Sex-specific development of cell-mediated immunity under experimentally altered rearing conditions in blue tit nestlings

In birds, poor rearing conditions usually have negative effects on T-cell-mediated immune response. However, earlier studies demonstrate that fitness-related traits such as body mass may show sex-specific patterns when subject to alteration of rearing conditions. Therefore, to investigate whether deterioration of rearing conditions influences the development of immune function differently in male and female nestlings, we performed brood size manipulation experiments on blue tit (Parus caeruleus) nestlings. To alter rearing conditions, some broods were increased by three nestlings soon after hatching, while other broods were left non-manipulated. Immune response was assessed as a hypersensitivity reaction to phytohaemagglutinin in 11-day-old nestlings. Additionally, we studied the consequences of brood size manipulation for fledgling body mass and tarsus length. The enlargement of brood size had different effects on the cellular immune responses of male and female nestlings, with males being more negatively affected than their female nest-mates. Sex-specific effects of poor rearing conditions were also recorded for tarsus length, such that tarsus growth was more retarded in female than in male nestlings. We discuss the effects of deterioration of rearing conditions on sex-specific development of cell-mediated immunity with respect to sexual dimorphism of size and developmental strategies in male and female nestlings.     

Detection of Methylated SEPT9 in Plasma Is a Reliable Screening Method for Both Left- and Right-Sided Colon Cancers

Background

Methylated Septin 9 (SEPT9) is a sensitive biomarker for colorectal cancer (CRC) from peripheral blood. However, its relationship to cancer localization, guaiac-based fecal occult blood test (gFOBT) and carcinoembryonic antigen (CEA) have not been described.

Methodology/Principal Findings

Plasma samples were collected for SEPT9 analysis from patients with no evidence of disease (NED) (n = 92) before colonoscopy and CRC (n = 92) before surgical treatment. DNA was isolated and bisulfite-converted using Epi proColon kit 2.0. Qualitative determination was performed using Epi proColon 2.0 RT-PCR assay. Samples for gFOBT and CEA analysis were collected from NED (n = 17 and 27, respectively) and CRC (n = 22 and 27, respectively). SEPT9 test was positive in 15.2% (14/92) of NED and 95.6% (88/92) of CRC, including 100% (67/67) from stage II to stage IV CRC and 84% (21/25) of stage I CRC when a sample was called positive if 1 out of 3 PCR replicates was positive. In a second analysis (2 out of 3 PCR replicates) specificity improved to 99% (91/92) of NEDs, at a sensitivity of 79.3% (73/92) of SEPT9 positives in CRC. gFOBT was positive in 29.4% (5/17) of NED and 68.2% (15/22) of CRC and elevated CEA levels were detected in 14.8% (4/27) of NED and 51.8% (14/27) of CRC. Both SEPT9 (84.8%) and CEA (85.2%) showed higher specificity than gFOBT (70.6%). SEPT9 was positive in 96.4% (54/56) of left-sided colon cancer (LSCC) cases and 94.4% (34/36) of right-sided colon cancer (RSCC) cases. gFOBT was positive in 83.3% (10/12) of cases with LSCC and 50% (5/10) of cases with RSCC, elevated CEA was detected 60% (9/15) of LSCC and 41.7% (5/12) of RSCC.

Conclusions/Significance

The high degree of sensitivity and specificity of SEPT9 in plasma makes it a better method to detect CRC than gFOBT and CEA, even for the more difficult to detect RSCC.     

Anticonvulsant Activity of Pterostilbene in Zebrafish and Mouse Acute Seizure Tests

Pterostilbene (PTE), a natural dimethylated analog of resveratrol, possesses numerous health-beneficial properties. The ability of PTE to cross the blood–brain barrier raised the possibility that this compound may modulate central nervous system functions, including seizure activity. The aim of our study was to investigate the activity of PTE in the larval zebrafish pentylenetetrazole (PTZ) seizure assay and three acute seizure tests in mice, i.e., in the maximal electroshock seizure threshold (MEST), 6 Hz-induced psychomotor seizure threshold and intravenous (iv) PTZ tests. Additionally, potential antidepressant activity of PTE was estimated in the forced swim test in mice. The chimney test was used to determine the influence of PTE on motor coordination in mice, while its influence on neuromuscular strength was assessed in the grip strength test in mice. Locomotor activity was determined to verify the results from the forced swim test. PTE revealed an evident anticonvulsant effect both in zebrafish larvae (10 µM; 2 h-incubation) and mice (at doses of 100 and 200 mg/kg, intraperitoneally) but it did not exhibit antidepressant potential in the forced swim test. Furthermore, it did not cause any statistically significant changes in motor coordination, neuromuscular strength and locomotor activity in mice. In conclusion, our present findings demonstrate for the first time the anticonvulsant potential of PTE. The aforementioned results suggest that it might be employed in epilepsy treatment, however, further precise studies are required to verify its activity in other experimental seizure and epilepsy models and its precise mechanism of action should be determined.

