Genetic Load of SARS-CoV-2 in Aerosols Collected in Operating Theaters.

After the outbreak of COVID-19, additional protocols have been established to prevent the transmission of the SARS-CoV-2 from the patient to the health personnel and vice versa in health care settings. However, in the case of emergency surgeries, it is not always possible to ensure that the patient is not infected with SARS-CoV-2, assuming a potential source of transmission of the virus to health personnel. This work aimed to evaluate the presence of the SARS-CoV-2 and quantify the viral load in indoor air samples collected inside operating rooms, where emergency and scheduled operations take place. Samples were collected for 3 weeks inside two operating rooms for 24 h at 38 L/min in quartz filters. RNA was extracted from the filters and analyzed using RT-qPCR targeting SARS-CoV-2 genes E, N1 and N2 regions. SARS-CoV-2 RNA was detected in 11.3% of aerosol samples collected in operating rooms, despite with low concentrations (not detected at 13.5 cg/m3 and 10.5 cg/m3 in the scheduled and emergency operating rooms, respectively). Potential sources of airborne SARS-CoV-2 could be aerosolization of the virus during aerosol-generating procedures and in open surgery from patients that might have been recently infected with the virus, despite presenting a negative COVID-19 test. Another source could be related to health care workers unknowingly infected with the virus and exhaling SARS-CoV-2 virions into the air. These results highlight the importance of reinforcing preventive measures against COVID-19 in operating rooms, such as the correct use of protective equipment, screening programs for health care workers, and information campaigns. IMPORTANCE Operating rooms are critical environments in which asepsis must be ensured. The COVID-19 pandemic entailed the implementation of additional preventative measures in health care settings, including operating theaters. Although one of the measures is to operate only COVID-19 free patients, this measure cannot be always implemented, especially in emergency interventions. Therefore, a surveillance campaign was conducted during 3 weeks in two operating rooms to assess the level of SARS-CoV-2 genetic material detected in operating theaters with the aim to assess the risk of COVID-19 transmission during operating procedures. SARS-CoV-2 genetic material was detected in 11% of aerosol samples collected in operating rooms, despite with low concentrations. Plausible SARS-CoV-2 sources have been discussed, including patients and health care personnel infected with the virus. These results highlight the importance of reinforcing preventive measures against COVID-19 in operating rooms, such as the correct use of protective equipment, screening programs for health care workers and information campaigns.

Motherhood-induced gene expression in the mouse medial amygdala: Changes induced by pregnancy and lactation but not by pup stimuli.

During lactation, adult female mice display aggressive responses toward male intruders, triggered by male-derived chemosensory signals. This aggressive behavior is not shown by pup-sensitized virgin females sharing pup care with dams. The genetic mechanisms underlying the switch from attraction to aggression are unknown. In this work, we investigate the differential gene expression in lactating females expressing maternal aggression compared to pup-sensitized virgin females in the medial amygdala (Me), a key neural structure integrating chemosensory and hormonal information. The results showed 197 genes upregulated in dams, including genes encoding hormones such as prolactin, growth hormone, or follicle-stimulating hormone, neuropeptides such as galanin, oxytocin, and pro-opiomelanocortin, and genes related to catecholaminergic and cholinergic neurotransmission. In contrast, 99 genes were downregulated in dams, among which we find those encoding for inhibins and transcription factors of the Fos and early growth response families. The gene set analysis revealed numerous Gene Ontology functional groups with higher expression in dams than in pup-sensitized virgin females, including those related with the regulation of the Jak/Stat cascade. Of note, a number of olfactory and vomeronasal receptor genes was expressed in the Me, although without differences between dams and virgins. For prolactin and growth hormone, a qPCR experiment comparing dams, pup-sensitized, and pup-naïve virgin females showed that dams expressed higher levels of both hormones than pup-naïve virgins, with pup-sensitized virgins showing intermediate levels. Altogether, the results show important gene expression changes in the Me, which may underlie some of the behavioral responses characterizing maternal behavior.

