High-throughput sequencing as a tool for monitoring prokaryote communities in a wastewater treatment plant.

In this study, the DNA metabarcoding technique was used to explore the prokaryote diversity and community structure in wastewater collected in spring and winter 2020-2021 as well as the efficiency of the treatment in a wastewater treatment plant (WWTP) in Ría de Vigo (NW Spain). The samplings included raw wastewater from the inlet stream (M1), the discharge water after the disinfection treatment (M3) and mussels used as bioindicators of possible contamination of the marine environment. Significant differences were discovered in the microbiome of each type of sample (M1, M3 and mussels), with 92 %, 45 % and 44 % of exclusive OTUs found in mussel, M3 and M1 samples respectively. Seasonal differences were also detected in wastewater samples, with which abiotic parameters (temperature, pH) could be strongly involved. Bacteria present in raw wastewater (M1) were associated with the human gut microbiome, and therefore, potential pathogens that could be circulating in the population in specific periods were detected (e.g., Arcobacter sp. and Clostridium sp.). A considerable decrease in putative pathogenic organisms from the M1 to M3 wastewater fractions and the scarce presence in mussels (<0.5 % total reads) confirmed the effectiveness of pathogen removal in the wastewater treatment plant. Our results showed the potential of the DNA metabarcoding technique for monitoring studies and confirmed its application in wastewater-based epidemiology (WBE) and environmental contamination studies. Although this technique cannot determine if the infective pathogens are present, it can characterize the microbial communities and the putative pathogens that are circulating through the population (microbiome of M1) and also confirm the efficacy of depuration treatment, which can directly affect the aquaculture sector and even human and veterinary health.

Transport of High-Risk Infectious Substances: Packaging for the Transport of Category A Infectious Specimens in Spain.

Infectious specimens and materials with pathogens included in Category A of the European Agreement concerning the International Carriage of Dangerous Goods by Road (ADR) must be transported following Packing Instruction P620. A triple packaging system must include leakproof receptacles and impact-resistant packaging to preserve the integrity of the samples and prevent the release of their content in any event during transport. ADR Packing Instruction P620 indicates that the primary receptacle or secondary packaging must withstand, without leakage, an internal pressure not less than 95 kPa at temperatures ranging from -40 °C to +55 °C. This study analyzes various packaging systems available in the Spanish market for the transportation of infectious samples to determine if they comply with the overpressure test, the most difficult to meet according to Packing Instruction P620. Five packaging systems were selected in this study. None of the secondary packaging tested showed adequate characteristics to withstand the pressure leakproof test. In this case, a primary receptacle (containing the sample directly) capable of withstanding an internal pressure of 95 kPa without leakage must be used (for example: test tubes with screw caps). However, manufacturer or distributor specifications are not always clear or readily available in this regard. Health, laboratory, and carrier personnel should be aware of the ADR regulation and packaging characteristics for safe and secure handling and transportation of high-risk Category A infectious materials.

Wastewater and marine bioindicators surveillance to anticipate COVID-19 prevalence and to explore SARS-CoV-2 diversity by next generation sequencing: One-year study.

This study presents the results of SARS-CoV-2 surveillance in sewage water of 11 municipalities and marine bioindicators in Galicia (NW of Spain) from May 2020 to May 2021. An integrated pipeline was developed including sampling, pre-treatment and biomarker quantification, RNA detection, SARS-CoV-2 sequencing, mechanistic mathematical modeling and forecasting. The viral load in the inlet stream to the wastewater treatment plants (WWTP) was used to detect new outbreaks of COVID-19, and the data of viral load in the wastewater in combination with data provided by the health system was used to predict the evolution of the pandemic in the municipalities under study within a time horizon of 7 days. Moreover, the study shows that the viral load was eliminated from the treated sewage water in the WWTP, mainly in the biological reactors and the disinfection system. As a result, we detected a minor impact of the virus in the marine environment through the analysis of seawater, marine sediments and, wild and aquacultured mussels in the final discharge point of the WWTP.

Enzymatic Functionalization of Wood as an Antifouling Strategy against the Marine Bacterium Cobetia marina.

