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The skeletal dysplasia spondyloepiphyseal dysplasia tarda (SEDT) is caused by mutations in the TRAPPC2 gene, which encodes Sedlin, a component of the trafficking protein particle (TRAPP) complex that we have shown previously to be required for the export of type II collagen (Col2) from the endoplasmic reticulum. No vertebrate model for SEDT has been generated thus far. To address this gap, we generated a Sedlin knockout animal by mutating the orthologous TRAPPC2 gene (olSedl) of Oryzias latipes (medaka) fish. OlSedl deficiency leads to embryonic defects, short size, diminished skeletal ossification and altered Col2 production and secretion, resembling human defects observed in SEDT patients. Moreover, SEDT knock-out animals display photoreceptor degeneration and gut morphogenesis defects, suggesting a key role for Sedlin in the development of these organs. Thus, by studying Sedlin function in vivo, we provide evidence for a mechanistic link between TRAPPC2-mediated membrane trafficking, Col2 export, and developmental disorders., (© 2023 The Authors. Traffic published by John Wiley & Sons Ltd.)

SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle to enable RNA replication 1 . The SARS-CoV-2 replication organelle is composed of double-membrane vesicles (DMVs) that are tethered to the endoplasmic reticulum (ER) by thin membrane connectors 2 , but the viral proteins and the host factors involved remain unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 replication organelle. NSP3 and NSP4 generate the DMVs, whereas NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6(ΔSGF) mutant, which arose independently in the Alpha, Beta, Gamma, Eta, Iota and Lambda variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: first, to act as a filter in communication between the replication organelle and the ER, by allowing lipid flow but restricting the access of ER luminal proteins to the DMVs; second, to position and organize DMV clusters; and third, to mediate contact with lipid droplets (LDs) through the LD-tethering complex DFCP1-RAB18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective means of refurbishing them with LD-derived lipids. Notably, both properly formed NSP6 connectors and LDs are required for the replication of SARS-CoV-2. Our findings provide insight into the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, and have the potential to fuel the search for broad antiviral agents., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Endogenous reactive oxygen species (ROS) are by-products of the aerobic metabolism of cells and have an important signalling role as secondary messengers in various physiological processes, including cell growth and development. However, the excessive production of ROS, as well as the exposure to exogenous ROS, can cause protein oxidation, lipid peroxidation and DNA damages leading to cell injuries. ROS accumulation has been associated to the development of health disorders such as neurodegenerative and cardiovascular diseases, inflammatory bowel disease and cancer. We report that spores of strain SF185, a human isolate of Bacillus megaterium, have antioxidant activity on Caco-2 cells exposed to hydrogen peroxide and on a murine model of dextran sodium sulfate-induced oxidative stress. In both model systems spores exert a protective state due to their scavenging action: on cells, spores reduce the amount of intracellular ROS, while in vivo the pre-treatment with spores protects mice from the chemically-induced damages. Overall, our results suggest that treatment with SF185 spores prevents or reduces the damages caused by oxidative stress. The human origin of SF185, its strong antioxidant activity, and its protective effects led to propose the spore of this strain as a new probiotic for gut health.

The survival of cells exposed to adverse environmental conditions entails various alterations in cellular function including major changes in the transcriptome as well as a radical reprogramming of protein translation. While in mammals this process has been extensively studied, stress responses in non-mammalian vertebrates remain poorly understood. One of the key cellular responses to many different types of stressors is the transient generation of structures called stress granules (SGs). These represent cytoplasmic foci where untranslated mRNAs are sorted or processed for re-initiation, degradation, or packaging into mRNPs. Here, using the evolutionarily conserved Y-box binding protein 1 (YB-1) and G3BP1 as markers, we have studied the formation of stress granules in zebrafish (D. rerio) in response to different environmental stressors. We show that following heat shock, zebrafish cells, like mammalian cells, form stress granules which contain both YB-1 and G3BP1 proteins. Moreover, zfYB-1 knockdown compromises cell viability, as well as recruitment of G3BP1 into SGs, under heat shock conditions highlighting the essential role played by YB-1 in SG assembly and cell survival. However, zebrafish PAC2 cells do not assemble YB-1-positive stress granules upon oxidative stress induced by arsenite, copper or hydrogen peroxide treatment. This contrasts with the situation in human cells where SG formation is robustly induced by exposure to oxidative stressors. Thus, our findings point to fundamental differences in the mechanisms whereby mammalian and zebrafish cells respond to oxidative stress.