     

Effects of amlodipine, diltiazem, and verapamil on the anticonvulsant action of topiramate against maximal electroshock-induced seizures in mice

To assess the effect of 3 calcium channel antagonists (amlodipine, diltiazem, and verapamil) on the anticonvulsant action of topiramate (a new generation antiepileptic drug) in the mouse maximal electroshock seizure (MES) model. Amlodipine (20 mg/kg) significantly enhanced the anticonvulsant activity of topiramate in the MES test in mice, reducing its ED50 value from 54.83 to 33.10 mg/kg (p < 0.05). Similarly, diltiazem (5 and 10 mg/kg) markedly potentiated the antiseizure action of topiramate against MES, lowering its ED50 value from 54.83 to 32.48 mg/kg (p < 0.05) and 28.68 mg/kg (p < 0.01), respectively. In contrast, lower doses of amlodipine (5 and 10 mg/kg) and diltiazem (2.5 mg/kg) and all doses of verapamil (5, 10, and 20 mg/kg) had no significant impact on the antiseizure action of topiramate. Pharmacokinetic verification of the interaction of topiramate with amlodipine and diltiazem revealed that neither amlodipine nor diltiazem affected total brain topiramate concentration in experimental animals, and thus, the observed interactions were concluded to be pharmacodynamic in nature. The favorable combinations of topiramate with amlodipine or diltiazem deserve more attention from a clinical viewpoint because the enhanced antiseizure action of topiramate was not associated with any pharmacokinetic changes in total brain topiramate concentration.     

The Use of Contact Mode Atomic Force Microscopy in Aqueous Medium for Structural Analysis of Spinach Photosynthetic Complexes

