Experimental Study on Differences in Clivus Chordoma Bone Invasion: An iTRAQ-Based Quantitative Proteomic Analysis

Although a bone tumor, significant differences in the extent of bone invasion exist in skull base chordoma, which directly affect the extent of surgical resection, and have an impact on its prognosis. However, the underlying mechanism of the phenomenon is not clearly understood. Therefore, we used an iTRAQ-based quantitative proteomics strategy to identify potential molecular signatures, and to find predictive markers of discrepancy in bone invasion of clivus chordoma. According to bone invasive classification criteria, 35 specimens of clivus chordoma were calssified to be either endophytic type (Type I) or exophytic type (Type II). An initial screening of six specimens of endophytic type and six of exophytic was performed, and 250 differentially expressed proteins were identified. Through the GO and IPA analysis, we found evidence that the expression of inflammatory activity-associated proteins up-regulated in endophytic type, whereas the expression of cell motility-associated proteins up-regulated in exophytic ones. Moreover, TGFβ1 and mTOR signal pathway seemed to be related with bone invasion. Thus, TGFβ1, PI3K, Akt, mTOR, and PTEN were validated in the following 23 samples by immune histochemistry and Western blot. The expression levels of TGFβ1 and PTEN were significantly lower in the endophytic type than in the exophytic ones. It was found that TGFβ1 may play an important role in its bone invasion. The mechanisms may be related with conducting an increased inflammatory cell response and a decline in cytoskeletal protein expression. PTEN is confirmed to be associated with the degree of bone invasion. The PI3K/AKT/mTOR signaling pathway might be associated with the bone invasion, but still needs a larger sample size to be verified These results, for the first time, not only demonstrate the biological changes that occur in different growth patterns from the perspective of proteomics, but also provide novel markers that may help to reveal the mechanisms behind clivus chordomas.     

中国红原鸡和泰国红原鸡遗传多样性分析

利用29个微卫星DNA标记对来自中国的红原鸡Gallus gallus spadiceus亚种和来自泰国的红原鸡Gallus gallus gallus亚种进行遗传多样性分析, 评估亚种内的遗传变异和亚种间的遗传分化, 结果表明: 共检测到168个等位基因, 每个位点的等位基因数从2到13不等, 所有位点平均的期望杂合度和PIC值分别为0.5780和0.53。中国和泰国红原鸡29个微卫星位点平均有效等位基因数分别为3.79和4.79, 平均基因杂合度为0.5379和0.6385, 两个红原鸡亚种均表现出较高的群体杂合度和丰富的遗传多样性。群体分化系数为19.4%(P<0.01), 两个红原鸡亚种间的Reynolds’遗传距离和Nm值分别为0.157和1.040。由此可见, Gallus gallus spadiceus亚种和Gallus gallus gallus亚种群体具有不同的群体遗传结构, 群体之间存在明显的遗传分化, 并不能将其认定为是同一亚种, 这也为中国家鸡具有独立的起源提供了一定的佐证。     

Quantitative genetics of ontogeny of sexual dimorphism in red junglefowl (Gallus gallus)

We studied phenotypic patterns and underlying quantitative genetics of development of sexual size dimorphism in red junglefowl (Gallus gallus). Using a multigenerational pedigree and the 'animal model' technique, we found significant heritability for many of the size and growth-related traits we examined, as well as significant genetic correlations among them. Despite sexual size dimorphism throughout posthatching ontogeny, the genetic correlation between males and females for all size measurements and growth parameters remained high. Significant positive phenotypic and genetic correlations between the fastest rate of growth and mass at week 26 (near asymptote) indicate that faster growth when young promotes larger adult size. However, age at which peak growth is reached does not appear to be phenotypically or genetically correlated with adult size. Positive genetic correlations within traits among ages were common, demonstrating that the genetic variance important to growth is relatively consistent among ages. However, male mass and tarsus length showed no genetic correlation between week 0 values and those from later ages. The body size traits of mass and tarsus length were genetically correlated with each other in females, but this pattern was not significant in males. Thus, despite striking sexual dimorphism in size and growth trajectories, size dimorphic traits in junglefowl show, with some exceptions, genetic integration between the sexes, among ages, and between traits.     

