Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication,System Setup,and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g. cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.     

Structure-Function Relationship of Substituted Bromomethylcoumarins in Nucleoside Specificity of RNA Alkylation

Selective alkylation of RNA nucleotides is an important field of RNA biochemistry, e.g. in applications of fluorescent labeling or in structural probing experiments, yet detailed structure-function studies of labeling agents are rare. Here, bromomethylcoumarins as reactive compounds for fluorescent labeling of RNA are developed as an attractive scaffold on which electronic properties can be modulated by varying the substituents. Six different 4-bromomethyl-coumarins of various substitution patterns were tested for nucleotide specificity of RNA alkylation using tRNA from Escherichia coli as substrate. Using semi-quantitative LC-MS/MS analysis, reactions at mildly acidic and slightly alkaline pH were compared. For all tested compounds, coumarin conjugates with 4-thiouridine, pseudouridine, guanosine, and uridine were identified, with the latter largely dominating. This data set shows that selectivity of ribonucleotide alkylation depends on the substitution pattern of the reactive dye, and even more strongly on the modulation of the reaction conditions. The latter should be therefore carefully optimized when striving to achieve selectivity. Interestingly, the highest selectivity for labeling of a modified nucleoside, namely of 4-thiouridine, was achieved with a compound whose selectivity was somewhat less dependent on reaction conditions than the other compounds. In summary, bromomethylcoumarin derivatives are a highly interesting class of compounds, since their selectivity for 4-thiouridine can be efficiently tuned by variation of substitution pattern and reaction conditions.     

Muscarinic Receptor Subclasses in the Chick Embryo Retina: Influence of Corticosterone Treatment

The present study was performed on retinas of chick embryos receiving at day 8 of incubation an intracerebral injection of 0.02 microgram of corticosterone. We had previously shown with the use of [3H]quinuclidinylbenzilate [( 3H]QNB) that such treatment induced the appearance of two muscarinic binding sites in the treated retinas, whereas only one was detectable in the controls. In the present study we investigated muscarinic cholinergic receptor subclasses with agonist and antagonist binding. Agonist binding was studied by varying the concentrations of carbachol and acetylcholine (10(-9) M-10(-5) M) in the presence of a constant concentration (0.2 nM) of [3H]QNB. Two subpopulations of receptors were revealed, a high- and a low-affinity receptor, in both treated and control retinas. However, in the hormone-treated retinas, the two subpopulations significantly differed from the controls in their affinity and in their relative percentage among the total receptor population. Moreover, using pirenzepine, an antagonist known to have the capacity to distinguish between muscarinic cholinergic subclasses, two receptor subpopulations were found to be present in the hormone-treated retinas but a single one in the controls. It is suggested that hormone treatment can either induce the appearance of a new subclass of muscarinic cholinergic receptors or favor the maturation of a population of retinal cells having these receptors. Pirenzepine binding in retinas from intact embryos of 7, 9, and 11 days of incubation revealed one receptor subpopulation. Thus, these findings are more consistent with the hypothesis that corticosterone effects the target cells, either inducing changes in muscarinic receptor and/or modifying the receptor environment.     

Uptake of Amino Acids by Brain Micro vessels Isolated from Rats with Experimental Chronic Renal Failure

The neurological disorders seen in patients with chronic renal failure and liver cirrhosis are analogous. Previous in vivo studies have shown that the impaired blood-brain amino acid transport seen in rats with chronic renal failure is similar to that of rats with portocaval anastomosis. To elucidate whether a comparable underlying pathogenic mechanism plays a role in both pathological conditions, blood and brain amino acid levels together with amino acid transport by isolated brain microvessels have been studied in rats with chronic renal failure and in sham-operated rats. Brain microvessels isolated from rats with experimental chronic renal failure showed that the uptake of labeled large neutral amino acid, i.e., leucine or phenylalanine, but not of lysine or alpha-methylaminoisobutyric acid, was significantly increased with respect to sham-operated rats; conversely, the uptake of glutamic acid in rats with chronic renal failure was significantly lower compared with values in controls. Kinetic analysis indicated that this was mainly due to increased exchange transport activity (Vmax) of the L-system, rather than to changes in the affinity (Km) of the carrier system for the relative substrate. These data, together with the significant rise of brain glutamine levels and an increased brain-to-plasma ratio of the sum of large neutral amino acids, are analogous to what was previously observed in rats with portocaval anastomosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Losing cichlid fish biodiversity: genetic and morphological homogenization of tilapia following colonization by introduced species

Among the many negative impacts of invasive species, hybridization with indigenous species has increasingly become recognized as a major issue. However, relatively few studies have characterized the phenotypic outcomes of hybridization following biological invasions. Here we investigate the genetic and morphological consequences of stocking invasive tilapia species in two water bodies in central Tanzania. We sampled individuals from the Mindu Reservoir on the Ruvu river system, and at Kidatu on the Great Ruaha–Rufiji river system. We screened individuals at 16 microsatellite loci, and quantified morphology using geometric morphometrics and linear measurements. In both the Mindu and Kidatu systems, we identified evidence of hybridization between indigenous Wami tilapia (Oreochromis urolepis) and the introduced Nile tilapia (Oreochromis niloticus) or blue-spotted tilapia (Oreochromis leucostictus). At both sites, purebred individuals could largely be separated using geometric morphometric variables, with hybrids occupying a broad morphospace among the parental species. Our data demonstrate that the gene pools and phenotypic identity of the indigenous O. urolepis have been severely impacted by the stocking of the invasive species. Given the lack of evidence for clear commercial benefits from stocking invasive tilapia species in waters already populated by indigenous congenerics, we suggest further spread of introduced species should be undertaken with considerable caution.     

