Genome-inferred spatio-temporal resolution of an uncultivated Roizmanbacterium reveals its ecological preferences in groundwater

Subsurface ecosystems like groundwater harbour diverse microbial communities, including small-sized, putatively symbiotic organisms of the Candidate Phyla Radiation, yet little is known about their ecological preferences and potential microbial partners. Here, we investigated a member of the superphylum Microgenomates (Cand. Roizmanbacterium ADI133) from oligotrophic groundwater using mini-metagenomics and monitored its spatio-temporal distribution using 16S rRNA gene analyses. A Roizmanbacteria-specific quantitative PCR assay allowed us to track its abundance over the course of 1 year within eight groundwater wells along a 5.4 km hillslope transect, where Roizmanbacteria reached maximum relative abundances of 2.3%. In-depth genomic analyses suggested that Cand. Roizmanbacterium ADI133 is a lactic acid fermenter, potentially able to utilize a range of complex carbon substrates, including cellulose. We hypothesize that it attaches to host cells using a trimeric autotransporter adhesin and inhibits their cell wall biosynthesis using a toxin–antitoxin system. Network analyses based on correlating Cand. Roizmanbacterium ADI133 abundances with amplicon sequencing-derived microbial community profiles suggested one potential host organism, classified as a member of the class Thermodesulfovibrionia (Nitrospirae). By providing lactate as an electron donor Cand. Roizmanbacterium ADI133 potentially mediates the transfer of carbon to other microorganisms and thereby is an important connector in the microbial community.     

CNF1 improves astrocytic ability to support neuronal growth and differentiation in vitro

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1β, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1β. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes.     

Single‐cell genomics based on Raman sorting reveals novel carotenoid‐containing bacteria in the Red Sea

Cell sorting coupled with single‐cell genomics is a powerful tool to circumvent cultivation of microorganisms and reveal microbial ‘dark matter’. Single‐cell Raman spectra (SCRSs) are label‐free biochemical ‘fingerprints’ of individual cells, which can link the sorted cells to their phenotypic information and ecological functions. We employed a novel Raman‐activated cell ejection (RACE) approach to sort single bacterial cells from a water sample in the Red Sea based on SCRS. Carotenoids are highly diverse pigments and play an important role in phototrophic bacteria, giving strong and distinctive Raman spectra. Here, we showed that individual carotenoid‐containing cells from a Red Sea sample were isolated based on the characteristic SCRS. RACE‐based single‐cell genomics revealed putative novel functional genes related to carotenoid and isoprenoid biosynthesis, as well as previously unknown phototrophic microorganisms including an unculturable Cyanobacteria spp. The potential of Raman sorting coupled to single‐cell genomics has been demonstrated.     

Impacts of inter-basin water transfer on the water quality of receiving reservoirs in a tropical semi-arid region

Inter-basin water transfer (IBWT) involves the transport of water from one geographically distinct basin to another to balance the distribution of water resources. Although the socio-economic benefits of implementing these projects are well recognized, little is known about the subsequent effects on the water quality of the receiving systems. We evaluated the effects of an IBWT on the water quality of three receiving reservoirs of an intermittent river in a semi-arid region. We compared the similarity among the reservoirs before and after the IBWT to assess how the reservoirs responded to the introduction of water. Although the last two reservoirs that have received water have become similar in terms of physical and chemical characteristics and algal biomass (chlorophyll-a), the first reservoir has not. The IBWT resulted in an improvement in the water quality of the first reservoir but a decrease in the water quality of the two successive reservoirs, along with a significant increase in algal biomass. Long river sections located upstream that were dry at the time of IBWT probably contributed nutrients to the water as it moved downstream and into the reservoirs. Significant differences in the water quality were observed for different sampling months after the IBWT, but not for different sampling depths. Before the IBWT, the predictor variables for algal biomass were basically transparency and non-algal turbidity, with which it established a positive relationship. After IBWT, however, algal biomass also showed a positive relationship with pH and temperature. We conclude that IBWT affects the water quality of receiving reservoirs and that the responses are reservoir specific. IBWT also increases the complexity of the correlations of physical and chemical variables with algal biomass.

     

Characterization,Toxicity, and Optimization for the Growth and Production of Bacteriocin-like Substances by Lactobacillus curvatus

Probiotics and Antimicrobial Proteins - This study aimed to characterize, evaluate toxicity and optimize the conditions for the growth and production of bacteriocin-like substances by Lactobacillus...     