Parameters of the chicken genome (Gallus gallus)

As more information on the chicken genome is gathered, it is becoming increasingly more important to be able to correlate genetic and physical maps. Quantitation of the chicken karyotype is important in establishing parameters which define the genome. Here we report on the physical lengths of the chicken macrochromosomes and establish the DNA content of each, thus identifying implicitly how much of the genome is represented by the microchromosomal component. For the first time, genetic and physical data on the chicken karyotype are presented in relation to one another.     

Analysis of chick (Gallus gallus) middle ear columella formation

Background

The chick middle ear bone, the columella, provides an accessible model in which to study the tissue and molecular interactions necessary for induction and patterning of the columella, as well as associated multiple aspects of endochondral ossification. These include mesenchymal condensation, chondrogenesis, ossification of the medial footplate and shaft, and joint formation between the persistent cartilage of the extracolumella and ossified columella. Middle and external ear defects are responsible for approximately 10% of congenital hearing defects. Thus, understanding the morphogenesis and the molecular mechanisms of the formation of the middle ear is important to understanding normal and abnormal development of this essential component of the hearing apparatus.

Results

The columella, which arises from proximal ectomesenchyme of the second pharyngeal arch, is induced and patterned in a dynamic multi-step process. From the footplate, which inserts into the inner ear oval window, the shaft spans the pneumatic middle ear cavity, and the extracolumella inserts into the tympanic membrane. Through marker gene and immunolabeling analysis, we have determined the onset of each stage in the columella's development, from condensation to ossification. Significantly, a single condensation with the putative shaft and extracolumella arms already distinguishable is observed shortly before initiation of five separate chondrogenic centers within these structures. Ossification begins later, with periosteum formation in the shaft and, unexpectedly, a separate periosteum in the footplate.

Conclusions

The data presented in this study document the spatiotemporal events leading to morphogenesis of the columella and middle ear structures and provide the first gene expression data for this region. These data identify candidate genes and facilitate future functional studies and elucidation of the molecular mechanisms of columella formation.     

Proteome changes underpin improved meat quality and yield of chickens (Gallus gallus) fed the probiotic Enterococcus faecium

Background

Supplementation of broiler chicken diets with probiotics may improve carcass characteristics and meat quality. However, the underlying molecular mechanism remains unclear. In the present study, 2D-DIGE-based proteomics was employed to investigate the proteome changes associated with improved carcass traits and meat quality of Arbor Acres broilers (Gallus gallus) fed the probiotic Enterococcus faecium.

Results

The probiotic significantly increased meat colour, water holding capacity and pH of pectoral muscle but decreased abdominal fat content. These meat quality changes were related to the altered abundance of 22 proteins in the pectoral muscle following E. faecium feeding. Of these, 17 proteins have central roles in regulating meat quality due to their biological interaction network. Altered cytoskeletal and chaperon protein expression also contribute to improved water holding capacity and colour of meat, which suggests that upregulation of chaperon proteins maintains cell integrity and prevents moisture loss by enhancing folding and recovery of the membrane and cytoskeletal proteins. The down-regulation of β-enolase and pyruvate kinase muscle isozymes suggests roles in increasing the pH of meat by decreasing the production of lactic acid. The validity of the proteomics results was further confirmed by qPCR.

Conclusions

This study reveals that improved meat quality of broilers fed probiotics is triggered by proteome alterations (especially the glycolytic proteins), and provides a new insight into the mechanism by which probiotics improve poultry production.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1167) contains supplementary material, which is available to authorized users.     

Neurogenic inflammation in the chicken (Gallus gallus var domesticus)

1. Neurogenic inflammation has been studied in the anaesthetized adult hen using a variety of different stimuli.2. Plasma extravasation was produced following antidromic stimulation of the external mandibular ramus of the trigeminal nerve which innervates the skin at the angle of the jaw and the anterior part of the wattle.3. Stimulation of the wattle by external application of mustard oil, thermal and mechanical stimuli, as well as intradermal injection of substance P and bradykinin, all produced plasma extravasation.4. These results demonstrate that, in contrast to previous findings in the pigeon, at least in the trigeminal of the chicken peripheral C-fibre nociceptors have similar physiological characteristics in relation to the neurogenic inflammatory mechanism to those seen in mammals.     

