Further studies on cortical tangential migration in wild type and Pax-6 mutant mice

In this study we present new data concerning the tangential migration from the medial and lateral ganglionic eminences (MGE and LGE) to the cerebral cortex during development. We have used Calbindin as a useful marker to follow the itinerary of tangential migratory cells during early developmental stages in wild-type and Pax-6 homozygous mutant mice. In the wild-type mice, at early developmental stages, migrating cells advance through the intermediate zone (IZ) and preplate (PP). At more advanced stages, migrating cells were present in the subplate (SP) and cortical plate (CP) to reach the entire developing cerebral cortex. We found that, in the homozygous mutant mice (Pax-6 ^Sey-Neu/Pax-6 ^Sey-Neu), this tangential migration is severely affected at early developmental stages: migrating cells were absent in the IZ, which were only found some days later, suggesting that in the mutant mice, there is a temporal delay in tangential migration. We have also defined some possible mechanisms to explain certain migratory routes from the basal telencephalon to the cerebral cortex. We describe the existence of two factors, which we consider to be essential for the normal migration; the first one is the cell adhesion molecule PSA-NCAM, whose role in other migratory systems is well known. The second factor is Robo-2, whose expression delimits a channel for the passage of migratory cells from the basal telencephalon to the cerebral cortex.     

Tangential migration in neocortical development

During cortical development, different cell populations arise in the basal telencephalon and subsequently migrate tangentially to the neocortex. However, it is not clear whether these cortical cells are generated in the lateral ganglionic eminence (LGE), the medial ganglionic eminence (MGE), or both. In this study, we have generated a three-dimensional reconstruction to study the morphological formation of the two ganglionic eminences and the interganglionic sulcus. As a result, we have demonstrated the importance of the development of these structures for this tangential migration to the neocortex. We have also used the tracers DiI and BDA in multiple experimental paradigms (whole embryo culture, in utero injections, and brain slice cultures) to analyze the routes of cell migration and to demonstrate the roles of both eminences in the development of the cerebral cortex. These results are further strengthened, confirming the importance of the MGE in this migration and demonstrating the early generation of tangential migratory cells in the LGE early in development. Finally, we show that the calcium-binding protein Calretinin is expressed in some of these tangentially migrating cells. Moreover, we describe the spatiotemporal sequence of GABA, Calbindin, and Calretinin expression, showing that these three markers are expressed in the cortical neuroepithelium over several embryonic days, suggesting that the cells migrating tangentially form a heterogeneous population.     

RGMa Regulates Cortical Interneuron Migration and Differentiation

The etiology of neuropsychiatric disorders, including schizophrenia and autism, has been linked to a failure to establish the intricate neural network comprising excitatory pyramidal and inhibitory interneurons during neocortex development. A large proportion of cortical inhibitory interneurons originate in the medial ganglionic eminence (MGE) of the ventral telencephalon and then migrate through the ventral subventricular zone, across the corticostriatal junction, into the embryonic cortex. Successful navigation of newborn interneurons through the complex environment of the ventral telencephalon is governed by spatiotemporally restricted deployment of both chemorepulsive and chemoattractive guidance cues which work in concert to create a migratory corridor. Despite the expanding list of interneuron guidance cues, cues responsible for preventing interneurons from re-entering the ventricular zone of the ganglionic eminences have not been well characterized. Here we provide evidence that the chemorepulsive axon guidance cue, RGMa (Repulsive Guidance Molecule a), may fulfill this function. The ventricular zone restricted expression of RGMa in the ganglionic eminences and the presence of its receptor, Neogenin, in the ventricular zone and on newborn and maturing MGE-derived interneurons implicates RGMa-Neogenin interactions in interneuron differentiation and migration. Using an in vitro approach, we show that RGMa promotes interneuron differentiation by potentiating neurite outgrowth. In addition, using in vitro explant and migration assays, we provide evidence that RGMa is a repulsive guidance cue for newborn interneurons migrating out of the ganglionic eminence ventricular zone. Intriguingly, the alternative Neogenin ligand, Netrin-1, had no effect on migration. However, we observed complete abrogation of RGMa-induced chemorepulsion when newborn interneurons were simultaneously exposed to RGMa and Netrin-1 gradients, suggesting a novel mechanism for the tight regulation of RGMa-guided interneuron migration. We propose that during peak neurogenesis, repulsive RGMa-Neogenin interactions drive interneurons into the migratory corridor and prevent re-entry into the ventricular zone of the ganglionic eminences.     