IGF-1R tyrosine kinase expression and dependency in clones of IGF-1R knockout cells (R-)

Insulin-like growth factor 1 receptor (IGF-1R) plays many crucial roles in cancer, like anti-apoptotic activity and necessity for transformation. IGF-1R knockout cells (R-) represent a useful tool for molecular mapping of biological properties of the receptor. R- cells have been shown to be refractory to transformation by viral and cellular oncogenes, highlighting the necessity of this receptor for transformation. Surprisingly, more recent studies have shown that these cells can undergo spontaneous transformation. This observation raises the question as whether R- cells over the years have acquired some properties mimicking those of IGF-1R. Using an IGF-1R inhibitor (cyclolignan PPP) we have identified clones of R- (R-s) that are sensitive to this compound. Since, PPP is closely related to podophyllotoxin, which is an efficient microtubule inhibitor, we first investigated if such a mechanism could explain the sensitivity to PPP. However, highly purified PPP showed no or very slight tubulin binding. Further analysis of R-s revealed expression of a 90 kDa protein being reactive to IGF-1R beta-subunit antibodies. This protein was weakly but constitutively tyrosine phosphorylated and was downregulated by siRNA targeting IGF-1R. This downregulation was paralleled by decreased R-s survival. Taken together, our study suggests that clones of R- express IGF-1R activity and dependency, which in turn may explain that R- can undergo spontaneous transformation.     

Optimization of <Emphasis Type="Italic">Curcuma</Emphasis> Oil/Quinine-Loaded Nanocapsules for Malaria Treatment

Quinine, a treatment used in chloroquine-resistant falciparum malaria, was loaded into poly(?-caprolactone) or Eudragit^® RS100 nanocapsules using Curcuma oil as the oil-based core. Until now, the effect of cationic nanocapsules on malaria has not been reported. A 2⁴ factorial design was adopted using, as independent variables, the concentration of Curcuma oil, presence of quinine, type of polymer, and aqueous surfactant. Diameter, zeta potential, and pH were the responses studied. The formulations were also evaluated for drug content, encapsulation efficiency, photostability, and antimalarial activity against Plasmodium berghei-infected mice. The type of polymer influenced all of the responses studied. Quinine-loaded Eudragit^® RS100 (F13) and PCL nanocapsules (F9), both with polysorbate 80 coating, showed nanometric particle size, positive zeta potential, neutral pH, high drug content, and quinine photoprotection ability; thus, these nanocapsules were selected for in vivo tests. Both formulations showed lower levels of parasitemia from the beginning of the experiment (5.78 ± 3.60 and 4.76 ± 3.46% for F9 and F13, respectively) and highest survival mean time (15.3 ± 2.0 and 14.9 ± 5.6 days for F9 and F13, respectively). F9 and F13 showed significant survival curve compared to saline, thus demonstrating that nanoencapsulation improved bioefficacy of QN and co-encapsulated curcuminoids, regardless of the surface charge.     

Morphometric Identification of Queens,Workers and Intermediates in In Vitro Reared Honey Bees (Apis mellifera)

In vitro rearing is an important and useful tool for honey bee (Apis mellifera L.) studies. However, it often results in intercastes between queens and workers, which are normally are not seen in hive-reared bees, except when larvae older than three days are grafted for queen rearing. Morphological classification (queen versus worker or intercastes) of bees produced by this method can be subjective and generally depends on size differences. Here, we propose an alternative method for caste classification of female honey bees reared in vitro, based on weight at emergence, ovariole number, spermatheca size and size and shape, and features of the head, mandible and basitarsus. Morphological measurements were made with both traditional morphometric and geometric morphometrics techniques. The classifications were performed by principal component analysis, using naturally developed queens and workers as controls. First, the analysis included all the characters. Subsequently, a new analysis was made without the information about ovariole number and spermatheca size. Geometric morphometrics was less dependent on ovariole number and spermatheca information for caste and intercaste identification. This is useful, since acquiring information concerning these reproductive structures requires time-consuming dissection and they are not accessible when abdomens have been removed for molecular assays or in dried specimens. Additionally, geometric morphometrics divided intercastes into more discrete phenotype subsets. We conclude that morphometric geometrics are superior to traditional morphometrics techniques for identification and classification of honey bee castes and intermediates.     

27-Nor-Δ4-dafachronic acid is a synthetic ligand of Caenorhabditis elegans DAF-12 receptor

27-Nor-Δ⁴-dafachronic acid was prepared in nine steps and 14% overall yield by two sequential 2-carbon homologations from 20β-carboxyaldehyde-4-pregnen-3-one. Its activity was evaluated in vivo, where it rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants and restored their normal resistance to oxidative stress. 27-Nor-Δ⁴-dafachronic acid was also able to directly bind and activate DAF-12 in a transactivation cell-based luciferase reporter assay, although it was less active than the corresponding 25R-and 25S dafachronic acids. The binding mode of the 27-Nor steroid was studied by molecular dynamics using a homology model of the CeDAF-12 receptor.