Development of drinking mechanisms in the chicken (Gallus gallus) (Aves)

Summary Development of the drinking mechanism from the day of hatching to the 4 week old chick was studied in relation to the growth of the oropharynx and drinking performance. Results indicate that due to scaling effects in the growing oropharynx, physical factors require the development of extra mechanisms. The swallowing mechanism to transport water from the pharynx into the esophagus during the tip up phase is already present in hatchlings, and does not change during ontogeny. The mechanism for water intake during the immersion phase changes considerably. Hatchlings display pecking behavior towards water. Week old chicks protrude the tongue far out, and the total volume ingested is sucked up by capillary action between the tongue, the floor, and the roof of the oropharynx. In 2 and 3 week old chicks the relative distance over which water is sucked up by capillarity decreases, whereas the volume of water pushed caudad by the tongue and bills increases. Laryngeal suction also develops in this period. At the age of 4 weeks chickens use the adult mechanism to drink. Most oropharyngeal elements increase two to three times in length and width during post hatching ontogeny. Due to these increases, water transport by capillarity alone does not suffice in the older age groups; therefore additional mechanisms such as pressure development by closing the bills and laryngeal suction must develop. Relative to the oropharyngeal length most elements grow isometrically. The width of the lingual alae and the larynx width, however, increase with positive allometry, which improves both capillary action as well as the transport of water during the tip up phase when the chicks grow older. The need to develop extra water transport mechanisms during growth due to scaling effects is shown to condition the radiation capacity of avian drinking mechanisms. Nidicolous birds are, however, not constrained by the need to have a properly operating drinking mechanism during the early ontogeny and may therefore have a better chance of developing alternativ drinking mechanisms then nidifugous birds.     

Morphology of congenital microphthalmia in chicks (Gallus gallus)

This study examines the morphology of sporadic congenital microphthalmia in 1-day-old chicks, with particular emphasis on the neural retina. On the basis of the size of the eyeball it is possible to classify microphthalmia into two groups, severe and mild. In severe microphthalmia (less than 5 mm in equatorial diameter), the eyeball is severely malformed, but in most cases it shows evidence of an organized neural retina. Although ganglion cells and an optic nerve head are present in a small proportion of these retinae, we could not trace an optic nerve projection to the brain. These results indicate that some ganglion cells are able to be sustained after the period of naturally occurring cell death, suggesting either that those ganglion cells have established some contact with the central nervous system or that the presence of their axons in a rudimentary optic nerve is adequate for survival. In mild microphthalmia (greater than 5 mm in equatorial diameter), the most consistent abnormality is a defect in the pecten, which together with other abnormalities such as orbital cysts and colobomas indicates that the major abnormality occurs in the region of the choroid fissure. Associated with these defects are abnormalities within the ganglion cell layer. In some cases the number of ganglion cells was reduced, and in others the numbers of both ganglion and displaced amacrine cells were reduced. Unexpectedly, there were localized regions completely devoid of cells in the ganglion cell layer. The timing of the congenital defect may provide some clue as to the presence of a critical period in which displaced amacrine cells are formed or are sensitive to events related to ganglion cell loss.     

Ontogeny of magnetoreception in chickens (Gallus gallus domesticus)

Chickens are precocial birds, capable of moving independently from within a few hours of hatching, and thus provide a useful model to investigate the development of magnetoreception in young birds. Chicks show some sharply-timed behavioural changes at around 11 days of age that may be indicative of improvements in navigation ability around this age. We trained Young (<10 days, n = 7) and Old (11 days or older, n = 6) chicks to find a hidden imprinting stimulus behind one of four screens in a square arena. Once criterion was reached, the directional choices of chicks were recorded in unrewarded tests in the geomagnetic field and in an experimental field shifted by 90° clockwise. These tests were separated with rewarded training trials to avoid extinction. In the first, and the first 3 tests, no indication was found that chicks chose the correct unimodal magnetic direction. Instead, in all tests, Old chicks preferred the screens in the same magnetic axis as the training direction in tests in the geomagnetic and shifted experimental fields significantly more than Young chicks (P < 0.05). Choices of Young chicks were no different to chance in tests, whereas choices by Old chicks were significantly different from chance (P < 0.05). Our findings support the hypothesis that magnetoreception appears in the second week of life in the chicken. It is, however, unclear when a truly directional magnetic response (i.e. an unimodal, rather than axial, response in our test) develops that could be used by the chicks for accurate navigation.     

iTRAQ-Based Quantitative Proteomic Analysis on S100 Calcium Binding Protein A2 in Metastasis of Laryngeal Cancer

