Quantification of Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and Nitrospira spp. from full-scale wastewater treatment plants by competitive PCR.

Utilizing the principle of competitive PCR, we developed two assays to enumerate Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and nitrite-oxidizing bacteria belonging to the genus NITROSPIRA: The specificities of two primer sets, which were designed for two target regions, the amoA gene and Nitrospira 16S ribosomal DNA (rDNA), were verified by DNA sequencing. Both assays were optimized and applied to full-scale, activated sludge wastewater treatment plant (WWTP) samples. If it was assumed that there was an average of 3.6 copies of 16S rDNA per cell in the total population and two copies of the amoA gene per ammonia-oxidizing bacterial cell, the ammonia oxidizers examined represented 0.0033% +/- 0.0022% of the total bacterial population in a municipal WWTP. N. oligotropha-like ammonia-oxidizing bacteria were not detected in an industrial WWTP. If it was assumed that there was one copy of the 16S rDNA gene per nitrite-oxidizing bacterial cell, Nitrospira spp. represented 0.39% +/- 0.28% of the biosludge population in the municipal WWTP and 0.37% +/- 0.23% of the population in the industrial WWTP. The number of Nitrospira sp. cells in the municipal WWTP was more than 62 times greater than the number of N. oligotropha-like cells, based on a competitive PCR analysis. The results of this study extended our knowledge of the comparative compositions of nitrifying bacterial populations in wastewater treatment systems. Importantly, they also demonstrated that we were able to quantify these populations, which ultimately will be required for accurate prediction of process performance and stability for cost-effective design and operation of WWTPs.     

Identifying Hendra virus diversity in pteropid bats

Hendra virus (HeV) causes a zoonotic disease with high mortality that is transmitted to humans from bats of the genus Pteropus (flying foxes) via an intermediary equine host. Factors promoting spillover from bats to horses are uncertain at this time, but plausibly encompass host and/or agent and/or environmental factors. There is a lack of HeV sequence information derived from the natural bat host, as previously sequences have only been obtained from horses or humans following spillover events. In order to obtain an insight into possible variants of HeV circulating in flying foxes, collection of urine was undertaken in multiple flying fox roosts in Queensland, Australia. HeV was found to be geographically widespread in flying foxes with a number of HeV variants circulating at the one time at multiple locations, while at times the same variant was found circulating at disparate locations. Sequence diversity within variants allowed differentiation on the basis of nucleotide changes, and hypervariable regions in the genome were identified that could be used to differentiate circulating variants. Further, during the study, HeV was isolated from the urine of flying foxes on four occasions from three different locations. The data indicates that spillover events do not correlate with particular HeV isolates, suggesting that host and/or environmental factors are the primary determinants of bat-horse spillover. Thus future spillover events are likely to occur, and there is an on-going need for effective risk management strategies for both human and animal health.     

Correction to: Preliminary insights into the population characteristics and distribution of reef (Mobula alfredi) and oceanic (M. birostris) manta rays in French Polynesia

Coral Reefs - This erratum has been initiated as many corrections which were submitted during proofing were overlooked by vendor.     

SERS‐based biosensor for Alzheimer disease evaluation through the fast analysis of human serum

Alzheimer disease (AD) is the most common form of dementia in the elderly, progressively affecting the cognitive functions with a complex diagnostic procedure that limits the time for a prompt intervention. In this study we optimized a reliable protocol for the analysis of AD patients and healthy subjects' serum using the Surface Enhanced Raman Spectroscopy (SERS), taking into consideration the effect of different variables on the final spectra, analyzed and compared through multivariate analysis and correlated with hippocampus volume. As results, we demonstrated a statistical difference between the spectra collected from the two investigated groups, with an accuracy, precision and specificity of respectively 83%, 86%, and 86%. The correlation of these data with those obtained from MRI, demonstrated a direct correlation between Raman spectra and hippocampus degeneration showing the Raman Spectroscopy (RS) as a potential tool for the monitoring of AD progression and rehabilitation treatments.     

HIV-1 integrase preassembled on donor DNA is refractory to activity stimulation by LEDGF/p75

LEDGF/p75 is known to enhance the integrase strand transfer activity in vitro, but the underlying mechanism is unclear. Using an integrase assay with a chemiluminescent readout adapted to a 96-well plate format, the effect of LEDGF/p75 on both the 3'-processing and strand transfer steps was analyzed. Integrase inhibitors of the strand transfer reaction remained active in the presence of LEDGF/p75, but displayed 3- to 7-fold higher IC50 values. Our analyses indicate that, in the presence of 150 nM LEDGF/p75, active integrase/donor DNA complexes were increased by 5.3-fold during the 3'-processing step. In addition, these integrase/donor DNA complexes showed a 4.5-fold greater affinity for the target DNA during the subsequent strand transfer step. We also observed a 3.7-fold increase in the rate constant of catalysis of the strand transfer step when 150 nM LEDGF/p75 was present during the 3'-processing step. In contrast, when LEDGF/p75 was added at the beginning of the strand transfer step, no increase in either the concentration of active integrase/donor DNA complex or its rate constant of strand transfer catalysis was observed. This observation suggested that the integrase/donor DNA formed in the absence of LEDGF/p75 became refractory to the stimulatory effect of LEDGF/p75. Instead, this LEDGF/p75 added at the start of the strand transfer step was able to promote the formation of a new cohort of active integrase/donor DNA complexes which became functional with a delay of 45 min after LEDGF/p75 addition. We propose a model whereby LEDGF/p75 can only bind integrase before the latter binds donor DNA whereas donor DNA can engage either free or LEDGF/p75-bound integrase.     