Arx acts as a regional key selector gene in the ventral telencephalon mainly through its transcriptional repression activity

The homeobox-containing gene Arx is expressed during ventral telencephalon development and required for correct GABAergic interneuron tangential migration from the ganglionic eminences to the olfactory bulbs, cerebral cortex and striatum. Its human ortholog is associated with a variety of neurological clinical manifestations whose symptoms are compatible with the loss of cortical interneurons and altered basal ganglia-related activities. Herein, we report the identification of a number of genes whose expression is consistently altered in Arx mutant ganglionic eminences. Our analyses revealed a striking ectopic expression in the ganglionic eminences of several of these genes normally at most marginally expressed in the ventral telencephalon. Among them, Ebf3 was functionally analyzed. Thus, its ectopic expression in ventral telencephalon was found to prevent neuronal tangential migration. Further, we showed that Arx is sufficient to repress Ebf3 endogenous expression and that its silencing in Arx mutant tissues partially rescues tangential cell movement. Together, these data provide new insights into the molecular pathways regulated by Arx during telencephalon development.     

Ephrins guide migrating cortical interneurons in the basal telencephalon

Cortical interneurons are born in the proliferative zones of the ganglionic eminences in the subpallium and migrate to the developing cortex along well-defined tangential routes. The mechanisms regulating interneuron migration are not completely understood. Here we examine the role of class-A members of the Eph/ephrin system in directing the migration of interneurons. In situ hybridizations demonstrated that ephrin-A3 is expressed in the developing striatum, an area that is strictly avoided by migrating cortical interneurons in vivo, which express the EphA4 receptor. We then examined interneuron migration in grafting experiments, where explants of the medial ganglionic eminence (MGE) from enhanced green fluorescent protein-expressing transgenic mice were homotopically grafted into host slices from wild-type littermate embryos. After blocking ephrin-A ligands, many interneurons invaded the striatal anlage. Moreover, stripe assay experiments revealed that ephrin-A3 acts as a repellent cue for neurons from the medial ganglionic eminence. Downregulation of the EphA4 receptor via siRNA transfection reduced the repulsive effect of ephrin-A3, indicating that EphA4 mediates at least in part the repulsive effect of ephrin-A3 on these cells. Together, these results suggest that ephrin-A3 acts as a repulsive cue that restricts cortical interneurons from entering inappropriate regions and thus contributes to define the migratory route of cortical interneurons.Key words: interneuron migration, cortical development, neuronal guidance cues, ephrin, Eph receptors, organotypic slice cultures     

