The Mycotoxin Fumonisin B1 Induces Metabolic and Molecular Perturbations Leading to Cell Death in Arabidopsis Cell Culture

Fumonisin B1 (FB1) is a fungal toxin produced by Fusarium spp. able to exert pleiotropic toxicity in plants. FB1 is known to be a strong inducer of the programmed cell death (PCD); however, the exact mechanism underling the plant-toxin interactions and the molecular events that lead to PCD are still unclear. Therefore, in this work we provided a comprehensive investigation of the response of the model organism Arabidopsis thaliana at the nuclear, transcriptional, and biochemical level after the treatment with FB1 at two different concentrations, namely 1 and 5µM during a time-course of 96 h. FB1 induced oxidative and nitrosative bursts and a rapid cell death in Arabidopsis cell cultures, which resembled a HR-like PCD event. Different genes involved in the regulation of PCD, antioxidant metabolism, photosynthesis, resistance, and sugar transport were upregulated, especially during the late treatment time and with higher FB1 concentration. Among the antioxidant enzymes and compounds studied, only glutathione appeared to be highly induced in both treatments, suggesting that it might be an important defense molecule induced during FB1 exposure. Collectively, these findings highlight the complexity of the defense network of A. thaliana and provide important information for the understanding of the physiological, molecular, and biochemical responses to counteract FB1-induced toxicity.

A LC–ESI–MS/MS analysis procedure coupled with solid phase extraction and MeOH extraction method for determination of pyrrolizidine alkaloids in Tussilago farfara and Lithospermi erythrorhzion

Pyrrolizidine alkaloids (PAs) that are plant toxin naturally produced for protection against herbivores in some plant families. They are associated with the potential hepatotoxic and carcinogenic diseases serious hepatic disease in humans and animals. As the concern of human health risk posed by exposure to PAs has been gradually increased, precise and reliable analysis is required for monitoring PAs. The present study developed a new and simple pretreatment using 50% MeOH (methanol) for quantification analysis of the PAs contained with high content in the herbal medicines. Another pretreatment method using cation-ion exchange solid-phase extraction (MCX-SPE) was employed for determining most of the PAs that are not contained in the herbal medicines. That is, the proposed LC–MS/MS method coupled with MCX-SPE extraction and 50% MeOH extraction method was developed. And to evaluate the reliability of its application for Farfarae Flos and Lithospermi Radix, a validation study was conducted. In addition, monitory study was performed with ten samples in each herbal medicine. As a result, the proposed method had good linearity with r2 ≥ 0.997. Also, the recoveries indicated to be in the ranges of 70.4–118.0% for the Farfarae Flos, 70.2–119.7% for the Lithospermi Radix. In two herbal medicines, the intra-day precision was revealed to satisfy the reference criteria in most of the PAs. In monitoring results, most of the PAs were not contained in two herbal medicines, whereas a part of PAs revealed to have high concentration in Farfarae Flos and Lithospermi Radix. The proposed method is considered as a simple and reliable method to quantify 28 PAs present in two herbal medicines. Especially, the simple MeOH extraction method seems to be available for quantification analysis of certain PAs in herbal medicines with high content.

Evolutionary and phylogenetic insights from a nuclear genome sequence of the extinct, giant, “subfossil” koala lemur Megaladapis edwardsi

No endemic Madagascar animal with body mass >10 kg survived a relatively recent wave of extinction on the island. From morphological and isotopic analyses of skeletal “subfossil” remains we can reconstruct some of the biology and behavioral ecology of giant lemurs (primates; up to ∼160 kg) and other extraordinary Malagasy megafauna that survived into the past millennium. Yet, much about the evolutionary biology of these now-extinct species remains unknown, along with persistent phylogenetic uncertainty in some cases. Thankfully, despite the challenges of DNA preservation in tropical and subtropical environments, technical advances have enabled the recovery of ancient DNA from some Malagasy subfossil specimens. Here, we present a nuclear genome sequence (∼2× coverage) for one of the largest extinct lemurs, the koala lemur Megaladapis edwardsi (∼85 kg). To support the testing of key phylogenetic and evolutionary hypotheses, we also generated high-coverage nuclear genomes for two extant lemurs, Eulemur rufifrons and Lepilemur mustelinus, and we aligned these sequences with previously published genomes for three other extant lemurs and 47 nonlemur vertebrates. Our phylogenetic results confirm that Megaladapis is most closely related to the extant Lemuridae (typified in our analysis by E. rufifrons) to the exclusion of L. mustelinus, which contradicts morphology-based phylogenies. Our evolutionary analyses identified significant convergent evolution between M. edwardsi and an extant folivore (a colobine monkey) and an herbivore (horse) in genes encoding proteins that function in plant toxin biodegradation and nutrient absorption. These results suggest that koala lemurs were highly adapted to a leaf-based diet, which may also explain their convergent craniodental morphology with the small-bodied folivore Lepilemur.

