Polypeptide N-acetylgalactosamine transferase 3: a post-translational writer on human health.

Polypeptide N-acetylgalactosamine transferase 3 (ppGalNAc-T3) is an enzyme involved in the initiation of O-GalNAc glycan biosynthesis. Acting as a writer of frequent post-translational modification (PTM) on human proteins, ppGalNAc-T3 has key functions in the homeostasis of human cells and tissues. We review the relevant roles of this molecule in the biosynthesis of O-GalNAc glycans, as well as in biological functions related to human physiological and pathological conditions. With main emphasis in ppGalNAc-T3, we draw attention to the different ways involved in the modulation of ppGalNAc-Ts enzymatic activity. In addition, we take notice on recent reports of ppGalNAc-T3 having different subcellular localizations, highlight critical intrinsic and extrinsic functions in cellular physiology that are exerted by ppGalNAc-T3-synthesized PTMs, and provide an update on several human pathologies associated with dysfunctional ppGalNAc-T3. Finally, we propose biotechnological tools as new therapeutic options for the treatment of pathologies related to altered ppGalNAc-T3. KEY MESSAGES: ppGalNAc-T3 is a key enzyme in the human O-GalNAc glycans biosynthesis. enzyme activity is regulated by PTMs, lectin domain and protein-protein interactions. ppGalNAc-T3 is located in human Golgi apparatus and cell nucleus. ppGalNAc-T3 has a central role in cell physiology as well as in several pathologies. Biotechnological tools for pathological management are proposed.

Aberrant Hoxa10 gene methylation as a mechanism for endosulfan-induced implantation failures in rats.

DNA methylation is a well-established epigenetic mechanism controlling gene expression. Environmental chemicals, such as pesticides have been shown to alter DNA methylation. We have previously shown that the insecticide endosulfan impairs female fertility in rats by increasing the rate of preimplantation embryo losses. In this study, we evaluated whether early postnatal exposure to endosulfan affects long-term transcriptional regulation of Homeobox A10 (Hoxa10) gene, which is a key marker of endometrial receptivity. Female rats were neonatally exposed to 6 or 600 µg/kg/day (ENDO6 and ENDO600, respectively) of endosulfan and uterine samples collected on gestational day (GD) 5. Hoxa10 protein and mRNA levels were assessed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), respectively. In silico analysis of enzyme-specific restriction sites and predicted transcription factors were performed to investigate the methylation status of the regulatory regions of Hoxa10 gene by methylation-sensitive restriction enzymes-PCR technique. The expression of the DNA methyltransferases (Dnmts) was also evaluated. ENDO600 showed a decreased uterine Hoxa10 expression at protein and transcript level, while ENDO6 decreased only the level of transcripts, during the receptive stage. In addition, endosulfan increased levels of Dnmt3a and Dnmt3b. Dysregulation of DNA methylation patterns of Hoxa10 regulatory regions was detected in ENDO6- and ENDO600-treated rats. All these results suggest that aberrant DNA methylation in Hoxa10 gene could be an underlining mechanism contributing to explain endosulfan-induced preimplantation losses.

Glyphosate Herbicide: Reproductive Outcomes and Multigenerational Effects.

Glyphosate base herbicides (GBHs) are the most widely applied pesticides in the world and are mainly used in association with GBH-tolerant crop varieties. Indiscriminate and negligent use of GBHs has promoted the emergence of glyphosate resistant weeds, and consequently the rise in the use of these herbicides. Glyphosate, the active ingredient of all GBHs, is combined with other chemicals known as co-formulants that enhance the herbicide action. Nowadays, the safety of glyphosate and its formulations remain to be a controversial issue, as evidence is not conclusive whether the adverse effects are caused by GBH or glyphosate, and little is known about the contribution of co-formulants to the toxicity of herbicides. Currently, alarmingly increased levels of glyphosate have been detected in different environmental matrixes and in foodstuff, becoming an issue of social concern. Some in vitro and in vivo studies have shown that glyphosate and its formulations exhibit estrogen-like properties, and growing evidence has indicated they may disrupt normal endocrine function, with adverse consequences for reproductive health. Moreover, multigenerational effects have been reported and epigenetic mechanisms have been proved to be involved in the alterations induced by the herbicide. In this review, we provide an overview of: i) the routes and levels of human exposure to GBHs, ii) the potential estrogenic effects of glyphosate and GBHs in cell culture and animal models, iii) their long-term effects on female fertility and mechanisms of action, and iv) the consequences on health of successive generations.