Formation and preservation of cortical layers in slice cultures

During cortical development, neurons generated at the same time in the ventricular zone migrate out into the cortical plate and form a cortical layer (Berry and Eayrs, 1963, Nature 197:984–985; Berry and Rogers, 1965, J. Anat. 99:691–709). We have been studying both the formation and maintenance of cortical layers in slice cultures from rat cortex. The bromodexyuridine (BrdU) method was used to label cortical neurons on their birthday in vivo. When slice cultures were prepared from animals at different embryonic and postnatal ages, all cortical layers that have already been established in vivo remained preserved for several weeks in vitro. In slice cultures prepared during migration in the cortex, cells contiuned to migrate towards the pial side of the cortical slice, however, migration ceased after about 1 week in culture. Thus, cortical cells reached their final laminar position only in slice cultures from postnatal animals, whereas in embryonic slices, migrating cells became scattered throughout the cortex. Previous studies demonstrated that radial glia fibers are the major substrate for migrating neurons (Rakic, 1972, J. Comp. Neurol. 145:61–84; Hatten and Mason, 1990, Experientia 46:907–916). Using antibodies directed against the intermediate filament Vimentin, radial glial cells were detected in all slice cutures where cell migration did occur. Comparable to the glia development in vivo, radial glial fibers disappeared and astrocytes containing the glia fibrillary-associated protein (GFAP) differentiated in slice cultures from postnatal cortex, after the neurons have completed their migration. In contrast, radial glial cells were detected over the whole culture period, and very few astrocytes differentiated in embryonic slices, where cortical neurons failed to finish their migration. The results of this study indicate that the local environment is sufficient to sustain the layered organization of the cortex and support the migration of cortical neurons. In addition, our results reveal a close relationship between cell migration and the developmental status of glial cells. © 1992 John Wiley & Sons, Inc.     

Ephrins guide migrating cortical interneurons in the basal telencephalon

Cortical interneurons are born in the proliferative zones of the ganglionic eminences in the subpallium and migrate to the developing cortex along well-defined tangential routes. The mechanisms regulating interneuron migration are not completely understood. Here we examine the role of class-A members of the Eph/ephrin system in directing the migration of interneurons. In situ hybridizations demonstrated that ephrin A3 is expressed in the developing striatum, an area that is strictly avoided by migrating cortical interneurons in vivo, which express the EphA4 receptor. We then examined interneuron migration in grafting experiments, where explants of the medial ganglionic eminence (MGE) from enhanced green fluorescent protein-expressing transgenic mice were homotopically grafted into host slices from wild-type littermate embryos. After blocking ephrin-A ligands, many interneurons invaded the striatal anlage. Moreover, stripe assay experiments revealed that ephrin-A3 acts as a repellent cue for neurons from the medial ganglionic eminence. Downregulation of the EphA4 receptor via siRNA transfection reduced the repulsive effect of ephrin-A3, indicating that EphA4 mediates at least in part the repulsive effect of ephrin A3 on these cells. Together, these results suggest that ephrin-A3 acts as a repulsive cue that restricts cortical interneurons from entering inappropriate regions and thus contributes to define the migratory route of cortical interneurons.     

The COUP-TF nuclear receptors regulate cell migration in the mammalian basal forebrain

Cells migrate via diverse pathways and in different modes to reach their final destinations during development. Tangential migration has been shown to contribute significantly to the generation of neuronal diversity in the mammalian telencephalon. GABAergic interneurons are the best-characterized neurons that migrate tangentially, from the ventral telencephalon, dorsally into the cortex. However, the molecular mechanisms and nature of these migratory pathways are only just beginning to be unravelled. In this study we have first identified a novel dorsal-to-ventral migratory route, in which cells migrate from the interganglionic sulcus, located in the basal telencephalon between the lateral and medial ganglionic eminences, towards the pre-optic area and anterior hypothalamus in the diencephalon. Next, with the help of transplantations and gain-of-function studies in organotypic cultures, we have shown that COUP-TFI and COUP-TFII are expressed in distinct and non-overlapping migratory routes. Ectopic expression of COUP-TFs induces an increased rate of cell migration and cell dispersal, suggesting roles in cellular adhesion and migration processes. Moreover, cells follow a distinct migratory path, dorsal versus ventral, which is dependent on the expression of COUP-TFI or COUP-TFII, suggesting an intrinsic role of COUP-TFs in guiding migrating neurons towards their target regions. Therefore, we propose that COUP-TFs are directly involved in tangential cell migration in the developing brain, through the regulation of short- and long-range guidance cues.     

