Fungal infection alters the selection,dispersal and drift processes structuring the amphibian skin microbiome

Symbiotic microbial communities are important for host health, but the processes shaping these communities are poorly understood. Understanding how community assembly processes jointly affect microbial community composition is limited because inflexible community models rely on rejecting dispersal and drift before considering selection. We developed a flexible community assembly model based on neutral theory to ask: How do dispersal, drift and selection concurrently affect the microbiome across environmental gradients? We applied this approach to examine how a fungal pathogen affected the assembly processes structuring the amphibian skin microbiome. We found that the rejection of neutrality for the amphibian microbiome across a fungal gradient was not strictly due to selection processes, but was also a result of species‐specific changes in dispersal and drift. Our modelling framework brings the qualitative recognition that niche and neutral processes jointly structure microbiomes into quantitative focus, allowing for improved predictions of microbial community turnover across environmental gradients.     

Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation

The human M(3) muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M(3) muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [(3)H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity.     

Vectors and P64k gene targeting for tandem affinity purification in Neisseria meningitidis

We present and describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Neisseria meningitidis. The tagging cassette is designed for carboxyl-terminal tagging of proteins and it contains only two repeats of IgG-binding units. P64k protein from N. meningitidis was chosen to fuse at these new affinity tags. This protein is well recognized in immunoassays by serum from human convalescent meningococcal disease and it is highly immunogenic in animals. To continue the characterization of this meningococcal antigen, we designed and constructed two vectors for use in TAP purification method. We also carried-out preliminary test to check the correct expression of the protein fused in these vectors.     

Evidence for the presence of immunoreactive histidyl-proline diketopiperazine [Cyclo (His-Pro)] in the adult human brain

The current study was undertaken to evaluate the presence of cyclo (His-Pro) in adult human brain tissues obtained at autopsy. We found evidence for immunoreactive cyclo (His-Pro), which diluted in parallel to the radioimmunoassay standard curve and which had mobility on HPLC that was similar to synthetic cyclo (His-Pro), in several regions of the adult human brain. Whereas the levels of cyclo (His-Pro) in the pituitary stalk-median eminence were high (2.2 ng/mg protein), the concentrations in the whole hypothalamus were much lower (0.105 ng/mg protein). Among the extrahypothalamic brain regions examined, the levels of cyclo (His-Pro) were highest in the cerebellar hemisphere (0.168 ng/mg protein) and olfactory bulbs (0.180 ng/mg protein) and were lowest in the hippocampus (0.080 ng/mg protein) and occipital cortex (0.079 ng/mg protein). Thus, immunoreactive cyclo (His-Pro) has widespread distribution in the adult human brain and the potential exists for this cyclic diepeptide to play a role in human brain function.     

The identification of gonadotropin-releasing hormone (GnRH) in hypothalamic and extrahypothalamic loci of the human nervous system.

Immunoreactive-like GnRH activity has been identified in 24 of 26 separate loci of the human central nervous system. Tissues, secured from 5 brains at autopsy, were dissected, extracted sequentially with 2N and glacial acetic acid, lyophilized, and eluted in buffered saline for GnRH determinations by specific radioimmunoassay. GnRH concentrations (ng/mg protein) ranged from 8.96 (infundibulum) to 0.001 (cerebellum. middle lobe). Highest extrahypothalamic concentrations of GnRH were found in mamillary body (0.076) and thalamus (0.002). Extrahypothalamic GnRH was identical to synthetic and hypothalamic GnRH by criteria of immunoidentity. No post-mortem GnRH peptidolysis, evaluated experimentally in rats, was evident between 0 and 16 hrs in intact tissues maintained at 4 degrees C. These data suggest that GnRH is distributed throughout regions of the human brain outside the hypothalamus and suggest new, non-endocrine functions for GnRH in the human CNS, analogous the those reported recently for GnRH in experimental animals.     

Thyrotropin-releasing hormone and cyclo (His-Pro)-like immunoreactivities in the cerebrospinal fluids of 'normal' infants and adults, and patients with various neuropsychiatric and neurologic disorders

Levels of thyrotropin-releasing hormone (TRH) - and cyclo(His-Pro) (CHP)-like immunoreactivities and the activity of enzyme Pyroglutamate aminopeptidase (PAPase) were measured in cerebrospinal fluid (CSF) of over 100 normal adults (NA) and infants, and adult patients with various neurologic and neuropsychiatric disorders (NNDA). Levels of TRH and CHP in CSF of over 70% of the NA group were below 50 and 500 pg/ml respectively. The TRH- and CHP-like immunoreactivities in the remainder of the 30% of NA specimens exhibiting higher peptide concentrations were enzymatically and chromatographically characterized and were found to behave like authentic peptides. The levels of both of these peptides were significantly elevated in the CSF of most of the NNDA patients. An elevation in the CSF level of CHP was significantly correlated with the level of TRH, but not PAPase. Results from this study suggest that CSF elevation of TRH level may be due to a nonspecific response to stress that may be associated with hospitalization, myelogram procedure, and/or the neurologic and neuropsychiatric diseases for which the patients were admitted.     

Harnessing a high cargo-capacity transposon for genetic applications in vertebrates

Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.     

Epidermal growth factor induces tumour marker AKR1B10 expression through activator protein-1 signalling in hepatocellular carcinoma cells

AKR1B10 (aldo-keto reductase 1B10) is overexpressed in liver and lung cancer, and plays a critical role in tumour development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumour marker; however, little is known about the regulatory mechanism of AKR1B10 expression. The present study showed that AKR1B10 is induced by mitogen EGF (epidermal growth factor) and insulin through the AP-1 (activator protein-1) signalling pathway. In human HCC (hepatocellular carcinoma) cells (HepG2 and Hep3B), EGF (50?ng/ml) and insulin (10?nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at bp -222 to -212. Deletion or mutation of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that the AP-1 proteins c-Fos and c-Jun were the predominant factors bound to the AP-1 consensus sequence, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA (short hairpin RNA) and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Taken together, these results suggest that AKR1B10 is up-regulated by EGF and insulin through AP-1 mitogenic signalling and may be implicated in hepatocarcinogenesis.