Understanding the pharmacokinetics of synthetic cathinones: Evaluation of the blood-brain barrier permeability of 13 related compounds in rats.

Synthetic cathinones are the second most commonly seized new psychoactive substance family in Europe. These compounds have been related to several intoxication cases, including fatalities. Although the pharmacological effects, metabolism, and pharmacokinetics of cathinones have been studied, there is little information about the permeability of these compounds through the blood-brain barrier (BBB). This is an important parameter to understand the behavior and potency of cathinones. In this work, 13 selected cathinones have been analyzed in telencephalon tissue from Sprague-Dawley rats intraperitoneally dosed at 3 mg/kg. Our results revealed a direct relationship between compound polarity and BBB permeability, with higher permeability for the more polar cathinones. The chemical moieties present in the cathinone had an important impact on the BBB permeability, with lengthening of the α-alkyl chain or functionalization of the aromatic ring with alkyl moieties resulting in lower concentration in telencephalon tissue. Our data suggest that transport of cathinones is a carrier-mediated process, similar to cocaine transport across the BBB.

Lack of GDAP1 induces neuronal calcium and mitochondrial defects in a knockout mouse model of charcot-marie-tooth neuropathy.

Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca2+, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria-endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.

Rapid degeneration of iPSC-derived motor neurons lacking Gdap1 engages a mitochondrial-sustained innate immune response.

Charcot-Marie-Tooth disease is a chronic hereditary motor and sensory polyneuropathy targeting Schwann cells and/or motor neurons. Its multifactorial and polygenic origin portrays a complex clinical phenotype of the disease with a wide range of genetic inheritance patterns. The disease-associated gene GDAP1 encodes for a mitochondrial outer membrane protein. Mouse and insect models with mutations in Gdap1 have reproduced several traits of the human disease. However, the precise function in the cell types affected by the disease remains unknown. Here, we use induced-pluripotent stem cells derived from a Gdap1 knockout mouse model to better understand the molecular and cellular phenotypes of the disease caused by the loss-of-function of this gene. Gdap1-null motor neurons display a fragile cell phenotype prone to early degeneration showing (1) altered mitochondrial morphology, with an increase in the fragmentation of these organelles, (2) activation of autophagy and mitophagy, (3) abnormal metabolism, characterized by a downregulation of Hexokinase 2 and ATP5b proteins, (4) increased reactive oxygen species and elevated mitochondrial membrane potential, and (5) increased innate immune response and p38 MAP kinase activation. Our data reveals the existence of an underlying Redox-inflammatory axis fueled by altered mitochondrial metabolism in the absence of Gdap1. As this biochemical axis encompasses a wide variety of druggable targets, our results may have implications for developing therapies using combinatorial pharmacological approaches and improving therefore human welfare. A Redox-immune axis underlying motor neuron degeneration caused by the absence of Gdap1. Our results show that Gdap1-/- motor neurons have a fragile cellular phenotype that is prone to degeneration. Gdap1-/- iPSCs differentiated into motor neurons showed an altered metabolic state: decreased glycolysis and increased OXPHOS. These alterations may lead to hyperpolarization of mitochondria and increased ROS levels. Excessive amounts of ROS might be the cause of increased mitophagy, p38 activation and inflammation as a cellular response to oxidative stress. The p38 MAPK pathway and the immune response may, in turn, have feedback mechanisms, leading to the induction of apoptosis and senescence, respectively. CAC, citric acid cycle; ETC, electronic transport chain; Glc, glucose; Lac, lactate; Pyr, pyruvate.

Becoming a mother shifts the activity of the social and motivation brain networks in mice.

