Distinct transthyretin oxidation isoform profile in spinal fluid from patients with Alzheimer’s disease and mild cognitive impairment

Background

Transthyretin (TTR), an abundant protein in cerebrospinal fluid (CSF), contains a free, oxidation-prone cysteine residue that gives rise to TTR isoforms. These isoforms may reflect conditions in vivo. Since increased oxidative stress has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD) it is of interest to characterize CSF-TTR isoform distribution in AD patients and controls. Here, TTR isoforms are profiled directly from CSF by an optimized immunoaffinity-mass spectrometry method in 76 samples from patients with AD (n = 37), mild cognitive impairment (MCI, n = 17)), and normal pressure hydrocephalus (NPH, n = 15), as well as healthy controls (HC, n = 7). Fractions of three specific oxidative modifications (S-cysteinylation, S-cysteinylglycinylation, and S-glutathionylation) were quantitated relative to the total TTR protein. Results were correlated with diagnostic information and with levels of CSF AD biomarkers tau, phosphorylated tau, and amyloid β_1-42 peptide.

Results

Preliminary data highlighted the high risk of artifactual TTR modification due to ex vivo oxidation and thus the samples for this study were all collected using strict and uniform guidelines. The results show that TTR is significantly more modified on Cys(10) in the AD and MCI groups than in controls (NPH and HC) (p ≤ 0.0012). Furthermore, the NPH group, while having normal TTR isoform distribution, had significantly decreased amyloid β peptide but normal tau values. No obvious correlations between levels of routine CSF biomarkers for AD and the degree of TTR modification were found.

Conclusions

AD and MCI patients display a significantly higher fraction of oxidatively modified TTR in CSF than the control groups of NPH patients and HC. Quantitation of CSF-TTR isoforms thus may provide diagnostic information in patients with dementia symptoms but this should be explored in larger studies including prospective studies of MCI patients. The development of methods for simple, robust, and reproducible inhibition of in vitro oxidation during CSF sampling and sample handling is highly warranted. In addition to the diagnostic information the possibility of using TTR as a CSF oxymeter is of potential value in studies monitoring disease activity and developing new drugs for neurodegenerative diseases.     

Combined Use of the Canine Adenovirus-2 and DREADD-Technology to Activate Specific Neural Pathways In Vivo

We here describe a technique to transiently activate specific neural pathways in vivo. It comprises the combined use of a CRE-recombinase expressing canine adenovirus-2 (CAV-2) and an adeno-associated virus (AAV-hSyn-DIO-hM₃D(G_q)-mCherry) that contains the floxed inverted sequence of the designer receptor exclusively activated by designer drugs (DREADD) hM₃D(G_q)-mCherry. CAV-2 retrogradely infects projection neurons, which allowed us to specifically express hM₃D(G_q)-mCherry in neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens (Acb), the majority of which were dopaminergic. Activation of hM₃D(G_q)-mCherry by intraperitoneal (i.p.) injections of clozapine-N-oxide (CNO) leads to increases in neuronal activity, which enabled us to specifically activate VTA to Acb projection neurons. The VTA to Acb pathway is part of the mesolimbic dopamine system and has been implicated in behavioral activation and the exertion of effort. Injections of all doses of CNO led to increases in progressive ratio (PR) performance. The effect of the lowest dose of CNO was suppressed by administration of a DRD1-antagonist, suggesting that CNO-induced increases in PR-performance are at least in part mediated by DRD1-signaling. We hereby validate the combined use of CAV-2 and DREADD-technology to activate specific neural pathways and determine consequent changes in behaviorally relevant paradigms.     

Hes1 Increases the Invasion Ability of Colorectal Cancer Cells via the STAT3-MMP14 Pathway

The Notch pathway contributes to self-renewal of tumor-initiating cell and inhibition of normal colonic epithelial cell differentiation. Deregulated expression of Notch1 and Jagged1 is observed in colorectal cancer. Hairy/enhancer of split (HES) family, the most characterized targets of Notch, involved in the development of many cancers. In this study, we explored the role of Hes1 in the tumorigenesis of colorectal cancer. Knocking down Hes1 induced CRC cell senescence and decreased the invasion ability, whereas over-expression of Hes1 increased STAT3 phosphorylation activity and up-regulated MMP14 protein level. We further explored the expression of Hes1 in human colorectal cancer and found high Hes1 mRNA expression is associated with poor prognosis in CRC patients. These findings suggest that Hes1 regulates the invasion ability through the STAT3-MMP14 pathway in CRC cells and high Hes1 expression is a predictor of poor prognosis of CRC.     

Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo

Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.     

Determining acetylcholinesterase inhibitory activity in plant extracts using a fluorimetric flow assay

A fluorometric assay for acetylcholinesterase inhibitory activity was developed in a flow system using the fluorogenic substrate 7-acetoxy-1-methyl quinolinium iodide which is hydrolysed to the highly fluorescent 7-hydroxy-1-methyl quinolinium iodide. The detection limit of galanthamine is 0.5 microM, which is about 20 times more sensitive than in the colorimetric flow assay. In the presence of 30% methanol or of 5% acetonitrile, about 70% of the enzyme activity could still be detected. Various plant extracts have been screened using the described system including bulbs of Galanthus nivalis, Eucharis amazonica (E. x grandiflora), Crinum powelli and Nerine bowdenii (all members of the Amaryllidaceae), which showed strong AchE inhibitory activity.     