Distinct cortical migrations from the medial and lateral ganglionic eminences

Recent evidence suggests that projection neurons and interneurons of the cerebral cortex are generally derived from distinct proliferative zones. Cortical projection neurons originate from the cortical ventricular zone (VZ), and then migrate radially into the cortical mantle, whereas most cortical interneurons originate from the basal telencephalon and migrate tangentially into the developing cortex. Previous studies using methods that label both proliferative and postmitotic cells have found that cortical interneurons migrate from two major subdivisions of the developing basal telencephalon: the medial and lateral ganglionic eminences (MGE and LGE). Since these studies labeled cells by methods that do not distinguish between the proliferating cells and those that may have originated elsewhere, we have studied the contribution of the MGE and LGE to cortical interneurons using fate mapping and genetic methods. Transplantation of BrdU-labeled MGE or LGE neuroepithelium into the basal telencephalon of unlabeled telencephalic slices enabled us to follow the fate of neurons derived from each of these primordia. We have determined that early in neurogenesis GABA-expressing cells from the MGE tangentially migrate into the cerebral cortex, primarily via the intermediate zone, whereas cells from the LGE do not. Later in neurogenesis, LGE-derived cells also migrate into the cortex, although this migration occurs primarily through the subventricular zone. Some of these LGE-derived cells invade the cortical plate and express GABA, while others remain within the cortical proliferative zone and appear to become mitotically active late in gestation. In addition, by comparing the phenotypes of mouse mutants with differential effects on MGE and LGE migration, we provide evidence that the MGE and LGE may give rise to different subtypes of cortical interneurons.     

Formation and preservation of cortical layers in slice cultures.

During cortical development, neurons generated at the same time in the ventricular zone migrate out into the cortical plate and form a cortical layer (Berry and Eayrs, 1963, Nature 197:984-985; Berry and Rogers, 1965, J. Anat. 99:691-709). We have been studying both the formation and maintenance of cortical layers in slice cultures from rat cortex. The bromodeoxyuridine (BrdU) method was used to label cortical neurons on their birthday in vivo. When slice cultures were prepared from animals at different embryonic and postnatal ages, all cortical layers that have already been established in vivo remained preserved for several weeks in vitro. In slice cultures prepared during migration in the cortex, cells continued to migrate towards the pial side of the cortical slice, however, migration ceased after about 1 week in culture. Thus, cortical cells reached their final laminar position only in slice cultures from postnatal animals, whereas in embryonic slice, migrating cells became scattered throughout the cortex. Previous studies demonstrated that radial glia fibers are the major substrate for migrating neurons (Rakic, 1972, J. Comp. Neurol. 145:61-84; Hatten and Mason, 1990, Experientia 46:907-916). Using antibodies directed against the intermediate filament Vimentin, radial glial cells were detected in all slice cultures where cell migration did occur. Comparable to the glia development in vivo, radial glial fibers disappeared and astrocytes containing the glia fibrillary-associated protein (GFAP) differentiated in slice cultures from postnatal cortex, after the neurons have completed their migration. In contrast, radial glial cells were detected over the whole culture period, and very few astrocytes differentiated in embryonic slices, where cortical neurons failed to finish their migration. The results of this study indicate that the local environment is sufficient to sustain the layered organization of the cortex and support the migration of cortical neurons. In addition, our results reveal a close relationship between cell migration and the developmental status of glial cells.     

The challenge of removing waste from wastewater: let technology use nature!

Tertiary treatments capable of removing chemical and biological contaminants of emerging concern have been successfully developed and implemented at full scale, opening the possibility of using wastewater treatment plants as recycling units, capable of producing wastewater that can be reused in various activities, such as agriculture irrigation; However, tertiary treatments remove only part of the wastewater microbiota, leaving the opportunity for regrowth and/or reactivation of potentially hazardous microorganisms, facilitated by the poor competition among the surviving microorganisms; Under the motto ‘added by technology, lead by nature’, the treatment and storage of treated wastewater must find the balance to develop a protection shield against the impoverishment the microbial quality and the development of potentially hazardous bacteria.