Up until the first half of the 20th century, silver found significant employment in medical applications, particularly in the healing of open wounds, thanks to its antibacterial and antifungal properties. Wound repair is a complex and dynamic biological process regulated by several pathways that cooperate to restore tissue integrity and homeostasis. To facilitate healing, injuries need to be promptly treated. Recently, the interest in alternatives to antibiotics has been raised given the widespread phenomenon of antibiotic resistance. Among these alternatives, the use of silver appears to be a valid option, so a resurgence in its use has been recently observed. In particular, in contrast to ionic silver, colloidal silver, a suspension of metallic silver particles, shows antibacterial activity displaying less or no toxicity. However, the human health risks associated with exposure to silver nanoparticles (NP) appear to be conflicted, and some studies have suggested that it could be toxic in different cellular contexts. These potentially harmful effects of silver NP depend on various parameters including NP size, which commonly range from 1 to 100 nm. In this study, we analyzed the effect of a colloidal silver preparation composed of very small and homogeneous nanoparticles of 0.62 nm size, smaller than those previously tested. We found no adverse effect on the cell proliferation of HaCaT cells, even at high NP concentration. Time-lapse microscopy and indirect immunofluorescence experiments demonstrated that this preparation of colloidal silver strongly increased cell migration, re-modeled the cytoskeleton, and caused recruitment of E-cadherin at cell-cell junctions of human cultured keratinocytes.

How the environment shapes the function and evolution of DNA repair systems is poorly understood. In a comparative study using zebrafish and the Somalian blind cavefish, Phreatichthys andruzzii, we reveal that during evolution for millions of years in continuous darkness, photoreactivation DNA repair function has been lost in P. andruzzii. We demonstrate that this loss results in part from loss-of-function mutations in pivotal DNA-repair genes. Specifically, C-terminal truncations in P. andruzzii DASH and 6-4 photolyase render these proteins predominantly cytoplasmic, with consequent loss in their functionality. In addition, we reveal a general absence of light-, UV-, and ROS-induced expression of P. andruzzii DNA-repair genes. This results from a loss of function of the D-box enhancer element, which coordinates and enhances DNA repair in response to sunlight. Our results point to P. andruzzii being the only species described, apart from placental mammals, that lacks the highly evolutionary conserved photoreactivation function. We predict that in the DNA repair systems of P. andruzzii, we may be witnessing the first stages in a process that previously occurred in the ancestors of placental mammals during the Mesozoic era., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The prototype cold-shock Y-box binding protein 1 (YB-1) is a multifunctional protein that regulates a variety of fundamental biological processes including cell proliferation and migration, DNA damage, matrix protein synthesis and chemotaxis. The plethora of functions assigned to YB-1 is strictly dependent on its subcellular localization. In resting cells, YB-1 localizes to cytoplasm where it is a component of messenger ribonucleoprotein particles. Under stress conditions, YB-1 contributes to the formation of stress granules (SGs), cytoplasmic foci where untranslated messenger RNAs (mRNAs) are sorted or processed for reinitiation, degradation, or packaging into ribonucleoprotein particles (mRNPs). Following DNA damage, YB-1 translocates to the nucleus and participates in DNA repair thereby enhancing cell survival. Recent data show that YB-1 can also be secreted and YB-1-derived polypeptides are found in plasma of patients with sepsis and malignancies. Here we show that in response to oxidative insults, YB-1 assembly in SGs is associated with an enhancement of YB-1 protein secretion. An enriched fraction of extracellular YB-1 (exYB-1) significantly inhibited proliferation of receiving cells and such inhibition was associated to a G2/M cell cycle arrest, induction of p21WAF and reduction of Np63 protein level. All together, these data show that acute oxidative stress causes sustained release of YB-1 as a paracrine/autocrine signal that stimulate cell cycle arrest., Competing Interests: The authors declare no conflicts of interest.

Y-box binding protein 1 (YBX-1 or YB-1) is an oncoprotein that promotes replicative immortality, tumor cell invasion and metastasis. The increase in the abundance of YB-1 in the cell or YB-1 translocation from the cytoplasm to the nucleus is characteristic of malignant cell growth. We have previously reported that ΔNp63α, a transcription factor that is known to play a pivotal role in keratinocyte proliferation and differentiation, promotes YB-1 nuclear accumulation. Here, we show that YB-1 is highly expressed in proliferating keratinocytes and is down-regulated during keratinocyte differentiation. ΔNp63α reduces YB-1 protein turnover and leads to accumulation of ubiquitin-conjugated YB-1 into the nucleus. Reduction of YB-1 protein level, following treatment with a DNA-damaging agent, is inhibited by ΔNp63α suggesting that YB-1 and ΔNp63α interplay can support keratinocyte proliferation and protect cells from apoptosis under genotoxic stress., (© 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Previous retrospective analyses have suggested a very positive correlation in toxic doses of antineoplastic agents between mice and humans. Additional toxicological information has now been accumulated and reveals a noticeable variability in the existing data base. Nevertheless, it is likely that mouse toxicological studies will become a principal determinant for estimating initial doses to be used in humans. Recognition of the factors responsible for differences in determinations of toxic dose levels in mice will enhance the proper utilization of this approach.