Epigenetic Changes Associated With Exposure to Glyphosate-Based Herbicides in Mammals.

Glyphosate is a phosphonomethyl amino acid derivative present in a number of non-selective and systemic herbicides. During the last years the use of glyphosate-based herbicide (GBH) has been increasing exponentially around the world, including Argentina. This fact added to the detection of glyphosate, and its main metabolite, amino methylphosphonic acid (AMPA), in environmental matrices such as soil, sediments, and food, has generated great concern about its risks for humans, animals, and environment. During the last years, there were controversy and intense debate regarding the toxicological effects of these compounds associated with the endocrine system, cancer, reproduction, and development. The mechanisms of action of GBH and their metabolites are still under investigation, although recent findings have shown that they could comprise epigenetic modifications. These are reversible mechanisms linked to tissue-specific silencing of gene expression, genomic imprinting, and tumor growth. Particularly, glyphosate, GBH, and AMPA have been reported to produce changes in global DNA methylation, methylation of specific genes, histone modification, and differential expression of non-coding RNAs in human cells and rodents. Importantly, the epigenome could be heritable and could lead to disease long after the exposure has ended. This mini-review summarizes the epigenetic changes produced by glyphosate, GBHs, and AMPA in humans and rodents and proposes it as a potential mechanism of action through which these chemical compounds could alter body functions.

Phonon Dephasing Dynamics in MoS2.

A variety of quantum degrees of freedom, e.g., spins, valleys, and localized emitters, in atomically thin van der Waals materials have been proposed for quantum information applications, and they inevitably couple to phonons. Here, we directly measure the intrinsic optical phonon decoherence in monolayer and bulk MoS2 by observing the temporal evolution of the spectral interference of Stokes photons generated by pairs of laser pulses. We find that a prominent optical phonon mode E2g exhibits a room-temperature dephasing time of ∼7 ps in both the monolayer and bulk. This dephasing time extends to ∼20 ps in the bulk crystal at ∼15 K, which is longer than previously thought possible. First-principles calculations suggest that optical phonons decay via two types of three-phonon processes, in which a pair of acoustic phonons with opposite momentum are generated.

Perinatal exposure to glyphosate or a glyphosate-based formulation disrupts hormonal and uterine milieu during the receptive state in rats.

We investigated the effects of perinatal exposure to a glyphosate-based herbicide (GBH) or glyphosate alone (Gly) on female fertility and the hormonal and uterine milieu during the preimplantation period. F0 pregnant rats orally received a GBH or Gly in a dose of 2 mg of glyphosate/kg/day from gestational day (GD) 9 until weaning. F1 females were evaluated to determine the reproductive performance on GD19; and the sex steroid serum levels, the expression of estrogen receptor alpha (ERα), progesterone receptor (PR) and implantation-related genes on GD5 (preimplantation period). GBH and Gly induced preimplantation losses in F1 rats. GBH and Gly groups exhibited higher 17ß-estradiol serum levels, without changes in progesterone. Both compounds increased the uterine ERα protein expression, with no differences at transcript level; and only Gly decreased PR mRNA expression. Also, GBH and Gly downregulated Hoxa10 and Lif genes, with no difference in Muc1 and Areg expression. To conclude, perinatal exposure to a GBH or Gly disrupted critical hormonal and uterine molecular targets during the receptive state, possibly associated with the implantation failures. Overall, similar results were found in GBH- and Gly-exposed rats, suggesting that the active principle might be the main responsible for the deleterious effects.

Correction to: Response to comments on: Perinatal exposure to a glyphosate-based herbicide impairs female reproductive outcomes and induces second-generation adverse effects in Wistar rats.

We report a mistake in the tables published in Milesi et al. (2019).

Anomalous spin-orbit torques in magnetic single-layer films.