Impaired AMPA signaling and cytoskeletal alterations induce early synaptic dysfunction in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a devastating neurodegenerative disorder that impairs memory and causes cognitive and psychiatric deficits. New evidences indicate that AD is conceptualized as a disease of synaptic failure, although the molecular and cellular mechanisms underlying these defects remain to be elucidated. Determining the timing and nature of the early synaptic deficits is critical for understanding the progression of the disease and for identifying effective targets for therapeutic intervention. Using single‐synapse functional and morphological analyses, we find that AMPA signaling, which mediates fast glutamatergic synaptic transmission in the central nervous system (CNS), is compromised early in the disease course in an AD mouse model. The decline in AMPA signaling is associated with changes in actin cytoskeleton integrity, which alters the number and the structure of dendritic spines. AMPA dysfunction and spine alteration correlate with the presence of soluble but not insoluble Aβ and tau species. In particular, we demonstrate that these synaptic impairments can be mitigated by Aβ immunotherapy. Together, our data suggest that alterations in AMPA signaling and cytoskeletal processes occur early in AD. Most important, these deficits are prevented by Aβ immunotherapy, suggesting that existing therapies, if administered earlier, could confer functional benefits.     

Impacts of soybean-induced defenses on <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> (Lepidoptera: Noctuidae) development

As soybean (Glycine max [L.] Merrill) is an important crop throughout the world, the action of herbivorous insects responsible for economic and productivity losses is the subject of constant research. Spodoptera frugiperda (J.E. Smith) caterpillars can cause extensive damage to soybean culture; this work investigated possible harmful effects on these caterpillars associated with the possible induced defenses of soybean plants. For this purpose, we assessed the biology of the insect (leaf consumption and performance traits) and chemical composition of the soybean leaves by ultraviolet–visible spectroscopy and chemometrics of three treatments: control, mechanically wounded soybean leaves, and S. frugiperda-damaged soybean leaves. The results reveal that both types of injuries induce changes in soybean metabolism regarding the production of phenolic substances, although only the herbivore-damaged plants provoke negative effects on insect biology. Variations in carotenoid production during the circadian cycle were also found in the control group. These results confirm that the soybean plants could endure and activate chemical defense mechanisms that impair the developmental lifecycle of the insect, suggesting possibilities for sustainable control strategies.     

The α1 and α6 Subunits Can Coexist in the Same Cerebellar GABAA Receptor Maintaining Their Individual Benzodiazepine-Binding Specificities

Abstract: Two GABA_A receptor subunit-specific antibodies anti-α₆ and anti-α₁ have been used for elucidating the relationship between the presence of α₁ and/or α₆ subunits in the cerebellar GABA_A receptors and the benzodiazepine-binding specificity. Receptor immunoprecipitation with the subunit-specific antibodies shows that 39% of the cerebellar GABA_A receptors have α₆, whereas 76% of the receptors have α₁ as determined by [³H]muscimol binding. Results show that 42–45% of the receptors having α₆ also have α₁, whereas 13–15% of the receptors that contain α₁ also have α₆. The immunoprecipitation results as well as immunopurification and immunoblotting experiments reveal the existence of three types of cerebellar GABA_A receptors; i.e., one has both α₁ and α₆ subunits, a second type has α₁ but not α₆, and a third type has α₆ but not α₁ subunits. The results also show that receptors where α₁ and α₆ subunits coexist have two pharmacologically different benzodiazepine-binding properties, each associated with a different α subunit. The α₁ subunit contributes the high-affinity binding of [³H]Ro 15-1788 (flumazenil) and the diazepam-sensitive binding of [³H]Ro 15-4513. The α₆ subunit contributes the diazepam-insensitive binding of [³H]Ro 15-4513, but it does not bind [³H]Ro 15-1788 with high affinity. Thus, in the cerebellar α₁–α₆ GABA_A receptors, there is no dominance of the pharmacology of one α subunit over the other.     

A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase

The narB gene from the cyanobacterium Synechococcus sp. PCC 7942 was cloned downstream from the LacI-regulated promoter P_trc in the Escherichia coli vector pTrc99A, rendering plasmid pCSLM1. Addition of isopropyl--D-thiogalactoside to E. coli (pCSLM1) resulted in the parallel expression of a 76 kDa polypeptide and a nitrate reductase activity with properties identical to those known for nitrate reductase isolated from Synechococcus cells. As is the case for nitrate reductase from Synechococcus cells, either reduced methyl viologen or reduced ferredoxin could be used as an electron donor for the reduction of nitrate catalyzed by E. coli (pCSLM1) extracts. This data shows that narB is a cyanobacterial structural gene for nitrate reductase.