The protection of wood in marine environments is a major challenge due to the high sensitivity of wood to both water and marine microorganisms. Besides, the environmental regulations are pushing the industry to develop novel effective and environmentally friendly treatments to protect wood in marine environments. The present study focused on the development of a new green methodology based on the laccase-assisted grafting of lauryl gallate (LG) onto wood to improve its marine antifouling properties. Initially, the enzymatic treatment conditions (laccase dose, time of reaction, LG concentration) and the effect of the wood specie (beech, pine, and eucalyptus) were assessed by water contact angle (WCA) measurements. The surface properties of the enzymatically modified wood veneers were assessed by X-ray photoelectron spectroscopy (XPS), Fourier transform-infrared spectroscopy (FTIR). Antifouling properties of the functionalized wood veneers against marine bacterium Cobetia marina were studied by scanning electron microscopy (SEM) and protein measurements. XPS and FTIR analysis suggested the stable grafting of LG onto the surface of wood veneers after laccase-assisted treatment. WCA measurements showed that the hydrophobicity of the wood veneers significantly increased after the enzymatic treatment. Protein measurements and SEM pictures showed that enzymatically-hydrophobized wood veneers modified the pattern of bacterial attachment and remarkably reduced the bacterium colonization. Thus, the results observed in the present study confirmed the potential efficiency of laccase-assisted treatments to improve the marine antifouling properties of wood.

Effects of Ectomycorrhizal Fungi and Heavy Metals (Pb, Zn, and Cd) on Growth and Mineral Nutrition of Pinus halepensis Seedlings in North Africa.

The pollution of soils by heavy metals resulting from mining activities is one of the major environmental problems in North Africa. Mycorrhizoremediation using mycorrhizal fungi and adapted plant species is emerging as one of the most innovative methods to remediate heavy metal pollution. This study aims to assess the growth and the nutritional status of ectomycorrhizal Pinus halepensis seedlings subjected to high concentrations of Pb, Zn, and Cd for possible integration in the restoration of heavy metals contaminated sites. Ectomycorrhizal and non-ectomycorrhizal P. halepensis seedlings were grown in uncontaminated (control) and contaminated soils for 12 months. Growth, mineral nutrition, and heavy metal content were assessed. Results showed that ectomycorrhizae significantly improved shoot and roots dry masses of P. halepensis seedlings, as well as nitrogen shoot content. The absorption of Pb, Zn, and Cd was much higher in the roots than in the shoots, and significantly more pronounced in ectomycorrhizal seedlings-especially for Zn and Cd. The presence of ectomycorrhizae significantly reduced the translocation factor of Zn and Cd and bioaccumulation factor of Pb and Cd, which enhanced the phytostabilizing potential of P. halepensis seedlings. These results support the use of ectomycorrhizal P. halepensis in the remediation of heavy metal contaminated sites.

Analysis and Optimization of Mn Removal from Contaminated Solid Matrixes by Electrokinetic Remediation.

Electrokinetic remediation is a useful technique for the removal of ionic contaminants in soils, sediments, sludges, and other solid porous matrixes. The efficiency of metal removal and the electricity consumption in the electrokinetic treatment of soils largely depend on electric and physicochemical conditions. This study analyzes the electrokinetic treatment of Mn contaminated kaolinite clay specimen and the influence of voltage, current intensity, moisture content, pH, and facilitating agents on metal removal and energy consumption. The objective of this study is to identify the influence of the typical variables used in electrokinetic remediation. The results showed that the operation at constant voltage or constant current intensity were equivalent in terms of metal removal and energy consumption, as long as the electric field intensity was kept low to minimize the consumption in parallel electrochemical reactions, especially the electrolysis of water. The moisture content had a significant influence on the Mn removal. Moisture content higher that 50 percent resulted in very effective Mn removal as compared with kaolinite specimens with lower moisture. The control of pH in the electrolyte solutions and the addition of facilitating agents (organic acids) enhanced the removal of Mn but increased the electric energy cost. Overall, the best conditions for Mn removal involved low to moderate electric potential difference (10 to 30 V), the use of citric acid as the facilitating agent, and the pH control in the cathode at a slightly acid pH. The electrokinetic treatment of a sludge from a water treatment plant contaminated with Mn was effective when pH control on the cathode was used. Mn and various metals (66% of Mn, 30% of Cu, 56% of Zn, 21% Sr, and 21% of Fe) were removed with moderate electricity and acid consumption.