No man ever steps in the same river twice, for it''s not the same river and he''s not the same man. Heraclitus

Access to wholesome drinking water is not only a major ambition but also a basic human right that since antiquity has called scientists, engineers and politicians for action. The recognition that human excreta compromise the quality of the sources of drinking water triggered the development of sewage drainage systems as far as 3500–2500 BC, in cities such as Ur and Babylon (Lofrano and Brown, 2010). Among these ancient cities, Rome, where the largest known ancient sewer (Cloaca Maxima) and the first roman aqueduct (Aqua Appia) were built (600–312 BC), stands up (Lofrano and Brown, 2010). Despite the unexpected regression observed during the Middle Ages, the rising of urban and industrial agglomerations, matched by a growing production of wastewater, has been triggering the development of wastewater treatment technologies since the industrial revolution (Lofrano and Brown, 2010).Unlike other industrial activities, whose high added value products enable high‐profit margins, wastewater treatment may be not prioritized, at least in world regions with limited income and capacity to invest in both infrastructure and operation systems. Consequently, most of the urban wastewater treatment plants (UWWTP) operating worldwide rely upon biological‐based low‐cost technologies. The conventional activated sludge (CAS) technology is one of the most commonly applied worldwide (Orhon, 2014). With a long development history itself, this aerobic biologic process, in full‐scale operation since 1914, is regarded as the conventional norm for wastewater treatment (Alleman and Prakasam, 1983; Orhon, 2014).A century ago the major challenge of environmental engineers was to develop a treatment system able to reduce the load of readily degradable organic matter and pathogens from sewage. CAS‐based treatment systems fully achieve these goals (Tchobanoglous et al., 2003). But more than one century of industrial innovation and development changed dramatically our lifestyle, and consequently, the type of pollutants discharged in wastewater. Nowadays, UWWTPs are also expected to remove excess of inorganic nitrogen (N) and phosphorus (P) nutrients, responsible for the eutrophication of the receptor water bodies, and a myriad of (potentially) hazardous chemical micropollutants, which may pose risk to the aquatic ecosystems and human health given their acute and chronic toxicity. These chemical micropollutants of emerging concern, which are found at very low concentrations (up to μg l⁻¹), include both natural and xenobiotic compounds such as pharmaceuticals, personal care products, steroid hormones, drugs of abuse, and pesticides, among others (European Commission, 2013; Ribeiro et al., 2015). In addition to the chemical micropollutants, UWWTPs are now also challenged to impede the release of high loads of biological contaminants of emerging concern, such as some pathogenic virus, protozoa, or bacteria in particular antibiotic‐resistant (ARB) harbouring antibiotic resistance genes (ARG), into the receptor water bodies (Dulio et al., 2018; European Commission, 2020).Effective wastewater treatment systems are indeed the primary and major barrier between human activities and the environment, with a pivotal role on the prevention of contamination of surface‐ and groundwater. Inevitably, water bodies such as rivers, lakes and aquifers bridge sectors of activity and geographies, for instance when used as sources of agriculture irrigation water, drinking water production or habitat and fountain for wildlife or food‐producing animals. Pressures to implement technologies able to efficiently remove both chemical and biological contaminants within the urban water cycle are exacerbated under the climate change scenario. Massive withdrawal and consumption coupled with unpredictable weather conditions, such as drought and flood events, has been leading not only to freshwater scarcity but also to the deterioration of water quality (WWAP, 2019; European Commission, 2020).Freshwater scarcity brought the new concept of UWWTPs as recycling units, capable of producing final effluents that can be safely and sustainably reused for different purposes, namely in agriculture, the sector with the largest consumption of freshwater (WWAP, 2019). But to be reused, treated wastewater must be safe. This means that the concentration of eventual chemical and/or biological pollutants in treated wastewater must not put at risk the environmental and human health. Hence, the degree of