Total synthesis of ent-pavettamine

Pavettamine, a plant toxin first isolated from Pavetta harborii in 1995, was previously identified as a polyamine with C2 symmetry and a 1,3-syn-diol moiety on a C10 carbon backbone – one of very few substituted polyamines to be isolated from nature. Its absolute configuration was later established by our first reported total synthesis in 2010. Herein we report the first total synthesis of the enantiomer of pavettamine, ent-pavettamine. The symmetrical structure of the molecule allows for the synthesis of a common C5 fragment that can be divergently transformed into two synthons for later convergent coupling to furnish the target carbon framework. Based on the success of the protocol we employed for the synthesis of the naturally occurring pavettamine, (S)-malic acid was again the starting material of choice for the synthesis of the two individual C5 fragments, with strategic differences in terminal-group manipulation allowing for the synthesis of ent-pavettamine rather than pavettamine. Chain extension and stereoselective ketone reduction were achieved using the (R)-methyl p-tolyl sulfoxide chiral auxiliary to give the desired 1,3-syn-diol C5 unit. A protecting-group strategy was also developed for the orthogonal protection of the alcohol and amine functional groups as they were unveiled. The functionalized C5 fragments were coupled via reductive amination revealing the C10 carbon backbone. Deprotection of the alcohol and amine functional groups successfully provided ent-pavettamine as a TFA salt.

Spiral Steroids During Pregnancy - It’s All About Potassium

This presentation has two parts. The 1st section describes processes during pregnancy with unknown, underlying biochemistry. The 2nd section shows the role of spiral steroids (SS) in these processes. 1] Fetal nutrition is provided through the placenta. Plasma electrolytes are 145 mM Na+ and 3-5 mM K+. Fetal K+ requirements reach a maximum during the 3rd trimester and must be pumped into cells against the plasma- intracellular gradient. 2] During the 3rd trimester, aldosterone is present in fetal plasma but the signal for endothelial sodium channel (ENaC) synthesis is blocked, leading to fetal Na+ wasting. The mechanism is unknown. 3] After parturition, infants are fed by nursing. Human milk contains 100 mM K+ and 10 mM Na+. Newborn infants are Na+ wasting, despite normal levels of aldosterone. Na+ wasting ends during the 2nd week post-partum without change in serum aldosterone levels. Infant physiology changes from K+ saving to Na+ saving by an unknown mechanism.4] Pre-eclampsia syndrome (proteinuria and hypertension during the 2nd half of pregnancy) affects 3-5 % of pregnant women. These symptoms usually resolve after parturition. 5] Women who have had pre-eclampsia have long-term, excess risk of cardiac and renal disorders. In 2018, we discovered Ionotropin, a SS. SS are phosphocholine esters of steroids with a lactone E-ring, similar to that of spironolactone. SS compounds function as aldosterone antagonists and regulate the NaK-ATPase. SS are involved in each of the 5 steps.1] SS are present in high levels in cord serum and were probably present in fetal plasma throughout gestation. SS stimulate the NaK-ATPase to pump K+ into cells against the gradient, just as does spironolactone.2] SS interfere with aldosterone signaling, just as does amiloride. This leads to increased fetal Na+ wasting, which becomes amniotic fluid.3] SS disappear from the infant circulation during the 1st week after parturition and decrease to adult levels during the 2nd week post-partum. Simultaneously, Na+ wasting ends and growth resumes.4] Women with pre-eclampsia have excess precursors for SS. These would be converted to SS in the fetal-placental unit and, during the 3rd trimester, diffuse into the maternal circulation and could cause pre-eclampsia.5] Ouabain, a plant toxin with a lactone E ring, causes renal and cardiac disorders in rat models. In women with pre-eclampsia, persistent excess SS may cause long-term damage. During gestation, the fetus requires K+ for growth. Our theory is, if (when) a fetus has inadequate K+ (hypokalemia), [a] the mother is signaled to produce SS precursors, [b] the feto-placental unit converts the precursors to SS, [c] fetal SS increase K+ transfer into tissues, and [d] excess SS transfuse back into the maternal circulation and damage maternal organs. We propose that pre-eclampsia is a side effect of fetal efforts to increase supply of K+. There are many possible origins of fetal hypokalemia.