The spin Hall effect couples charge and spin transport1-3, enabling electrical control of magnetization4,5. A quintessential example of spin-Hall-related transport is the anomalous Hall effect (AHE)6, first observed in 1880, in which an electric current perpendicular to the magnetization in a magnetic film generates charge accumulation on the surfaces. Here, we report the observation of a counterpart of the AHE that we term the anomalous spin-orbit torque (ASOT), wherein an electric current parallel to the magnetization generates opposite spin-orbit torques on the surfaces of the magnetic film. We interpret the ASOT as being due to a spin-Hall-like current generated with an efficiency of 0.053 ± 0.003 in Ni80Fe20, comparable to the spin Hall angle of Pt7. Similar effects are also observed in other common ferromagnetic metals, including Co, Ni and Fe. First-principles calculations corroborate the order of magnitude of the measured values. This work suggests that a strong spin current with spin polarization transverse to the magnetization can be generated within a ferromagnet, despite spin dephasing8. The large magnitude of the ASOT should be taken into consideration when investigating spin-orbit torques in ferromagnetic/non-magnetic bilayers.

Epigenetic disruption of estrogen receptor alpha is induced by a glyphosate-based herbicide in the preimplantation uterus of rats.

Previously, we have shown that perinatal exposure to a glyphosate-based herbicide (GBH) induces implantation failures in rats. Estrogen receptor alpha (ERα) is critical for successful implantation. ERα transcription is under the control of five promoters (E1, OT, O, ON, and OS), which yield different transcripts. Here, we studied whether perinatal exposure to a GBH alters uterine ERα gene expression and prompts epigenetic modifications in its regulatory regions during the preimplantation period. Pregnant rats (F0) were orally treated with 350 mg glyphosate/kg bw/day through food from gestational day (GD) 9 until weaning. F1 females were bred, and uterine samples were collected on GD5 (preimplantation period). ERα mRNA levels and its transcript variants were evaluated by RT-qPCR. Enzyme-specific restriction sites and predicted transcription factors were searched in silico in the ERα promoter regions to assess the methylation status using the methylation-sensitive restriction enzymes-PCR technique. Post-translational modifications of histones were studied by the chromatin immunoprecipitation assay. GBH upregulated the expression of total ERα mRNA by increasing the abundance of the ERα-O transcript variant. In addition, different epigenetic changes were detected in the O promoter. A decrease in DNA methylation was observed in one of the three sites evaluated in the O promoter. Moreover, histone H4 acetylation and histone H3 lysine 9 trimethylation (H3K9me3) were enriched in the O promoter in GBH-exposed rats, whereas H3K27me3 was decreased. All these alterations could account for the increase in ERα gene expression. Our findings show that perinatal exposure to a GBH causes long-term epigenetic disruption of the uterine ERα gene, which could be associated with the GBH-induced implantation failures.

Biosynthesis of O-N-acetylgalactosamine glycans in the human cell nucleus.

Biological functions of nuclear proteins are regulated by post-translational modifications (PTMs) that modulate gene expression and cellular physiology. However, the role of O-linked glycosylation (O-GalNAc) as a PTM of nuclear proteins in the human cell has not been previously reported. Here, we examined in detail the initiation of O-GalNAc glycan biosynthesis, representing a novel PTM of nuclear proteins in the nucleus of human cells, with an emphasis on HeLa cells. Using soluble nuclear fractions from purified nuclei, enzymatic assays, fluorescence microscopy, affinity chromatography, MS, and FRET analyses, we identified all factors required for biosynthesis of O-GalNAc glycans in nuclei: the donor substrate (UDP-GalNAc), nuclear polypeptide GalNAc -transferase activity, and a GalNAc transferase (polypeptide GalNAc-T3). Moreover, we identified O-GalNAc glycosylated proteins in the nucleus and present solid evidence for O-GalNAc glycan synthesis in this organelle. The demonstration of O-GalNAc glycosylation of nuclear proteins in mammalian cells reported here has important implications for cell and chemical biology.

An Update on Addison's Disease.

Addison's disease - the traditional term for primary adrenal insufficiency (PAI) - is defined as the clinical manifestation of chronic glucocorticoid- and/or mineralocorticoid deficiency due to failure of the adrenal cortex which may result in an adrenal crisis with potentially life-threatening consequences. Even though efficient and safe pharmaceutical preparations for the substitution of endogenous gluco- and mineralocorticoids are established in therapy, the mortality in patients with PAI is still increased and the health-related quality of life (HRQoL) is often reduced.PAI is a rare disease but recent data report an increasing prevalence. In addition to the common "classical" causes of PAI like autoimmune, infectious, neoplastic and genetic disorders, other iatrogenic conditions - mostly pharmacological side effects (e. g., adrenal haemorrhage associated with anticoagulants, drugs affecting glucocorticoid synthesis, action or metabolism and some of the novel anti-cancer checkpoint inhibitors) are contributing factors to this phenomenon.Due to the rarity of the disease and often non-specific symptoms at least in the early stages, PAI is frequently not considered resulting in a delayed diagnosis. Successful therapy is mainly based on adequate patient education as a cornerstone in the prevention and management of adrenal crisis. A focus of current research is in the development of pharmacokinetically optimized glucocorticoid preparations as well as regenerative therapies.

