The Anatomical Record Advances in Integrative Anatomy and Evolutionary Biology

We addressed the brain drainage system as inferred by the endocranial morphology of the occipito-temporal region of the El Sidrón Neandertal specimen SD-1219. Morphological details of the endocranial surface and its anatomical implications were analyzed for the reconstruction of the dural sinus drainage pattern and its comparison with Neandertals and other hominids. The specimen SD-1219 shows a pattern in which the superior sagittal sinus goes into the right transverse sinus. Comparative analyses with a large sample of fossil hominids reveal a pattern of the SD-1219 fossil that is typical for Neandertals. The analysis of the proportions of the occipital lobes prints within the occipital fossae reveals that the left occipital pole projects toward the right. This possibly indicates brain asymmetry (petalia) in this Neandertal individual, similar to that observed in some modern human brains. Conversely, no such asymmetry was observed in the cerebellar fossae. A particular feature of this fossil is the presence of two crests, located at the middle of the left cerebellar fossa that can be related to either an imprinting of a cerebellar fissure or some bone response to mechanical influence on internal bone surface morphology during cerebellar development. Specific aspects of the paleoneurology of Neandertals are discussed. Further quantitative studies on the endocranial morphology of the occipito-temporal and -mastoid region will shed light on the paleoneurological significance of this important anatomical region for the understanding of human evolution.

Tudor-SN is a multifunctional protein that is highly expressed in multiple cancers including breast cancer. Tudor-SN, as a component in RNA-induced splicing complex, was recently reported to regulate gene expression in a microRNA (miRNA)-dependent manner, such as let-7, miR-34a and miR-221. However, how Tudor-SN is associated with cancer development still remains largely elusive. In the present study, we explored the role of Tudor-SN in breast cancer. Stable knockdown of endogenous Tudor-SN, performed on the breast cancer cell line MDA-MB-231 by small hairpin RNA expression vectors, suppressed the in vitro migration and invasion ability of the metastatic breast cancer cell line. Interestingly, we found Tudor-SN as a miRNA regulator according to microarray analysis, and further identified that Tudor-SN negatively regulated the expression of miR-127, and consequently increased the expression of the proto-oncogene BCL6 which was a convincing target of miR-127. Moreover, overexpression of miR-127 reduced the in vitro migration and proliferation ability of breast cancer cell MDA-MB-231. Collectively, our results suggested a novel mechanism that Tudor-SN promoted metastasis and proliferation of breast cancer cells via downregulating the miR-127 expression. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.

The functional significance of shape differences between modern human and australopithecine distal femora remains unclear. Here, we examine the morphological component of the effective mechanical advantage (EMA) of the quadriceps muscle group in a sample of hominins that includes the fossil A.L. 129-1a (Australopithecus afarensis) and modern humans. Quadriceps muscle moment arms were calculated from three-dimensional computer models of specimens through a range of knee flexion. All hominins were compared using the same limb positions to allow us to examine, in isolation, the morphological component of the lengths of the pertinent moment arms. After taking into account the differences in bicondylar angle, the morphological component of the EMA was calculated as the ratio of the quadriceps muscle and ground reaction force moment arms. Our analyses reveal that A.L. 129-1a would have possessed a morphological component of the quadriceps muscle EMA expected for a hominin of its body mass. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.

Interleukin 13 (IL-13) is a key cytokine involved in the regulation of inflammatory, immune responses, and cell differentiation. The present study was to investigate the effect of IL-13 on the expression of glycoprotein IIb (GPIIb), a megakaryocytic gene, in Dami cells (human megakaryoblastic leukemia cell line) and HEL cells (human erythroleukemic cell line, which has both erythroid and megakaryocytic markers). Furthermore, it addresses the mechanisms governing the regulation of GPIIb expression by IL-13. The molecular responses of Dami cells and HEL cells to IL-13 treatment were analyzed by RT-PCR, Western blot, chromatin immunoprecipitation (ChIP) and flow cytometry analysis. We show that IL-13Rα1 and IL-4Rα are expressed in Dami cells and HEL cells. The expression of GPIIb was significantly upregulated at the mRNA and protein levels by treatment with IL-13. Moreover, IL-13 induced phosphorylation of signal transducer and activator of transcription 6(STAT6). By using a STAT6-specific antibody and PCR primers designed to yield a product, which encompasses the STAT6 binding site of the GPIIb promoter, we have shown the binding of the IL-13-mediated activation of STAT6 to the promoter of GPIIb gene. These results broaden the involvement of IL-13 into megakaryocyte differentiation by STAT6 pathway.