To investigate the dynamics of photosynthetic pigment-protein complexes in vascular plants at high resolution in an aqueous environment, membrane-protruding oxygen-evolving complexes (OECs) associated with photosystem II (PSII) on spinach (Spinacia oleracea) grana membranes were examined using contact mode atomic force microscopy. This study represents, to our knowledge, the first use of atomic force microscopy to distinguish the putative large extrinsic loop of Photosystem II CP47 reaction center protein (CP47) from the putative oxygen-evolving enhancer proteins 1, 2, and 3 (PsbO, PsbP, and PsbQ) and large extrinsic loop of Photosystem II CP43 reaction center protein (CP43) in the PSII-OEC extrinsic domains of grana membranes under conditions resulting in the disordered arrangement of PSII-OEC particles. Moreover, we observed uncharacterized membrane particles that, based on their physical characteristics and electrophoretic analysis of the polypeptides associated with the grana samples, are hypothesized to be a domain of photosystem I that protrudes from the stromal face of single thylakoid bilayers. Our results are interpreted in the context of the results of others that were obtained using cryo-electron microscopy (and single particle analysis), negative staining and freeze-fracture electron microscopy, as well as previous atomic force microscopy studies.Oxygenic photosynthesis supports most life on Earth through the absorption of solar energy, which powers the extraction of electrons from water and the subsequent use of those electrons to convert CO₂ into organic compounds (Nelson and Ben-Shem, 2004; Merchant and Sawaya, 2005; Nelson, 2011). The light-dependent reactions of photosynthesis occur within photosynthetic or thylakoid membranes and are catalyzed by two reaction centers, PSI and PSII. Both photosystems have associated light-harvesting complexes (LHCI and LHCII) that act as antenna to efficiently capture light energy. The oxygen-evolving complex (OEC) is an integral component of PSII, catalyzing the extraction of electrons from water. The two photosystems are connected through an intersystem electron transport chain that includes the hydrophobic electron carrier plastoquinone, the membrane-bound cytochrome b₆f complex (cyt b₆f), and the mobile electron carrier plastocyanin. The electrochemical gradient generated during light-driven electron flow is used in the synthesis of ATP by the ATP synthase complex. Components of the photosynthetic apparatus vary among photosynthetic organisms and under different environmental conditions, especially for proteins associated with light-harvesting complexes (Liu and Scheuring, 2013). However, investigations of the mechanisms associated with the dynamic acclimation of photosynthetic electron transport and light harvesting to environmental cues require real-time observations that are difficult to achieve because of limitations in our ability to view such changes (e.g. difficulties in tagging proteins with fluorophores and resolving fluorescent images; Zaks et al., 2013).In vascular plants, thylakoid membranes form a network of interconnected tubular structures enclosing a lumenal space. This membrane system can be divided into two morphologically distinct regions: the grana, which are formed by stacks of appressed membranes; and the stroma lamellae, which are unappressed membranes that form connections between grana stacks. These distinct thylakoid regions are enriched for specific photosynthetic complexes. The major complexes in grana are PSII and LHCII, which can interact and form a variety of PSII-LHCII supercomplexes (Dekker and Boekema, 2005; Kouřil et al., 2012), as well as cyt b₆f (Johnson et al., 2014). Grana stacks are also the site of water oxidation and oxygen evolution; the Mn₄CaO₅ cluster is the PSII cofactor that catalyzes this process (Umena et al., 2011). This cluster resides between the transmembrane subunits of the PSII core (formed by PSII proteins D1 and D2 and their associated pigment cofactors, with PSII reaction center proteins CP43 and CP47, α- and β-subunits of cytochrome b559, and PSII reaction center protein I [PsbI]) and the lumenal, peripheral membrane proteins of the OEC. The OEC is composed of extrinsic membrane polypeptides of 33 kD, 23 kD, and 17 kD, designated oxygen-evolving enhancer protein 1, 2, and 3 (PsbO, PsbP, and PsbQ) that protrude into the thylakoid lumen in vascular and nonvascular plants as well as in green algae. Cyanobacteria also have PsbO, with PsbV and PsbU serving as functional analogs of plant PsbP and PsbQ, respectively (Dekker and Boekema, 2005). Based on removal/reconstitution experiments, these subunits have been shown to be critical for PSII stability and oxygen evolution activity (Kuwabara and Murata, 1983; Ljungberg et al., 1983; Ghanotakis et al., 1984). They may also impact the association of Ca²⁺ and Cl⁻ with PSII, the polypeptide conformation around the manganese cluster, and the formation of channels within PSII that allow access of water to the catalytic site and the exit of protons from the complex as water is oxidized (Bricker et al., 2012).The x-ray crystal structures of purified PsbP (Kohoutová et al., 2009) and PsbQ (Balsera et al., 2005) from spinach (Spinacia oleracea) have been determined. For cyanobacteria, PSII crystals have been used to establish high-resolution structures for PsbO, PsbV, and PsbU (Umena et al., 2011), with more recent analyses at room temperature (Kern et al., 2013). To study the bound state of these peripheral proteins in spinach, electron density maps were established based on cryo-electron microscopy (cryo-EM) and single particle analysis of purified PSII-LHCII supercomplexes, with structural verification based on the removal of extrinsic polypeptides of the complexes from the membranes (Nield et al., 2002). In these structures, determined at less than 2 nm (or 20 Å) resolution, the PsbP and PsbQ subunits of OEC were assigned to a single membrane protrusion, with a second membrane protrusion assigned to PsbO (Boekema et al., 2000a); these topological structures are the most prominent protruding features of the reaction center-containing membrane protein complexes. Since two PSII reaction centers associate to form a dimeric PSII-LHCII supercomplex (Bumba and Vácha, 2003), the six OEC subunits (two PsbO, two PsbP, and two PsbQ) are visualized as four protrusions associated with each supercomplex. However, after the cyanobacterial PSII core structure was solved (including the positions of the extrinsic subunits) and aligned to the cryo-EM of PSII-LHCII supercomplexes (Nield and Barber, 2006), the structure was reevaluated. The PSII lumenal small protruding mass was assigned to the large extrinsic loop of CP47 (encoded by psbB), while the larger protrusion was assigned to PsbO, PsbP, PsbQ, and the large extrinsic loop associated with CP43 (encoded by psbC).Attempts have been made to visualize PSII complexes and proteins in their native membrane environment using transmission electron microscopy (TEM) in conjunction with freeze fracture (Johnson et al., 2011) and negative staining of grana membranes from both spinach (Boekema et al., 2000b) and Arabidopsis (Arabidopsis thaliana; Betterle et al., 2009; Wientjes et al., 2013). These techniques provide little information regarding the extent of the protrusion of the polypeptide subunits out of the plane of the membranes. Even though cryo-electron tomography of isolated chloroplasts and plunge-frozen thylakoid membranes (Daum et al., 2010) and grana stacks (Kouřil et al., 2011) can preserve sample hydration using a vitrification process during freezing, it is difficult to determine the height of the extrinsic thylakoid protein protrusions from the membrane surface. Furthermore, at the resolution obtained with these techniques, the small and large protrusions of each PSII monomer may appear merged into a single structure. This would result in the visualization of only two distinguishable topological entities for each PSII-OEC dimer.The generation of images by atomic force microscopy (AFM), which involves raster scanning by a sharp tip that is in contact with the sample, complements other structural determination methods. The vertical position of the tip is controlled in order to maintain a constant imaging force (balancing interaction forces between the tip and the scanned structure). Control is implemented by a feedback loop that continuously monitors the force with a highly sensitive force sensor that activates a high-precision actuator. Logging the vertical position of the piezoelectric actuator that controls the vertical position of the tip can provide particle height relative to the membrane at high vertical resolution; this is done concurrently with logging the lateral position of each pixel to generate the image (Bippes and Muller, 2011). Probing samples with AFM in air has been employed to image spinach grana membranes (Kirchhoff et al., 2008) to elucidate the arrangement of Arabidopsis PSII-LHCII supercomplexes associated with nonphotochemical quenching (Onoa et al., 2014) and to determine the areal density of Arabidopsis PSII-OEC during the PSII repair cycle (Puthiyaveetil et al., 2014).Most AFM studies of grana have been performed with membrane surfaces exposed to air. This raises issues concerning the extent to which membrane properties are altered during measurements in a nonaqueous environment (Zaks et al., 2013), where it may be impossible to maintain appropriate hydration and ionic conditions. However, AFM has also been used in aqueous medium to establish high-resolution topography images of membrane proteins (Bippes and Muller, 2011) and specifically to characterize the PSII-OEC, which was previously observed as an ordered array within spinach grana membranes (Sznee et al., 2011). In recent studies, a map of the lumenal surface of grana membranes was generated in aqueous medium that distinguishes cyt b₆f dimers from PSII-OEC (Johnson et al., 2014). However, the potential of AFM imaging in a liquid environment has not been realized for the high-resolution analysis of features associated with thylakoid membranes and the PSII-OEC dimer. We used contact mode atomic force microscopy (CM-AFM) to (1) image PSII-OEC topology in liquid medium at high resolution, (2) identify other features/particles associated with grana membranes, and (3) optimize the use of AFM for monitoring the dynamics of thylakoid membrane complexes as the conditions of the environment are modulated (e.g. light, specific ions, and temperature).     