Laryngeal cancer is the most frequent neoplasm in the head and neck region, with the vast majority of tumors originating from squamous cells. The survival rate of patients with laryngeal cancer has not improved substantially over the past 25 years. To acquire further knowledge regarding the molecules responsible for laryngeal cancer oncogenesis and, in turn, to improve target therapy，iTRAQ and mass spectrometry analysis were utilized to detect differences in protein expression from 15 paired laryngeal cancer and adjacent non-cancerous tissue samples. Using mass spectrometry analysis, the expression levels of 100 proteins in laryngeal cancer samples were distinct from the non-tumor, non-cancerous samples. Further validation of the differentially expressed proteins S100A2, KRT16, FGB and HSPB1 were carried out using quantitative real-time RT-PCR, immunoblot and immunohistochemistry. Functional analysis of one of the highly expressed proteins, S100 calcium binding protein A2 (S100A2), was performed using RNA interference. As a consequence, attenuated S100A2 expression enhanced the ability of HEp-2 cell lines to migrate and invade in vitro. Our investigation complements the current understanding of laryngeal cancer progression. Furthermore, this study supports the concept that enhanced expression of S100A2 may be a promising strategy in developing novel cancer therapeutic drugs.     

Flexibility of the drinking mechanism in adult chickens (Gallus gallus) (Aves)

Summary Drinking was studied in adult chickens by cinematography and radiography. Three subsequent behavioral phases occur. (1) Water is transported from the water box into the oropharynx when the beak is immersed. Then delicately tuned cyclic motion patterns of beaks, tongue, and larynx transport water by capillarity, squeezing, and suction. (2) During the elevation of the head the tongue is elevated and the larynx is depressed to keep the water in the pharynx against the gravitational and centrifugal forces that result from the upward swing of the head. (3) During the tip up phase gravity transports the water to the esophagus, while the adhering water is pushed and squeezed caudad by tongue and larynx movements. Flexibility in the adult drinking mechanism was analyzed by comparing the drinking of normal and beak-trimmed chickens under normal drinking conditions, as well as while drinking small drops. Three modes of behavioral flexibility were discussed: conservative, regressive, and progressive flexibility. Most behavioral elements of the modal action pattern in drinking are so flexible that a chicken can reorganize the movement patterns of jaws, tongue, larynx, and head to adapt the mechanism to external (drop drinking) or internal (beak-trimming) changes. However, in drop drinking, the normal chicken relies upon a regressive takeover by an ontogenetically earlier developed pattern in the craniocervical motion system. Presence of the observed progressive flexibility in lingual and cervical motion patterns is shown to be a precondition for the avian drinking mechanism to keep up with dominant evolutionary changes in feeding mechanisms.     

Histamine-induced potentiation of cholinergic transmission in chick oesophagus (Gallus gallus)

1. The effects of histamine on cholinergic nerve transmission were investigated in the oesophagus of the chick (0-29 days old). 2. Histamine potentiated the contractile responses produced by vagal or transmural nerve stimulation and by the ganglionic stimulant (DMPP) with the increase of tonus of oesophagus. On the other hand, a selective H2 agonist, 4-methylhistamine, did not have any effect. 3. The sensitivity of oesophagus to ACh did not change in the presence of histamine (0.2-2 microM). 4. Mepyramine but not metiamide antagonized the contractile and potentiating effects of histamine. 5. From these findings, it is concluded that histamine preferentially acts on H1-receptors located in cholinergic neurones to facilitate cholinergic transmission in the chick oesophagus, potentiating the nerve-mediated contraction.     

Development of an auditory discrimination in domestic chicks (Gallus gallus)

Ninety-two chicks were exposed to two different sounds, one of which was paired during training with a conspicuous visual stimulus. Visual stimuli maintained subsequent responses to the auditory stimuli, whereas responses to discriminably different sounds that were not paired with visual stimuli habituated. Auditory discriminations were learned at 1 day of age both by chicks that were previously ‘imprinted’ to the visual stimulus and by those that were not. Only the visually imprinted chicks performed significantly better than random when trained at 2 or 3 days, indicating an early critical period for visual, but not auditory, stimuli. It is suggested that visual stimuli enhance responses to species-typical and individually distinctive vocalizations.     