During pregnancy hormones increase motivated pup-directed behaviors. We here analyze hormone-induced changes in brain activity, by comparing cFos-immunoreactivity in the sociosexual (SBN) and motivation brain networks (including medial preoptic area, MPO) of virgin versus late-pregnant pup-naïve female mice exposed to pups or buttons (control). Pups activate more the SBN than buttons in both late-pregnant and virgin females. By contrast, pregnancy increases pup-elicited activity in the motivation circuitry (e.g. accumbens core) but reduces button-induced activity and, consequently, button investigation. Principal components analysis supports the identity of the social and motivation brain circuits, placing the periaqueductal gray between both systems. Linear discriminant analysis of cFos-immunoreactivity in the socio-motivational brain network predicts the kind of female and stimulus better than the activity of the MPO alone; this suggests that the neuroendocrinological basis of social (e.g. maternal) behaviors conforms to a neural network model, rather than to distinct hierarchical linear pathways for different behaviors.

In-depth comparison of the metabolic and pharmacokinetic behaviour of the structurally related synthetic cannabinoids AMB-FUBINACA and AMB-CHMICA in rats.

Synthetic cannabinoids receptor agonists (SCRAs) are often almost completely metabolised, and hence their pharmacokinetics should be carefully evaluated for determining the most adequate biomarker in toxicological analysis. Two structurally related SCRAs, AMB-FUBINACA and AMB-CHMICA, were selected to evaluate their in vivo metabolism and pharmacokinetics using male Sprague-Dawley rats. Brain, liver, kidney, blood (serum) and urine samples were collected at different times to assess the differences in metabolism, metabolic reactions, tissue distribution and excretion. Both compounds experimented O-demethyl reaction, which occurred more rapidly for AMB-FUBINACA. The parent compounds and O-demethyl metabolites were highly bioaccumulated in liver, and were still detected in this tissue 48 h after injection. The different indazole/indole N-functionalisation produced diverse metabolic reactions in this moiety and thus, different urinary metabolites were formed. Out of the two compounds, AMB-FUBINACA seemed to easily cross the blood-brain barrier, presenting higher brain/serum concentrations ratio than AMB-CHMICA.

SARS-CoV-2 Prevalence in Laparoscopic Surgery Filters. Analysis in Patients with Negative Oropharyngeal RT-qPCR in a Pandemic Context: A Cross-Sectional Study.

OBJECTIVE: Surgical societies of different specialties have lately demonstrated a growing concern regarding the potential risk of SARS-CoV-2 transmission during surgery, mainly via aerosols carrying SARS-CoV-2 particles during laparoscopy smoke evacuation. Since there is not sufficient scientific evidence to rule out this hypothesis, our study aimed to evaluate the prevalence of the appearance of SARS-CoV-2 genetic material in the in-filter membrane of the smoke filter systems, used in laparoscopic surgery, in a tertiary referral hospital during the peak phases of the pandemic. METHODS: During the highest incidence of the pandemic outbreak, 180 laparoscopic smoke evacuation systems were collected from laparoscopies performed between April 2020 and May 2021 in University General Hospital of Castellón. As part of the safety protocol established as a result of the pandemic, an oropharyngeal reverse-transcription polymerase chain reaction (RT-PCR) was performed before surgery. We performed RT-qPCR tests for the detection and quantification of SARS-CoV-2 genetic material in the in-filter membranes extracted from the smoke evacuation systems. RESULTS: We found two RT-qPCR positive in-filters from a sample of 128 patients with SARS-CoV-2-negative results in their oropharyngeal RT-qPCR, i.e., 1.6% (95% CI: 0.5-5.5%). From this estimation, the predictive posterior probabilities of finding n cases of negative oropharyngeal COVID-19 patients with positive filters increases with the increasing number of surgeries performed. CONCLUSIONS: This cross-sectional study provides evidence suggesting that airborne transmission of SARS-CoV-2 particles from smoke evacuation of aerosols carrying viral particles during laparoscopy should not be ruled out.

Pregnancy Changes the Response of the Vomeronasal and Olfactory Systems to Pups in Mice.