Perinatal exposure to a glyphosate-based herbicide impairs female reproductive outcomes and induces second-generation adverse effects in Wistar rats.

Glyphosate-based herbicides (GBHs) are the most globally used herbicides raising the risk of environmental exposition. Here, we investigated whether perinatal exposure to low doses of a GBH alters the female reproductive performance, and/or induced second-generation effects related to congenital anomalies or growth alterations. Pregnant rats (F0) received a GBH through food, in a dose of 2 mg (GBH-LD: GBH-low dose group) or 200 mg (GBH-HD: GBH-high dose group) of glyphosate/kg bw/day from gestational day (GD) 9 until weaning. Body weight gain and vaginal canal-opening of F1 females were recorded. Sexually mature F1 females were mated to evaluate their reproductive performance by assessing the pregnancy rate, and on GD19, the number of corpora lutea, the implantation sites (IS) and resorption sites. To analyze second-generation effects on F2 offspring, we analyzed the fetal morphology on GD19, and assessed the fetal length and weight, and the placental weight. GBH exposure neither altered the body weight gain of F1 females, nor vaginal opening onset. Although all GBH-exposed F1 rats became pregnant, a lower number of IS was detected. F2 offspring from both GBH groups showed delayed growth, evidenced by lower fetal weight and length, associated with a higher incidence of small for gestational age fetuses. In addition, higher placental weight and placental index were found in F2 offspring from GBH-HD dams. Surprisingly, structural congenital anomalies (conjoined fetuses and abnormally developed limbs) were detected in the F2 offspring from GBH-HD group. In conclusion, perinatal exposure to low doses of a GBH impaired female reproductive performance and induced fetal growth retardation and structural congenital anomalies in F2 offspring.

Lipidomic Changes in Skeletal Muscle in Patients after Biliopancreatic Diversion.

The mechanisms behind the fast improvements of insulin sensitivity and release of the diabetic metabolic state after bariatric surgery are still not completely understood. To further elucidate the effects on the individual cellular level, we applied mass spectrometry to investigate the changes in the lipidomic profile of skeletal muscle cells before and after biliopancreatic diversion in six patients. We found a decrease in lipid storage species, mainly triacylglycerides (e. g., TAG 52:2 from 19.84 to 13.26 mol%; p=0.028), and an increase in structural and signaling lipids, including phosphatidylcholines [PC 36:2 (18:1/18:1) from 0.12 to 0.65 mol%; p=0.046], phosphatidylinositols (PI 36:2 from 0.008 to 0.039 mol%; p=0.046), and cardiolipins (CL 72:8 from 0.16 to 1.22 mol%; p=0.043). The proportional increase in structural lipids was directly and the decrease in TAGs was inversely correlated to improved post-operative insulin sensitivity, measured by euglycemic hyperinsulinemic clamp. Thus, short-term recovery of insulin sensitivity after biliopancreatic diversion may, beside gut hormonal adaptation, mechanical factors, shifts in the gut microbiome, and changes in bile acid and phospholipid metabolism, additionally be attributed to a metabolic recovery of skeletal muscle cells, reflected by normalization of the cellular lipidomic profile. Further studies are needed to investigate whether improved insulin sensitivity of skeletal muscle might be directly associated with the degradation of ectopic triglycerides, thereby reducing the reservoir of lipotoxic intermediates, which might interfere with insulin signaling and hamper mitochondrial metabolism.

Functional control of polypeptide GalNAc-transferase 3 through an acetylation site in the C-terminal lectin domain.