A histochemical study of keratinization in the domestic fowl (Gallus gallus)

The microanatomy of the epidermis of the domestic fowl is described and related to the distribution of various histochemical constituents involved in keratinization.
The avian horny layer over the back is composed of a loose network of structurally solid horny cells. This is in contrast to most mammalian epidermal horny cells in which structural keratin is found only in the peripheral cytoplasm, and the interior of the keratinocyte contains soluble products of cytolysis with possibly some free keratin filaments dispersed in the fluid material.
The avian tarsal epidermal horny scales show similarities to both the scales of lizards and snakes and to mammalian tail scales which appear to be homologous structures.
It is suggested that a thin layer of cells containing no detectable disulphide bonds, found in the tarsal scale region of the young chick, is probably mechanically weak and may function as a fission plane for sloughing of the horny layer. A specialized epidermis and thickened horny layer is developed in the fowl on the plantar underside of the toes, but this is quite different in structure from the mammalian plantar epidermis.
The overlapping of zones rich in ribonucleic acid (RNA) and bound cysteine (SH) in the growing feather suggests that protein synthesis and the preparatory stages to keratin disulphide bonding normally occur concurrently in feather formation. This is in contrast to the growing hair which has a region rich in RNA followed immediately before it becomes keratinized by a discrete keratogenous zone weak in RNA but rich in bound cysteine.     

Myocardial contractility in chickens (Gallus gallus): analysis of systolic time intervals

The avian cardiovascular system is of special interest because avian hearts are relatively larger than mammalian hearts, and activation of ventricular myocardium in birds has a "flash" pattern. Systolic time intervals and indices of myocardial contractility were examined in anaesthetized open-chest chickens by polycardiography, including synchronous recordings of electrocardiogram, phonocardiogram, and apex cardiogram. The asynchronous contraction time, isometric contraction time, pre-ejection period and ejection time were 26 +/- 3 (Mean +/- SD), 21 +/- 9, 47 +/- 12, and 83 +/- 23 ms, respectively, for heart rates of 260 +/- 57 bpm. The myocardial tension index, isometric contraction index and the pre-ejection period/ejection time ratio were 0.39 +/- 0.11, 0.42 +/- 0.10, and 0.54 +/- 0.14, respectively. A "flash" pattern of ventricular myocardial depolarization causes more rapid excitation and as a consequence shorter asynchronous contraction time of relatively larger chicken hearts compared with rabbit hearts. Inverse relation (P < 0.05) of the asynchronous contraction time to the heart rate in chickens is probably associated with the specific activation pattern of avian ventricles. Establishment of the values of systolic time intervals will facilitate a better understanding of cardiac function in birds. The obtained results are discussed in comparison with the rabbit. The indices calculated from the systolic time intervals show disadvantageous contractile function of chicken heart compared to rabbit heart.     

Maturation of glomerular size distribution profiles in domestic fowl (Gallus gallus)

Previous histological evaluations of chick kidneys indicated nephrons continue to develop from embryonic foci for up to 6 weeks after hatching. The present study was conducted using an in vivo alcian blue staining technique to quantify posthatch changes in glomerular numbers and sizes in female domestic fowl at 1, 3, 5, 9, 12, 21, and 30 weeks of age. Changes in glomerular size distributions reflect changes in the heterogeneous nephron populations of avian kidneys. Foci of embryonic tissue were observed at the periphery of renal lobules up to 12 weeks of age. Glomerular numbers increased from 69,800/kidney at 1 week to 586,000/kidney at 12 weeks, with no further significant increase up to 30 weeks (599,000/kidney). The increase in glomerular number per gram kidney weight remained constant as kidney mass increased up to 12 weeks of age, after which the number of glomeruli per gram kidney weight declined significantly as kidney size increased without further addition of new nephrons. Glomerular size distribution profiles were constructed using eleven circumference categories. The peak number of glomeruli fell within the 0.11-0.14 mm category at 1 and 3 weeks; within the 0.15-0.18 mm category at 5, 9, and 12 weeks; and within the 0.19-0.22 mm category at 21 and 30 weeks. One and 3-week-old chicks had no glomeruli within the largest (greater than or equal to 0.35 mm circumference) size categories, and 9-12-week-old birds had significantly fewer glomeruli in these categories than 21-30-week-old birds. These results demonstrate that posthatch renal maturation in domestic fowl involves the ongoing formation of new nephrons up to 12 weeks of age, with subsequent kidney growth (12-30 weeks of age) accomplished by enlargement of existing nephrons (nephron hypertrophy). The cumulative evidence indicates that nephrons destined to develop loops of Henle (mammalian-type) develop first, with shorter (reptilian-type) nephrons developing later as the kidneys enlarge.