of HGA chemical oxidation, mostly based on structural similarity between HGA and hydroquinone that is known to be oxidized to the corresponding para-benzoquinone. To test such correlation, a comprehensive, comparative investigation on HGA and BQA chemical behaviours was carried out by a combined approach of spectroscopic techniques (UV spectrometry, Nuclear Magnetic Resonance, Electron Paramagnetic Resonance, Dynamic Light Scattering) under acid/base titration both in solution and in biofluids. New insights on the process leading from HGA to ochronotic pigment have been obtained, spotting out the central role of radical species as intermediates not reported so far. Such evidence opens the way for molecular investigation of HGA fate in cells and tissue aiming to find new targets for Alkaptonuria therapy.

Implantálható kardioverter-defibrillátor által sikeresen megszüntetett malignus kamrai ritmuszavar dystrophia myotonicában szenvedő betegben

Összefoglaló. A dystrophia myotonica (DM) multiszisztémás, autoszomális domináns módon öröklődő, többségében felnőttkori izombetegség, melynek incidenciája 1 : 8000. A betegség kapcsán fellépő izomszöveti degeneráció a harántcsíkolt izomszövet átépülése mellett a szívizomszövetet is érinti, ami fontos oki szerepet játszik az érintett betegek csökkent várható élettartamában. A DM-ben szenvedők halálozásának közel egyharmadáért a cardiovascularis okok tehetők felelőssé. Esetriportunkban egy 52 éves, korábban kritikus bradycardia és I. fokú atrioventricularis blokk miatt pacemakerimplantáción átesett, DM-mel diagnosztizált nőbeteg kardiológiai utánkövetését mutatjuk be. A hirtelen szívhalál rizikóstratifikációja céljából elvégzett invazív elektrofiziológiai vizsgálat során kamrafibrilláció lépett fel, így a korábban implantált pacemakerelektródák mellé sokkelektróda került beültetésre, a pacemakerkészüléket implantálható kardioverter-defibrillátorra (ICD) cseréltük. Az 1 éves ICD-kontrollvizsgálat során azt találtuk, hogy a beültetés óta 22, tartós kamrai tachycardiával járó epizód lépett fel, melyek közül a készülék valamennyit sikeresen terminálta. Az eset bemutatásával szeretnénk rámutatni arra, hogy a magas cardiovascularis rizikócsoportba tartozó DM-betegek azonosítása kiemelkedő fontosságú lehet a hirtelen szívhalál megelőzése érdekében. Orv Hetil. 2021; 162(46): 1856–1858. Summary. Myotonic dystrophy (DM) is one of the most frequent adulthood diseases of the skeletal muscles, which develops multisystemic features and shows autosomal dominant trait. In DM, tissue degeneration affects not only the skeletal, but the cardiac muscle, too. In one third of the patients, the cause of death is of cardiac origin. We report on our patient’s case, who was diagnosed with DM at the age of 52, in whom episodes of critical bradycardia with first-degree atrioventricular block was detected, resulting in a pacemaker implantation. Invasive cardiac electrophysiological study was performed, during which ventricular fibrillation was registered. A shock electrode was added to the previously implanted pacemaker, enabling defibrillation in case of detection of a sustained ventricular arrhythmia. During the 1-year follow-up, 22 episodes of sustained ventricular tachycardia were identified, with the device successfully terminating the malignant arrhythmias. Our case shows that electrophysiological study and the succeeding implantation of an implantable cardiac defibrillator is highly important in identifying and terminating ventricular arrhythmias in high-risk DM patients. Orv Hetil. 2021; 162(46): 1856–1858.

Platelet extracellular vesicles enhance the proangiogenic potential of adipose-derived stem cells in vivo and in vitro

Background Adipose-derived mesenchymal stem cells (ADSC)-based therapy is an outstanding treatment strategy for ischaemic disease. However, the therapeutic efficacy of this strategy is not ideal due to the poor paracrine function and low survival rate of ADSCs in target regions. Platelet extracellular vesicles (PEVs) are nanoparticles derived from activated platelets that can participate in communication between cells. Accumulating evidence indicates that PEVs can regulate the biological functions of several cell lines. In the present study, we aimed to investigate whether PEVs can modulate the proangiogenic potential of ADSCs in vitro and in vivo. Methods PEVs were identified using scanning electron microscope (SEM), flow cytometry (FCM) and nanoparticle tracking analysis (NTA). The CCK8 assay was performed to detect proliferation of cells. Transwell and wound healing assays were performed to verify migration capacity of cells. AnnexinV-FITC/PI apoptosis kit and live/dead assay were performed to assess ADSCs apoptosis under Cocl2-induced hypoxia condition. The underlying mechanisms by which PEVs affected ADSCs were explored using real time-PCR(RT-PCR) and Western blot. In addition, matrigel plug assays were conducted and mouse hindlimb ischaemic models were established to investigate the proangiogenic potential of PEV-treated ADSCs in vivo. Results We demonstrated that ADSC could internalize PEVs, which lead to a series of biological reactions. In vitro, dose-dependent effects of PEVs on ADSC proliferation, migration and antiapoptotic capacity were observed. Western blotting results suggested that multiple proteins such as ERK, AKT, FAK, Src and PLCγ1 kinase may contribute to these changes. Furthermore, PEVs induced upregulation of several growth factors expression in ADSCs and amplified the proliferation, migration and tube formation of HUVECs induced by ADSC conditioned medium (CM). In in vivo experiments, compared with control ADSCs, the injection of PEV-treated ADSCs resulted in more vascularization in matrigel plugs, attenuated tissue degeneration and increased blood flow and capillary density in ischaemic hindlimb tissues. Conclusion Our data demonstrated that PEVs could enhance the proangiogenic potential of ADSCs in mouse hindlimb ischaemia. The major mechanisms of this effect included the promotion of ADSC proliferation, migration, anti-apoptosis ability and paracrine secretion.

Epigenetic features in regulation of telomeres and telomerase in stem cells

The epigenetic nature of telomeres is still controversial and different human cell lines might show diverse histone marks at telomeres. Epigenetic modifications regulate telomere length and telomerase activity that influence telomere structure and maintenance. Telomerase is responsible for telomere elongation and maintenance and is minimally composed of the catalytic protein component, telomerase reverse transcriptase (TERT) and template forming RNA component, telomerase RNA (TERC). TERT promoter mutations may underpin some telomerase activation but regulation of the gene is not completely understood due to the complex interplay of epigenetic, transcriptional, and posttranscriptional modifications. Pluripotent stem cells (PSCs) can maintain an indefinite, immortal, proliferation potential through their endogenous telomerase activity, maintenance of telomere length, and a bypass of replicative senescence in vitro. Differentiation of PSCs results in silencing of the TERT gene and an overall reversion to a mortal, somatic cell phenotype. The precise mechanisms for this controlled transcriptional silencing are complex. Promoter methylation has been suggested to be associated with epigenetic control of telomerase regulation which presents an important prospect for understanding cancer and stem cell biology. Control of down-regulation of telomerase during differentiation of PSCs provides a convenient model for the study of its endogenous regulation. Telomerase reactivation has the potential to reverse tissue degeneration, drive repair, and form a component of future tissue engineering strategies. Taken together it becomes clear that PSCs provide a unique system to understand telomerase regulation fully and drive this knowledge forward into aging and therapeutic application.