Motherhood entails changes in behavior with increased motivation for pups, induced in part by pregnancy hormones acting upon the brain. This work explores whether this alters sensory processing of pup-derived chemosignals. To do so, we analyse the expression of immediate early genes (IEGs) in the vomeronasal organ (VNO; Egr1) and centers of the olfactory and vomeronasal brain pathways (cFos) in virgin and late-pregnant females exposed to pups, as compared to buttons (socially neutral control). In pup-exposed females, we quantified diverse behaviors including pup retrieval, sniffing, pup-directed attack, nest building and time in nest or on nest, as well as time off nest. Pups induce Egr1 expression in the VNO of females, irrespective of their physiological condition, thus suggesting the existence of VNO-detected pup chemosignals. A similar situation is found in the accessory olfactory bulb (AOB) and posteromedial part of the medial bed nucleus of the stria terminalis (BSTMPM). By contrast, in the medial amygdala and posteromedial cortical amygdala (PMCo), responses to pups-vs-buttons are different in virgin and late-pregnant females, thus suggesting altered sensory processing during late pregnancy. The olfactory system also shows changes in sensory processing with pregnancy. In the main olfactory bulbs, as well as the anterior and posterior piriform cortex, buttons activate cFos expression in virgins more than in pregnant females. By contrast, in the anterior and especially posterior piriform cortex, pregnant females show more activation by pups than buttons. Correlation between IEGs expression and behavior suggests the existence of two vomeronasal subsystems: one associated to pup care (with PMCo as its main center) and another related to pup-directed aggression observed in some pregnant females (with the BSTMPM as the main nucleus). Our data also suggest a coactivation of the olfactory and vomeronasal systems during interaction with pups in pregnant females.

Dysfunctional mitochondrial fission impairs cell reprogramming.

We have recently shown that mitochondrial fission is induced early in reprogramming in a Drp1-dependent manner; however, the identity of the factors controlling Drp1 recruitment to mitochondria was unexplored. To investigate this, we used a panel of RNAi targeting factors involved in the regulation of mitochondrial dynamics and we observed that MiD51, Gdap1 and, to a lesser extent, Mff were found to play key roles in this process. Cells derived from Gdap1-null mice were used to further explore the role of this factor in cell reprogramming. Microarray data revealed a prominent down-regulation of cell cycle pathways in Gdap1-null cells early in reprogramming and cell cycle profiling uncovered a G2/M growth arrest in Gdap1-null cells undergoing reprogramming. High-Content analysis showed that this growth arrest was DNA damage-independent. We propose that lack of efficient mitochondrial fission impairs cell reprogramming by interfering with cell cycle progression in a DNA damage-independent manner.

Nonsense-mediated mRNA decay controls the changes in yeast ribosomal protein pre-mRNAs levels upon osmotic stress.

The expression of ribosomal protein (RP) genes requires a substantial part of cellular transcription, processing and translation resources. Thus, the RP expression must be tightly regulated in response to conditions that compromise cell survival. In Saccharomyces cerevisiae cells, regulation of the RP gene expression at the transcriptional, mature mRNA stability and translational levels during the response to osmotic stress has been reported. Reprogramming global protein synthesis upon osmotic shock includes the movement of ribosomes from RP transcripts to stress-induced mRNAs. Using tiling arrays, we show that osmotic stress yields a drop in the levels of RP pre-mRNAs in S. cerevisiae cells. An analysis of the tiling array data, together with transcription rates data, shows a poor correlation, indicating that the drop in the RP pre-mRNA levels is not merely a result of the lowered RP transcription rates. A kinetic study using quantitative RT-PCR confirmed the decrease in the levels of several RP-unspliced transcripts during the first 15 minutes of osmotic stress, which seems independent of MAP kinase Hog1. Moreover, we found that the mutations in the components of the nonsense-mediated mRNA decay (NMD), Upf1, Upf2, Upf3 or in exonuclease Xrn1, eliminate the osmotic stress-induced drop in RP pre-mRNAs. Altogether, our results indicate that the degradation of yeast RP unspliced transcripts by NMD increases during osmotic stress, and suggest that this might be another mechanism to control RP synthesis during the stress response.