Development of a Novel Pipette Tip-Aided Cell Cloning Method for The Effective Isolation of Genome-Edited Porcine Cell

Direct colony cloning of adherent mammalian cells using rings or dilution cloning has been used frequently for obtaining stable transfectants after gene delivery. As an alternative to these methods, successful isolation of the cells in a single colony is possible by placing a trypsin-immersed small paper disk onto the colony and subsequently picking up the paper with the assumption that it carries the trypsinized cells. However, the cloning success using this technique largely relies on the cell type used. In the present study, a novel, simple, and non-invasive technique for the isolation of cells from single colonies using a disposable pipette tip was developed. Using this technique, success was achieved in isolating the clonal populations of genome-edited porcine fibroblastic cells with 100% efficiency after co-transfection with the clustered, regularly interspaced, short palindromic repeats-CRISPR associated protein 9 (CRISPR/Cas9)-based genome editing components [for targeting the porcine GGTA1 that encodes α-1,3-galactosyltransferase (α-GalT)] and the piggyBac-based gene delivery components [to enable efficient chromosomal integration of the transgene carrying the cDNA of enhanced green fluorescent protein (EGFP)]. A toxin-based, drug-free selection system involving saporin (plant toxin)-conjugated BS-I-B4 lectin (IB4SAP) was employed in the present study. Since IB4SAP binds specifically to the cell-surface α-Gal epitope (synthesized by α-GalT), it is supposed that treatment with IB4SAP theoretically eliminates the untransfected or genome-edited porcine cells with a mono-allelic knockout (KO) phenotype, while all the surviving clones have a bi-allelic GGTA1 mutation. A total of 16 clones were isolated in the present study, all of which exhibited loss of the α-Gal epitope (a cell-surface carbohydrate synthesized by α-GalT), suggesting that all the clones had a bi-allelic KO phenotype. Moreover, 75% of these clones expressed EGFP uniformly, while the remainder had mosaic or no EGFP expression. These findings indicate the fidelity of the developed pipette tip-aided cell cloning approach for the efficient isolation of genome-edited porcine fibroblast clones.

Effectiveness of the toxin-degrading strain screening strategy: Deficiency and improvement in screening the gossypol-degrading microorganism Aspergillus niger

Background The gossypol, as an antinutritional factor or natural plant toxin, has antifertility effects on humans and animals, exists in cottonseed oil and meal, and is a typical food or feed contaminant. It is the classic screening method to using acetate gossypol as the sole carbon sources to prepare selective medium and screening gossypol-degrading microorganisms. By the classic screening strategy, many researchers have discovered that some microorganisms, and Aspergillus niger is the most reported microorganism. A strain of A. niger which can grow in an agar solid medium with gossypol as the sole carbon source is isolated from cattle rumen liquid was obtained by classic screening strategy. The effectiveness of the classic screening strategy was verified, by duplicating and verifying the degradation of gossypol by the A. niger. Results the A. niger can reduce the free gossypol (FG) content through biosorption but has no effect on the total gossypol (TG) content. And it can secrete agarase, utilise agar as carbon source. Conclusions The A. niger can secrete agarase, but cannot effectively degrade gossypol, In this case, it will mislead researchers and lead them to make wrong judgments. The usual methods of previous screening strategies are not rigorous enough, the classic screening method has defect in screening toxin-degrading strain, so agar control group should be added.

Mucoricin is a Ricin-Like Toxin that is Critical for the Pathogenesis of Mucormycosis

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We now demonstrate that Mucorales fungi produce a toxin that plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro, and prevent hypovolemic shock, organ necrosis, and death in mice with mucormycosis. RNAi inhibition of the toxin in Rhizopus delemar, compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin, ricin, including the ability to inhibit protein synthesis by its N-glycosylase activity, the existence of a motif that mediates vascular leak, and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross-react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name “mucoricin” for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin- like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.