Benefits of phytoremediation amended with DC electric field. Application to soils contaminated with heavy metals.

The objective of this study was to determine the influence of the DC electric current in the physicochemical properties of soil. The electric current may induce changes in pH and electric conductivity that will be reflected in the distribution of the electric potential in the soil specimen. This information will be used for the development of a phytoremediation technology amended with electric current. The results showed that low or moderate voltage gradients (0.67 V/cm) induced small changes in physicochemical properties of soil that do not compromise plant survival. The selected voltage gradient was used in electro-phytoremediation tests in soil contaminated with heavy metals (Cd, Co, Cr, Cu, Pb and Zn). Two plants species adapted to the soil and climate conditions were selected (Brassica rapa L. subsp. rapa and Lolium perenne L.). The electric field enhanced the plant growing, mainly in L. perenne, and increased the phytoremediation of the 6 metals. Mixed cultures of the two plant species showed interesting results for large scale applications.

Phytoremediation of mixed contaminated soil enhanced with electric current.

Brassica rapa is a plant species that can germinate and grow in mixed contaminated soil with PAH and metals (Cr, Pb and Cd). This plant was selected among 14 plant species for electro-phytoremediation tests because its fast germination and growth in contaminated soil. The influence of type of the electric field (AC, DC) and mode of application (continuous, periodic and polarity inversion) was studied in the electro-phytoremediation tests. The application of 1 ACV/cm potential gradient around B. rapa resulted in the effective elimination of anthracene and phenanthrene, but only minor metal removal. The results of this work suggest that alternating current (AC) may be the most suitable electric field for large scale applications. The spatial configuration of electrodes affects the distribution of the electric field in the soil. Various spatial distribution of electrodes have been tested and it has been identified that parallel anodes and cathodes on the soil surface are the most appropriate configuration for field scale applications. Other configurations can be used to concentrate the contaminant around the growing pant or to transport the contaminants from deep soil layers to the rhizosphere.

PLK4 is a microtubule-associated protein that self-assembles promoting de novo MTOC formation.

The centrosome is an important microtubule-organising centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), which nucleates microtubules. Centrosomes can form close to an existing structure (canonical duplication) or de novo How centrosomes form de novo is not known. The master driver of centrosome biogenesis, PLK4, is critical for the recruitment of several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where PLK4 can induce de novo MTOC formation ( Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro, PLK4 can self-assemble into condensates that recruit α- and ß-tubulins. In Xenopus extracts, PLK4 assemblies additionally recruit STIL, a substrate of PLK4, and the microtubule nucleator γ-tubulin, forming acentriolar MTOCs de novo The assembly of these robust microtubule asters is independent of dynein, similar to what is found for centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organising catalytic scaffold that recruits centriole components, PCM factors and α- and ß-tubulins, leading to MTOC formation.This article has an associated First Person interview with the first author of the paper.

Green Binder Based on Enzymatically Polymerized Eucalypt Kraft Lignin for Fiberboard Manufacturing: A Preliminary Study.

The capability of laccase from Myceliophthora thermophila to drive oxidative polymerization of Eucalyptus globulus Kraft lignin (KL) was studied as a previous step before applying this biotechnological approach for the manufacturing of medium-density fiberboards (MDF) at a pilot scale. This method, which improves the self-bonding capacity of wood fibers by lignin enzymatic cross-linking, mimics the natural process of lignification in living plants and trees. An interesting pathway to promote these interactions could be the addition of lignin to the system. The characterization of E. globulus KL after enzymatic treatment showed a decrease of phenolic groups as well as the aromatic protons without loss of aromaticity. There was also an extensive oxidative polymerization of the biomolecule. In the manufacture of self-bonded MDF, the synergy generated by the added lignin and laccase provided promising results. Thus, whenever laccase was present in the treatment, MDF showed an increase in mechanical and dimensional stability for increasing amounts of lignin. In a pilot scale, this method produced MDF that meets the requirements of the European standards for both thickness swell (TS) and internal bonding (IB) for indoor applications.

CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis.

Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation.

Electrokinetic treatment of an agricultural soil contaminated with heavy metals.

The high organic matter content in agricultural soils tends to complex and retain contaminants such as heavy metals. Electrokinetic remediation was tested in an agricultural soil contaminated with Co(+2), Zn(+2), Cd(+2), Cu(+2), Cr(VI), Pb(+2) and Hg(+2). The unenhanced electrokinetic treatment was not able to remove heavy metals from the soil due to the formation of precipitates in the alkaline environment in the soil section close to the cathode. Moreover, the interaction between metals and organic matter probably limited metal transportation under the effect of the electric field. Citric acid and ethylenediaminetetraacetic acid (EDTA) were used in the catholyte as complexing agents in order to enhance the extractability and removal of heavy metals from soil. These complexing agents formed negatively charged complexes that migrated towards the anode. The acid front electrogenerated at the anode favored the dissolution of heavy metals that were transported towards the cathode. The combined effect of the soil pH and the complexing agents resulted in the accumulation of heavy metals in the center of the soil specimen.

A Proteome-wide screen for mammalian SxIP motif-containing microtubule plus-end tracking proteins.

Microtubule plus-end tracking proteins (+TIPs) are structurally and functionally diverse factors that accumulate at the growing microtubule plus-ends, connect them to various cellular structures, and control microtubule dynamics [1, 2]. EB1 and its homologs are +TIPs that can autonomously recognize growing microtubule ends and recruit to them a variety of other proteins. Numerous +TIPs bind to end binding (EB) proteins through natively unstructured basic and serine-rich polypeptide regions containing a core SxIP motif (serine-any amino acid-isoleucine-proline) [3]. The SxIP consensus sequence is short, and the surrounding sequences show high variability, raising the possibility that undiscovered SxIP containing +TIPs are encoded in mammalian genomes. Here, we performed a proteome-wide search for mammalian SxIP-containing +TIPs by combining biochemical and bioinformatics approaches. We have identified a set of previously uncharacterized EB partners that have the capacity to accumulate at the growing microtubule ends, including protein kinases, a small GTPase, centriole-, membrane-, and actin-associated proteins. We show that one of the newly identified +TIPs, CEP104, interacts with CP110 and CEP97 at the centriole and is required for ciliogenesis. Our study reveals the complexity of the mammalian +TIP interactome and provides a basis for investigating the molecular crosstalk between microtubule ends and other cellular structures.

BLD10/CEP135 is a microtubule-associated protein that controls the formation of the flagellum central microtubule pair.

Cilia and flagella are involved in a variety of processes and human diseases, including ciliopathies and sterility. Their motility is often controlled by a central microtubule (MT) pair localized within the ciliary MT-based skeleton, the axoneme. We characterized the formation of the motility apparatus in detail in Drosophila spermatogenesis. We show that assembly of the central MT pair starts prior to the meiotic divisions, with nucleation of a singlet MT within the basal body of a small cilium, and that the second MT of the pair only assembles much later, upon flagella formation. BLD10/CEP135, a conserved player in centriole and flagella biogenesis, can bind and stabilize MTs and is required for the early steps of central MT pair formation. This work describes a genetically tractable system to study motile cilia formation and provides an explanation for BLD10/CEP135's role in assembling highly stable MT-based structures, such as motile axonemes and centrioles.

Deconstructing the centriole: structure and number control.

Centrioles are very small microtubule-based organelles essential for centrosome, cilia and flagella assembly, which are involved in a variety of cellular and developmental processes. Although the centriole was first described almost a century ago, the knowledge on its assembly mechanism remains poor. In the past decade, forefront functional studies have provided important data on the different players involved in centriole biogenesis. Centriole research has now started to profit from highly sensitive structural, imaging, and biochemical techniques that are unveiling how those players contribute to assemble such a small and complex structure. We will review those studies and discuss how this field will increasingly benefit from the newborn and exciting era of super resolution analyses.

NMDA receptor activation suppresses microtubule growth and spine entry.