O-GalNAc glycans are important structures in cellular homeostasis. Their biosynthesis is initiated by members of the polypeptide GalNAc-transferase (ppGalNAc-T) enzyme family. Mutations in ppGalNAc-T3 isoform cause diseases (congenital disorders of glycosylation) in humans. The K626 residue located in the C-terminal ß-trefoil fold of ppGalNAc-T3 was predicted to be a site with high likelihood of acetylation by CBP/p300 acetyltransferase. We used a site-directed mutagenesis approach to evaluate the role of this acetylation site in biological properties of the enzyme. Two K626 mutants of ppGalNAc-T3 (T3K626Q and T3K626A) had GalNAc-T activities lower than that of wild-type enzyme. Direct and competitive interaction assays revealed that GalNAc recognition by the lectin domain was altered in the mutants. The presence of GlcNAc glycosides affected the interaction of the three enzymes with mucin-derived peptides. In GalNAc-T activity assays, the presence of GlcNAc glycosides significantly inhibited activity of the mutant (T3K626Q) that mimicked acetylation. Our findings, taken together, reveal the crucial role of the K626 residue in the C-terminal ß-trefoil fold in biological properties of human ppGalNAc-T3. We propose that acetylated residues on ppGalNAc-T3 function as control points for enzyme activity, and high level of GlcNAc glycosides promote a synergistic regulatory mechanism, leading to a metabolically disordered state.

Extrinsic Functions of Lectin Domains in O-N-Acetylgalactosamine Glycan Biosynthesis.

Glycan biosynthesis occurs mainly in Golgi. Molecular organization and functional regulation of this process are not well understood. We evaluated the extrinsic effect of lectin domains (ß-trefoil fold) of polypeptide GalNAc-transferases (ppGalNAc-Ts) on catalytic activity of glycosyltransferases during O-GalNAc glycan biosynthesis. The presence of lectin domain T3lec or T4lec during ppGalNAc-T2 and ppGalNAc-T3 catalytic reaction had a clear inhibitory effect on GalNAc-T activity. Interaction of T3lec or T4lec with ppGalNAc-T2 catalytic domain was not mediated by carbohydrate. T3lec, but not T2lec and T4lec, had a clear activating effect on Drosophila melanogaster core 1 galactosyltransferase enzyme activity and a predominant inhibitory effect on in vivo human core 1 glycan biosynthesis. The regulatory role of the ß-trefoil fold of ppGalNAc-Ts in enzymatic activity of glycosyltransferases involved in the O-glycan biosynthesis pathway, described here for the first time, helps clarify the mechanism of biosynthesis of complex biopolymers (such as glycans) that is not template-driven.

Insulin Resistance, Microbiota, and Fat Distribution Changes by a New Model of Vertical Sleeve Gastrectomy in Obese Rats.

Metabolic surgery improves insulin resistance and type 2 diabetes possibly because of weight loss. We performed a novel sleeve gastrectomy in rats that resects ∼80% of the glandular portion, leaving the forestomach almost intact (glandular gastrectomy [GG]) and compared subsequent metabolic remodeling with a sham operation. GG did not affect body weight, at least after 10 weeks; improved hepatic and peripheral insulin sensitivity likely through increased Akt, glycogen synthase kinase 3, and AMPK phosphorylation; and reduced ectopic fat deposition and hepatic glycogen overaccumulation. Body adipose tissue was redistributed, with reduction of intraabdominal fat. We found a reduction of circulating ghrelin levels, increased GLP-1 plasma concentration, and remodeling of gut microbiome diversity characterized by a lower relative abundance of Ruminococcus and a higher relative abundance of Lactobacillus and Collinsella These data suggest that at least in rat, the glandular stomach plays a central role in the improvement of insulin resistance, even if obesity persists. GG provides a new model of the metabolically healthy obese phenotype.

Quantifying interface and bulk contributions to spin-orbit torque in magnetic bilayers.

Spin-orbit interaction-driven phenomena such as the spin Hall and Rashba effect in ferromagnetic/heavy metal bilayers enables efficient manipulation of the magnetization via electric current. However, the underlying mechanism for the spin-orbit interaction-driven phenomena remains unsettled. Here we develop a sensitive spin-orbit torque magnetometer based on the magneto-optic Kerr effect that measures the spin-orbit torque vectors for cobalt iron boron/platinum bilayers over a wide thickness range. We observe that the Slonczewski-like torque inversely scales with the ferromagnet thickness, and the field-like torque has a threshold effect that appears only when the ferromagnetic layer is thinner than 1 nm. Through a thickness-dependence study with an additional copper insertion layer at the interface, we conclude that the dominant mechanism for the spin-orbit interaction-driven phenomena in this system is the spin Hall effect. However, there is also a distinct interface contribution, which may be because of the Rashba effect.