contamination of the treated wastewater determines its end use or site of discharge (European Commission, 1991, 2020; Becerra‐Castro et al., 2015).Upgrading technologies capable of removal of N and P nutrients from wastewater have been successfully developed and implemented. Nowadays, full‐scale UWWTP with trains favouring the recirculation of the mixed liquor between aerobic and anoxic tanks, where ammonification of organic‐N, nitrification and denitrification occur according to the oxygen availability in each compartment are commonly found; and an increasing number of UWWTP where, in addition to the trains referred to above, recirculation includes anaerobic reactors favouring P granules accumulation are also operating worldwide (Tchobanoglous et al., 2003). More recently, the simultaneous C, N, and P removal is assured through the aerobic granular sludge technology, given the spatial distribution of the microorganisms of the different metabolic groups in the different micro‐environments of the granules (Nancharaiah and Reddy, 2018).In contrast with the C, N and P removal, the biological removal of chemical micropollutants seems to be less efficient. Despite the ability of a vast number of microorganisms to degrade a wide diversity of micropollutants, the low concentration of these compounds in wastewater may contribute for their low bioavailability in the biological reactors. Consequently, the secondary final effluents of CAS‐based UWWTPs still contain numerous micropollutants at environmental worrisome concentrations (McEachran et al., 2018).Advanced Oxidation Technologies (AOTs) have been recommended among the best solutions for the removal of chemical micropollutants from the secondary effluents of CAS‐based UWWTPs. A vast number of scientific studies has been conducted in this area, in order to develop and optimize tertiary processes capable of the efficient removal of these contaminants from the effluents before discharge into the receptor water bodies (Ribeiro et al., 2015). Among these technologies, ozonation has high visibility, being implemented in full‐scale UWWTPs, for instance in Switzerland, a country that recently implemented legislation recommending advanced treatment of wastewater aiming at protecting the environment (Rizzo et al., 2019).One of the advantages of AOTs is their capacity to disinfect water (Rizzo et al., 2020). Hence, besides degrading undesirable chemical micropollutants, numerous scientific bench studies demonstrated that the mechanisms for microbial inactivation used by AOTs, such as the oxidative stress as it is generated by ozonation, are also capable of reducing the microbial load of wastewater, including ARB&ARGs (e.g. Rizzo et al., 2020). Such promising results opened the possibility of upgrading CAS‐based UWWTPs with a final AOT polishing step and using the facilities as recycling units of urban wastewater. Additional treatment may be required in a reuse scenario, and in that cases, the final treated wastewater may need to undergo an adsorption post‐AOT treatment step to eventually remove toxic degradation products (Rizzo et al., 2019) and to be stored for periods that may vary between few hours to some days, depending on the needs. Hence, some bench and full‐scale studies have been conducted to assess the microbiological quality of the wastewater after the final AOT treatment.Consistently, studies focused on the effect of AOTs conclude that the microbiota, including ARB&ARGs, surviving AOT treatment is capable of re‐regrowth during the storage period, sometimes to values reaching or surpassing those measured in the untreated secondary effluent (Zimmermann et al., 2011; Becerra‐Castro et al., 2016; Czekalski et al., 2016; Sousa et al., 2017; Moreira et al., 2018; Biancullo et al., 2019; Iakovides et al., 2019). Moreover, re‐regrowth is accompanied by the disturbance of the microbial community, with possible implications on the decrease of diversity, and the overgrowth of Proteobacteria (Becerra‐Castro et al., 2016; Moreira et al., 2018). Among these, bacterial groups described as potential vectors of antibiotic resistance, such as Pseudomonas, have been detected at high relative abundance (Alexander et al., 2016; Jäger et al., 2018; Moreira et al., 2018).The same phenomena occur when other technologies are applied in the wastewater treatment. Comparatively milder processes such as UV_{254 nm} irradiation or even coagulation lead to similar disturbances (Becerra‐Castro et al., 2016; Grehs et al., 2019). When comparing different technologies, a positive correlation between disinfection