Dynamic microtubules are important to maintain neuronal morphology and function, but whether neuronal activity affects the organization of dynamic microtubules is unknown. Here, we show that a protocol to induce NMDA-dependent long-term depression (LTD) rapidly attenuates microtubule dynamics in primary rat hippocampal neurons, removing the microtubule-binding protein EB3 from the growing microtubule plus-ends in dendrites. This effect requires the entry of calcium and is mediated by activation of NR2B-containing NMDA-type glutamate receptor. The rapid NMDA effect is followed by a second, more prolonged response, during which EB3 accumulates along MAP2-positive microtubule bundles in the dendritic shaft. MAP2 is both required and sufficient for this activity-dependent redistribution of EB3. Importantly, NMDA receptor activation suppresses microtubule entry in dendritic spines, whereas overexpression of EB3-GFP prevents NMDA-induced spine shrinkage. These results suggest that short-lasting and long-lasting changes in dendritic microtubule dynamics are important determinants for NMDA-induced LTD.

SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase.

The ends of growing microtubules (MTs) accumulate a set of diverse factors known as MT plus end-tracking proteins (+TIPs), which control microtubule dynamics and organization. In this paper, we identify SLAIN2 as a key component of +TIP interaction networks. We showed that the C-terminal part of SLAIN2 bound to end-binding proteins (EBs), cytoplasmic linker proteins (CLIPs), and CLIP-associated proteins and characterized in detail the interaction of SLAIN2 with EB1 and CLIP-170. Furthermore, we found that the N-terminal part of SLAIN2 interacted with ch-TOG, the mammalian homologue of the MT polymerase XMAP215. Through its multiple interactions, SLAIN2 enhanced ch-TOG accumulation at MT plus ends and, as a consequence, strongly stimulated processive MT polymerization in interphase cells. Depletion or disruption of the SLAIN2-ch-TOG complex led to disorganization of the radial MT array. During mitosis, SLAIN2 became highly phosphorylated, and its interaction with EBs and ch-TOG was inhibited. Our study provides new insights into the molecular mechanisms underlying cell cycle-specific regulation of MT polymerization and the organization of the MT network.

Cell and molecular biology of microtubule plus end tracking proteins: end binding proteins and their partners.

The microtubule plus end is a crucial site for the regulation of microtubule dynamics and microtubule association with different cellular organelles and macromolecular complexes. Several evolutionarily conserved groups of proteins form comet-like accumulations at the growing microtubule plus ends. These proteins belong to functionally diverse and structurally unrelated families: they include motors, nonmotor proteins, microtubule polymerases, and depolymerases as well as regulatory and adaptor proteins. Here, we provide an overview of microtubule plus end binding proteins, describe what is known about the mechanisms of their association with growing microtubule tips, and discuss their functional properties in relation to microtubule plus end accumulation.

In vitro reconstitution of the functional interplay between MCAK and EB3 at microtubule plus ends.

The kinesin-13 family member mitotic centromere-associated kinesin (MCAK) is a potent microtubule depolymerase. Paradoxically, in cells it accumulates at the growing, rather than the shortening, microtubule plus ends. This plus-end tracking behavior requires the interaction between MCAK and members of the end-binding protein (EB) family, but the effect of EBs on the microtubule-destabilizing activity of MCAK and the functional significance of MCAK accumulation at the growing microtubule tips have so far remained elusive. Here, we dissect the functional interplay between MCAK and EB3 by reconstituting EB3-dependent MCAK activity on dynamic microtubules in vitro. Whereas MCAK alone efficiently blocks microtubule assembly, the addition of EB3 restores robust microtubule growth, an effect that is not dependent on the binding of MCAK to EB3. At the same time, EB3 targets MCAK to growing microtubule ends by increasing its association rate with microtubule tips, a process that requires direct interaction between the two proteins. This EB3-dependent microtubule plus-end accumulation does not affect the velocity of microtubule growth or shortening but enhances the capacity of MCAK to induce catastrophes. The combination of MCAK and EB3 thus promotes rapid switching between microtubule growth and shortening, which can be important for remodeling of the microtubule cytoskeleton.