Elevated yolk androgen levels benefit offspring development in a between-clutch context

The field of androgen deposition in avian eggs and its consequencesfor offspring development has received a lot of attention inrecent research. However, although variation within clutchesin yolk androgens is relatively well understood, the adaptivesignificance of patterns of variation between clutches remainsrather unclear. Furthermore, it has been hypothesized that yolkandrogens act as a means of an adaptive maternal effect to adjustoffspring to a given posthatching environment. Thus, the consequencesof maternal yolk androgens for offspring development are likelyto depend on the specific environment of a given brood. We experimentallymanipulated yolk androgen concentrations in spotless starlingeggs, using a between-brood design in which full broods weremanipulated applying either an androgen or sham treatment, inorder to test the effects of between-brood variation in yolkandrogen levels. We also included in the analysis several femalecharacters that have been shown to affect androgen deposition.Androgen-chicks tended to gain more mass, a similar effect tothat shown in previous studies where direct competition betweenchicks belonging to different treatments was allowed, but didnot gain a survival benefit. Androgen-chicks had wider beakflanges, an effect that has not been described previously andcould play an important role in food acquisition. In addition,androgen-chicks had higher endogenous plasma levels of androgens,which could induce higher begging intensity. We hypothesizethat these effects are an important mechanistic link for ourunderstanding of how yolk androgens exert their effects on offspringdevelopment after hatching. Contrary to our predictions, wefound no evidence that the observed effects depended on theenvironment under which it was investigated. Because high yolkandrogen levels seem to be beneficial for nestlings in thisspecies, we hypothesize that yolk hormone deposition could becostly for females or alternatively that potential negativeeffects later in adulthood may constrain maternal hormone allocation.     