Amino acid sequence versus morphological data and the interordinal relationships of mammals

To a large extent, the mutual affinities of the mammalian orders continue to puzzle systematists, even though comparative anatomy and amino acid sequencing offer a massive data base from which these relationships could potentially be adduced. In the present paper the consistency index--the number of character states less the number of characters in a data set, divided by the total number of changes in the character states on a cladogram--was used to examine the relative resolving powers of recently published morphological and molecular- sequence data. Consistency indices were calculated for previously published alpha crystallin A chain and myoglobin amino acid-sequence cladograms and for four original amino acid-sequence cladograms (alpha crystallin A, myoglobin, and alpha and beta hemoglobin); these were found to be comparable to the consistency indices of morphologically based cladograms. Qualitative comparisons between the morphologically based and molecularly based trees were also made; only moderate congruence between the two was observed. Moreover, there was a general lack of congruence between the cladograms specified by each of the four proteins. Amino acid-sequence and morphological data agreed on the placement of edentates as an early eutherian offshoot and on the grouping of hyracoids, proboscideans, and sirenians. Otherwise there was only limited congruence: morphology strongly supported the grouping of lagomorphs and rodents and the alliance of pholidotes and edentates, but sequence analyses did not. The placement of tubulidentates differed widely among proteins. Morphology indicated the close association of sirenians with proboscideans; proteins suggested a pairing of sirenians with hyracoids. Sequence data did not identify many (morphologically well-diagnosed) orders as monophyletic (e.g., Lagomorpha).(ABSTRACT TRUNCATED AT 250 WORDS)      

Histological and histochemical study of the uropygial gland of chimango caracara (Milvago chimango vieillot, 1816)

The uropygial glands of birds are sebaceous organs that contribute to the water-repellent properties of the feather coat. We studied the histological and histochemical characteristics of the uropygial gland of chimango caracara using hematoxylin and eosin (H & E), Gomori´s trichrome, orcein, Gomori´s reticulin, periodic acid-Schiff (PAS), Alcian blue (AB) and a variety of lectins. The gland is composed of two lobes and a papilla with 20 downy feathers. It is surrounded by a capsule of dense connective tissue that contains elastic, reticular and smooth muscle fibers. The papilla is delicate and has two excretory ducts. The gland mass relative to body mass was 0.143%. Both adenomer cells and their secretions were stained with Sudan IV, PAS and AB, and were positive for numerous lectins that indicated the presence of lipids and carbohydrates. Immunohistochemical techniques to detect PCNA confirmed cell proliferation in the basal stratum of the adenomer cells. The lipids and glycoconjugates secreted by the uropygial gland serve numerous functions including protection against microorganisms.     

Effect of Tetracosactid on Post Partum Cyclicity in Cows after Induction of Parturition with PGF2α

Parturition and retention of fetal membranes were induced with PGF_2α in 3 primiparous dairy cows. Starting on day 12 post partum (PP) the cows were treated with 500 μ g i.m. of ACTH-analogue (tetracosactid) every 6 h for 6 times. Changes in plasma concentrations of cortisol, progesterone and 15-ketodihydro-PGF_2α were evaluated immediately after treatment. The effects on the resumption of ovarian activity were evaluated by clinical and ultrasound examinations and by progesterone and 15-ketodihydro-PGF_2α analyses for 56 days after parturition. Treatment was able to induce a statistically significant (p < 0.01) similar increase in cortisol and progesterone after both the 1^st and the 6^th injections, in all cows. No changes in 15-ketodihydro-PGF_2αconcentrations were seen after any of the injections of ACTH-analogue. The first corpus luteum (CL) was seen on day 18 PP (cow A), and 28 (cow B) and in both cases it was followed by a normal ovarian cyclicity. No CL was observed during the whole period of study in cow C. Progesterone profiles confirmed these clinical and ultrasonographic findings. The steroid output, especially progesterone, induced by the ACTH-analogue might be a stimulus for the onset of ovarian cyclicity, since 2 of the 3 animals ovulated earlier than expected. These findings point to the fact that interference with the stress system might have a positive effect on ovarian cyclicity. The different pattern of response does however demand further studies.     

Tumor‐associated macrophages (TAMs) depend on ZEB1 for their cancer‐promoting roles

Accumulation of tumor‐associated macrophages (TAMs) associates with malignant progression in cancer. However, the mechanisms that drive the pro‐tumor functions of TAMs are not fully understood. ZEB1 is best known for driving an epithelial‐to‐mesenchymal transition (EMT) in cancer cells to promote tumor progression. However, a role for ZEB1 in macrophages and TAMs has not been studied. Here we describe that TAMs require ZEB1 for their tumor‐promoting and chemotherapy resistance functions in a mouse model of ovarian cancer. Only TAMs that expressed full levels of Zeb1 accelerated tumor growth. Mechanistically, ZEB1 expression in TAMs induced their polarization toward an F4/80^low pro‐tumor phenotype, including direct activation of Ccr2. In turn, expression of ZEB1 by TAMs induced Ccl2, Cd74, and a mesenchymal/stem‐like phenotype in cancer cells. In human ovarian carcinomas, TAM infiltration and CCR2 expression correlated with ZEB1 in tumor cells, where along with CCL2 and CD74 determined poorer prognosis. Importantly, ZEB1 in TAMs was a factor of poorer survival in human ovarian carcinomas. These data establish ZEB1 as a key factor in the tumor microenvironment and for maintaining TAMs’ tumor‐promoting functions.