efficacy and the predominance of ubiquitous, potentially hazardous, bacteria in the treated stored wastewater seems to occur (Becerra‐Castro et al., 2016). Interestingly, clean built environments, where asepsis and frequent disinfection are the rule, are characterized by the predominance of Proteobacteria (Mahnert et al., 2019). Moreover, cleaning with aggressive agents seems to favour microbiomes encoding functions related with virulence, multi‐drug efflux, oxidative stress, as well as membrane transport and secretion, which empower cells to acquire nutrients in highly competitive nutrient‐poor environments (Mahnert et al., 2019).Such results are not unexpected. Any process reducing the diversity and abundance of microorganisms in a given ecosystem, through physical removal of the cells or physical and/or chemical inactivation of macromolecules or cellular processes, is expected to generate a habitat where intercellular competition for space and nutrients is reduced, offering the opportunity for those that randomly survived the process and that are most versatile and fast to grow, to proliferate. Therefore, among the survivors, those with high capacity to grow under the conditions prevailing in the disinfected or cleaned system will thrive. Conversely, the microorganisms with specific requirements (e.g. nutritional) or with slower grow rates will be outcompeted. Proteobacteria are well known for their genomic plasticity. Some proteobacterial species, such as Pseudomonas aeruginosa, colonize a wide diversity of environmental compartments, including mineral water, chlorinated drinking water, surface water and soils, and even human bodies (Grobe et al., 2001; Naze et al., 2010; Palleroni, 2015). Part of the success of this ubiquitous opportunistic pathogenic species rely upon its capacity to exchange genetic information through horizontal gene transfer (Kung et al., 2010). Hence, Pseudomonas aeruginosa harbour genetic information which allows cell development in a wide diversity of environmental conditions, including in the presence of a vast array of antimicrobial compounds. Therefore, besides carrying intrinsic antimicrobial resistance, P. aeruginosa strains are excellent vectors of ARG dissemination (Manaia, 2017). The predominance of microorganisms with these type of features in treated wastewater is thus not desirable, mainly if its further use in agriculture irrigation is envisaged, given the possibility of contamination of the food chain.In this context, it may be argued that the upgrading UWWTPs with a final disinfection step is not enough to transform these facilities into wastewater recycling units, and more studies should be carried out to design and implement storage systems capable of attenuating the imbalance of the bacterial community before reuse of the stored treated wastewater.Measures to restore the microbial richness and diversity of the disinfected wastewater would prevent the overgrowth of hazardous bacteria fitted to couple with very clean oligotrophic environments, such as P. aeruginosa, through competition. Such measures might include the inoculation of the disinfected wastewater with balanced natural microbial communities, with a rich and diverse phylogenetic and functional assembly of microorganisms (van Bruggen et al., 2019). In these communities, organisms belonging to a wide variety of species interact through complex relationships (mutualism, commensalism, competition, predation, parasitism) assuring metabolic redundancy and the integrity of nutrient cycles and energy flows (van Bruggen et al., 2019). Such communities are stable and resilient, that is, show little disturbance and restore rapidly upon alteration of the environmental conditions or invasion (van Bruggen et al., 2019). Hence, procedures such as diluting disinfected wastewater with non‐polluted surface water, mixing with pristine sediments or soils or discharge in wetlands would introduce a healthy microbiome in the treated wastewater. Under this circumstance, the exogenous microbiome would act as a protection shield for the proliferation of the hazardous microorganism surviving the disinfection process, in a similar way of the natural human microbiota, our first line of defence against the invasion of pathogens.Definitely, microbes must have a say on removing waste from wastewater. The next research steps should be oriented towards a better understanding of the biotic relationships occurring in the treated wastewater and technological implementation of systems that are able to nurture these important artisan communities.     