The effect of different doses of cisplatin on the pharmacokinetic parameters of cefepime in mice

The simulation of human serum levels is essential in animal models to extrapolate the experimental results to clinical practice. Administration of a nephrotoxic drug such as cisplatin can be used to cause renal dysfunction as an approach to mimic human serum levels of renally excreted drugs. We aimed to determine the dose of cisplatin that did not affect the survival rate of mice and to achieve human-like serum concentrations of cefepime. Different doses of cisplatin (0, 10, 14, 18, 22 and 26 mg/kg) were given by intraperitoneal (i.p.) injection to mice three days prior to the i.p. administration of 80 mg/kg cefepime. With cisplatin doses of 18 and 22 mg/kg, the half-life of cefepime was significantly prolonged (P < 0.001) and all mice survived. The pretreatment with 26 mg/kg cisplatin significantly decreased survival (P = 0.001), but the half-life of cefepime was not significantly longer than of 18 mg/kg cisplatin. Serum levels of cefepime after the pretreatment with 18 mg/kg cisplatin were comparable to published human data. The administration of cisplatin appears to be a suitable method in mice for simulating human serum concentrations of renally excreted drugs.     

3,6-Diazaphenothiazines as potential lead molecules – synthesis,characterization and anticancer activity

3,6-Diazaphenothiazines were obtained in cyclization of 3-amino-3′-nitro-2,4′-dipyridinyl sulfide and the reaction of sodium 3-amino-2-pyridinethiolate with 4-chloro-3-nitropyridine followed by alkylation and heteroarylation. The thiazine ring formation ran via the Smiles rearrangement. The structure elucidation was based on 2D NMR and X-ray analysis of N-methylated product. 3,6-Diazaphenothiazines were investigated for antitumor activity using glioblastoma SNB-19, melanoma C-32 and breast cancer MCF-7 cells. 10H-3,6-diazaphenothiazine was 10 times more active (IC_50?<_?0.72?μg/mL) than cisplatin. Two diazaphenothiazines with the 2-pyrimidinyl and dimethylaminopropyl substituents were selectively active against MCF-7 and C-32 cells. The expressions of H3 (proliferation marker), TP53, CDKN1A (cell cycle regulators), BAX and BCL-2 (proapoptopic and antiapoptopic genes) were detected by RT-QPCR method. The expression analysis suggests the cell cycle arrest and the mitochondrial apoptosis pathway activation in MCF-7 and SNB-19 cells.     

Filamentous Aggregation of Sequestosome-1/p62 in Brain Neurons and Neuroepithelial Cells upon <Emphasis Type="Italic">Tyr-Cre</Emphasis>-Mediated Deletion of the Autophagy Gene <Emphasis Type="Italic">Atg7</Emphasis>

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7^f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7^f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7^f/f Tyr-Cre mice relative to Atg7^f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7^f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7^f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells.     

Development and application of a next-generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases

Background

Inherited retinal disorders are clinically and genetically heterogeneous with more than 150 gene defects accounting for the diversity of disease phenotypes. So far, mutation detection was mainly performed by APEX technology and direct Sanger sequencing of known genes. However, these methods are time consuming, expensive and unable to provide a result if the patient carries a new gene mutation. In addition, multiplicity of phenotypes associated with the same gene defect may be overlooked.

Methods

To overcome these challenges, we designed an exon sequencing array to target 254 known and candidate genes using Agilent capture. Subsequently, 20 DNA samples from 17 different families, including four patients with known mutations were sequenced using Illumina Genome Analyzer IIx next-generation-sequencing (NGS) platform. Different filtering approaches were applied to identify the genetic defect. The most likely disease causing variants were analyzed by Sanger sequencing. Co-segregation and sequencing analysis of control samples validated the pathogenicity of the observed variants.

Results

The phenotype of the patients included retinitis pigmentosa, congenital stationary night blindness, Best disease, early-onset cone dystrophy and Stargardt disease. In three of four control samples with known genotypes NGS detected the expected mutations. Three known and five novel mutations were identified in NR2E3, PRPF3, EYS, PRPF8, CRB1, TRPM1 and CACNA1F. One of the control samples with a known genotype belongs to a family with two clinical phenotypes (Best and CSNB), where a novel mutation was identified for CSNB. In six families the disease associated mutations were not found, indicating that novel gene defects remain to be identified.

Conclusions

In summary, this unbiased and time-efficient NGS approach allowed mutation detection in 75% of control cases and in 57% of test cases. Furthermore, it has the possibility of associating known gene defects with novel phenotypes and mode of inheritance.