The large body of literature and techniques generated by mammalian toxicity studies provides a conceptual and technical framework within which the absorption, fate, and disposition of xenobiotics in aquatic organisms can be studied. This review emphasizes the similarities and differences between mammalian and aquatic systems, e.g., lung vs. gill as site of absorption and toxicity. These must be taken into consideration when designing aquatic toxicity studies. Studies of phenol red in dogfish shark as an example show physiologic-based pharmacokinetic modeling to be a useful tool for investigating and eventually predicting species differences in xenobiotic disposition and drug differences within the same species. This discussion demonstrates that both laboratory and modeling procedures are now available to carry out sophisticated studies of xenobiotic fate and disposition in fish. Such studies are needed to pinpoint sites and mechanisms of pollutant toxicity in aquatic organisms.

cis-Diamminedichloroplatinum(II) is an antineoplastic drug that is undergoing a renewed clinical interest as a drug for use in combination regimens. In order to increase the understanding of the pharmacokinetics of this drug, the plasma clearance and organ distribution of platinum were followed in female beagle dogs treated with a single i.v. dose of cis-diamminedichloroplatinum(II). Plasma levels of platinum were determined by flameless atomic absorption spectrometry and showed a distinctly biphasic clearance pattern with a rapid-phase half-time of considerably less than 1 hr and a slow-phase half-time of nearly 5 days. During the first 4 hr after treatment, plasma levels fell by 90% while 60 to 70% of the applied dose was recovered in the urine. Sixteen tissues plus plasma, bile, and urine were routinely analyzed for platinum content. An easily measurable plasma concentration of platinum was still detectable 12 days after treatment, with no significant change in plasma concentration between Days 4 and 12. Initial concentrations of platinum were highest in organs of excretion, gonads, spleen, and adrenals but remained significantly elevated only in kidney, liver, ovary, and uterus, where a tissue: plasma ratio of 3 to 4 was maintained for as long as 6 days posttreatment. The apparent in vitro binding of platinum to dog plasma and to bovine serum albumin was studied by ultrafiltration and increased progressively during 48 hr of incubation at 37 degrees.

The possibility that the mass spectrum and pharmacological activity of a compound may be directly related has been explored with the help of various computer-based pattern-recognition techniques. The relationship appears to hold at least for tranquilizers and sedatives, and compounds with one or the other of these two pharmacological activities can thus be classified from their mass spectra with a high degree of accuracy.

1. Changes in certain kinetic properties (V(max.) and apparent K(m)) of hepatic microsomal mixed-function oxidases have been studied as a function of postnatal development and maturation in male rats. 2. Microsomal cytochrome P-450 content changed only slightly between 1 and 12 weeks of age. 3. Aniline hydroxylase activity (V(max.)) increased abruptly between 1 and 2 weeks of age to greater than adult activities and then returned to a plateau value between 4(1/2) and 12 weeks of age. Ethylmorphine demethylase activity remained low and relatively constant between 1 and 3 weeks of age and then increased markedly ( approximately 100%) between 3 and 4(1/2) weeks. 4. The apparent Michaelis constant (K(m)) for aniline hydroxylation increased almost linearly with time between 1 and 6 weeks of age and tended to reach a plateau value thereafter. The apparent K(m) for ethylmorphine demethylation increased between 1 and 3 weeks of age and then decreased abruptly to a constant value between 6 and 12 weeks. 5. The data indicate that developmental changes in the activity of these microsomal oxidases do not correlate temporally with each other or with changes in microsomal cytochrome P-450 content. 6. The most dramatic changes in enzyme activity were associated with early development (1-3 weeks) and weaning (3-4 weeks). 7. Changes in weight of seminal vesicle, a criterion of sexual maturation in male rats, were most prominent between 6 and 8 weeks of age and thus appeared to be separated in time from the prominent changes in enzyme activity.