Physical studies of RNA involvement in bacteriophage lambda DNA replication and prophage excision

Non-diffusible genetic elements in bacteriophage λ DNA replication and λ prophage excision have been analyzed by the DNA-cutting assay of Freifelder and Kirschner (1971) and Freifelder et al. (1972). The mutant ti12, which affects a unique site for replication in or near the origin of replication (Dove et al., 1971), makes λ DNA partially refractory to replicative DNA-cutting. RNA synthesis in the vicinity of the origin, of replication seems to control the susceptibility of λ DNA to replicative DNA-cutting (Dove et al., 1969). Analogously, RNA synthesis in the vicinity of the left-hand prophage terminus seems to control excisional DNA-cutting of derepressed λ DNA, as predicted by the studies of Davies et al. (1972). These physical studies confirm previous genetic analyses and imply that the elements involved act at a very early stage in replication and in excision.     

Macrophage to dendritic cell transitioning induced by Toxoplasma

Toxoplasma gondii exploits the migratory properties of monocytes and dendritic cells to promote tissue dissemination. Recently, ten Hoeve et al. reported that the parasite effector protein GRA28 conspires with host chromatin modifiers to confer dendritic cell-like features that convert sessile macrophages into migratory cells that transport infection to distal organs.     

Interneurons in the developing human neocortex

Cortical interneurons play a crucial role in the functioning of cortical microcircuitry as they provide inhibitory input to projection (pyramidal) neurons. Despite their involvement in various neurological and psychiatric disorders, our knowledge about their development in human cerebral cortex is still incomplete. Here we demonstrate that at the beginning of corticogenesis, at embryonic 5 gestation weeks (gw, Carnegie stage 16) in human, early neurons could be labeled with calretinin, calbindin, and GABA antibodies. These immunolabeled cells show a gradient from the ganglionic eminences (GE) toward the neocortex, suggesting that GE is a well conserved source of early born cortical interneurons from rodents to human. At mid-term (20 gw), however, a subset of calretinin(+) cells proliferates in the cortical subventricular zone (SVZ), suggesting a second set of interneuron progenitors that have neocortical origin. Neuropeptide Y, somatostatin, or parvalbumin cells are sparse in mid-term cerebral cortex. In addition to the early source of cortical interneurons in the GE and later in the neocortical SVZ, other regions, such as the subpial granular layer, may also contribute to the population of human cortical interneurons. In conclusion, our findings from cryosections and previous in vitro results suggest that cortical interneuron progenitor population is more complex in humans relative to rodents. The increased complexity of progenitors is probably evolutionary adaptation necessary for development of the higher brain functions characteristic to humans.     

Developmental mechanisms for the generation of telencephalic interneurons

Interneurons, which release the neurotransmitter γ-aminobutyric acid (GABA), are the major inhibitory cells of the central nervous system (CNS). Despite comprising only 20-30% of the cerebral cortical neuronal population, these cells play an essential and powerful role in modulating the electrical activity of the excitatory pyramidal cells onto which they synapse. Although interneurons are present in all regions of the mature telencephalon, during embryogenesis these cells are generated in specific compartments of the ventral (subpallial) telencephalon known as ganglionic eminences. To reach their final destinations in the mature brain, immature interneurons migrate from the ganglionic eminences to developing telencephalic structures that are both near and far from their site of origin. The specification and migration of these cells is a complex but precisely orchestrated process that is regulated by a combination of intrinsic and extrinsic signals. The final outcome of which is the wiring together of excitatory and inhibitory neurons that were born in separate regions of the developing telencephalon. Disruption of any aspect of this sequence of events during development, either from an environmental insult or due to genetic mutations, can have devastating consequences on normal brain function.     

Electrophorus electricus acetylcholinesterases; separation and selective modification by collagenase.

Abstract— The multiple forms of acetylcholinesterase found in the electric organ of the eel Electrophorus electricus have been fractionated by differential solubilization from an ammonium sulfate precipitate by means of a column elution procedure (King , 1972). This procedure cleanly separates ‘native’ forms from ‘degraded’ forms, and subsequent sedimentation reveals three native and two degraded forms. All three native forms, in distinction to the degraded ones, are insoluble at low ionic strength and are shifted to higher sedimentation constants by limited collagenase treatment. These results suggest that the long (500 Á) tail seen previously on the native forms of this enzyme (Dudai et al., 1973; Rieger et al., 1973a, b) may contain collagen.