Over-expression and refolding of MAP kinase phosphatase 3

MAP kinase phosphatase 3 (MKP3, also known as DUSP6 and PYST1) is involved in extracellular signal receptor kinase (ERK) regulation and functions as a specific phosphatase to the activated (phosphorylated) forms of ERK1 and ERK2. MKP3 displays allosteric activation, which aids in tightly regulating its function to ERK substrates, but not other related MAPKs. Due to MKP3's specificity for the ERK signaling pathway, the development of specific activators or inhibitors to the enzyme have been suggested in order to expressly influence the ERK1 and ERK2 pathways. To produce the high yields of MKP3 protein necessary for physico-chemical characterization of MKP3 and for high throughput screening of its small-molecule activators and inhibitors, we have cloned, purified and, and identified refolding conditions suitable for producing full-length, human MKP3 from Escherichia coli inclusion bodies. Furthermore, we demonstrate the use of a 96-well plate format refolding assay in which the ERK-induced activity of MKP3 is simulated by 33% DMSO. The assay allowed for rapid detection of MKP3's function following a refolding screen in the absence of ERK and thus provides quick and inexpensive testing of MKP3 activity. Following screening, the refolded product was confirmed to be correctly folded by steady-state kinetic analysis and by the CD spectroscopy-determined secondary structure content. CD data were consistent with 36% helix and 14% sheet, which compared to an expected 32.9% helix and 12.4% sheet. These data indicated that MKP3 was properly folded, making it a suitable protein for use in functional studies.     

Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy

Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.     

Distinct intracellular vesicle transport mechanisms are selectively modified by spastin and spastin mutations

Spastin is a microtubule severing ATPase that regulates intracellular and axonal transport of vesicles. Intracellular vesicle trafficking was analyzed in differentiated SH‐SY5Y‐neuroblastoma cells, transfected with spastin wild‐type and three spastin mutations (ΔN, K388R, S44L) to investigate spastin‐mediated effects on the velocity of vesicles, stained with LysoTracker Red®. The vesicle velocity varied considerably between mutations and detailed analysis revealed up to five distinct velocity classes. Microtubule severing by overexpressed wild‐type spastin caused reduced vesicle velocity. S44L and ΔN mutations, which were functionally impaired, showed similar velocities as control cells. K388R‐transfected cells exhibited an intermediate velocity profile. The results support the idea that spastin mutations not only alter axonal transport, but in addition regulate intracellular trafficking in the cell soma as well. J. Cell. Physiol. 226: 362–368, 2011. © 2010 Wiley‐Liss, Inc.     

X-chromosome polysomy in the male

Summary A review of 569 male patients with X-chromosome polysomies (544 Klinefelter and 25 patients with other types of X-chromosome polysomy) is presented here. These patients were detected among the 77000 persons karyotyped in the Leuven cytogenetic center between the years 1966 and 1987. In the group of 544 Klinefelter patients special attention was paid to (1) the age at diagnosis, (2) social and marital status of the postpubertal males, (3) physical and intellectual abilities of the prepubertal boys, (4) delineation of the concurrence of Klinefelter syndrome and fragile X syndrome, and (5) the frequency of malignancies. In 25 patients with other X-chromosome polysomies (2 n48 chromosomes) genotype/phenotype correlation is reviewed, especially for the patients with 48,XXYY and 49,XXXXY karyotypes. Finally, double aneuploidy and rare structural X-chromosome aberrations are briefly discussed.     

Extracellular Amino Acid Concentrations in the Dorsal Spinal Cord of Freely Moving Rats Following Veratridine and Nociceptive Stimulation

In vivo microdialysis was used to sample extracellular concentrations of amino acids in the dorsal lumbar spinal cord of freely moving rats. Changes in the extracellular concentrations of amino acids were measured in response to infusion of veratridine (180 microM), a sodium channel activator, as well as during acute noxious stimulation by an injection of 5% formalin into the metatarsal region of the hindleg. Veratridine produced a tetrodotoxin (TTX)-sensitive increase in the extracellular concentration of Glu. Concentrations of Asp, taurine, Ala, Asn, and Gly were not significantly elevated following veratridine stimulation. Intradermal injection of formalin produced a TTX-sensitive increase in Asp concentration and a non-TTX-sensitive increase in Glu concentration. These data support the hypothesis that Glu and Asp are dorsal horn neurotransmitters involved in nociception.