The study examined the expression of matrix metalloproteinases (MMPs), type I collagen and osteocalcin during bone healing in a rat calvarial experimental defect model. Twelve-week-old male Wistar rats were used. A full-thickness standardized trephine defect was made in the parietal bone, with the rat under anesthesia. RNA was extracted from tissue that filled the original bone defect on days 1 and 3 and in weeks 1, 2, 3, 5, 8, 10, 12, 18, and 24 and processed for quantitative analysis of expression of type I collagen, osteocalcin and matrix metalloproteinases (MMPs) 2, 8, and 13 by using real-time polymerase chain reaction. Alternatively, the rats were fixed by perfusion through the aorta and resected calvaria were processed for in situ hybridization for these molecules. The expression of type I collagen, osteocalcin and MMPs 2 and 13 increased toward week 2 and decreased thereafter, whereas the expression of MMP 8 was the highest on day 1. The mRNA transcripts of type I collagen and osteocalcin were localized in osteoblasts and osteocytes, some of which expressed MMPs 2, 8, and 13. Osteoblasts and osteocytes may play a role in the remodeling of extracellular matrices with MMPs during healing of a defect in bone.

The aim of this study is to investigate whether BMP-2 regulates the oral sulcus formation of mouse embryonic tongue by modifying the expression of TIMP and MMP. The BMP-2 siRNA induced a 180% increase in the depth of oral sulcus cavity (P < 0.01) by stimulating the invagination of oral sulcus into the mesenchymal tissues consisting of tongue floor, whereas the recombinant BMP-2 suppressed the process in the organ culture system of mouse embryonic tongue. The BMP-2 siRNA induced a 60% decrease in the expression of TIMP-1 mRNA (P < 0.05) and a drastic decline in TIMP-1 protein was observed around the oral sulcus in the BMP-2 siRNA treated mandibles. The recombinant BMP-2 induced a 220% increases in the expression of TIMP-1 mRNA and the area of the immunostaining for TIMP-1 around the oral sulcus was larger in the mandibles treated with the recombinant BMP-2 than the vehicle. The BMP-2 siRNA induced a 60% increase in the expression of MMP-13 protein and a marked increase in the staining intensity for MMP-13 was observed in the epithelial region of the BMP-2 siRNA treated mandibles. The recombinant BMP-2 induced a 70% decrease in the expression of MMP-13 mRNA and the decrease was mainly observed in the tissues around oral sulcus. The expressions of BMP-2, TIMP-1, and MMP-13 were verified in the tissues around in vivo developing oral sulcus at E11, 12, and 13 by immunohistochemistry. These results suggest that BMP-2 regulates the formation of oral sulcus by altering the balance between TIMP-1 and MMP-13.

During the 1947 fieldwork season, Member 4 (2-3 My) of the South African Sterkfontein site yielded two important Australopithecus africanus fossils: a cranium popularly nicknamed "Mrs. Ples" (Sts 5), and a partial skeleton (Sts 14). Previous reports have proposed that Sts 5 was a nonfully grown adolescent individual (Thackeray et al., S Afr J Sci 2002a;98:21-22), and that Sts 14 was a sub-adult specimen (according to various signs of immaturity in the skeleton) (Berge and Gommery, C R Acad Sci Paris, Sciences de la terre et des planètes 1999;329:227-232; Häusler and Berger, J Hum Evol, 2001;40:411-417; Thackeray et al., S Afr J Sci, 2002b;98:211-212). It was subsequently proposed that these fossils actually belonged to the same individual (Thackeray et al., S Afr J Sci, 2002b;98:211-212), a proposition supported by their spatial positions within the site. The present work attempts to revise these different assertions. The results obtained: (i) show that the Sts 5 fossil represents a fully grown adult cranium; (ii) provide new evidence of immaturity in the Sts 14 skeletal elements (sustaining the proposed young adult age of this specimen), and (iii) suggest that although the revised ages-at-death for these fossils are partially compatible, there is no evidence to support the idea that they represent a single individual. Finally, the encephalization quotient associated with a hypothetical union of Sts 5 and Sts 14 (calculated using data from both specimens) lies between the upper and lower limits of the currently estimated range for this species and H. habilis, respectively.

MicroRNAs (miRNAs) are novel small noncoding RNA molecules that regulate gene expression at the post transcriptional level. Compelling evidence shows that there are causative links between miRNAs deregulation and cancer development and progression. This study aims to explore the functions of miR-16-1 on proliferation, apoptosis, motility, and invasion of glioma cells. Quantitative real-time PCR (qRT-PCR) was performed to detect the expression of miR-16-1 in normal brain tissues and two glioma cell lines, including U251 and U87. CCK-8, Annexin V/FITC (fluorescein isothiocyanate), wound healing, and transwell assays were used to evaluate the functions of miR-16-1 that involves cell proliferation, apoptosis, motility, and invasion. In addition, we conducted qRT-PCR to examine mRNA expression levels of Zyxin, one of putative target genes of miR-16-1, in U251 glioma cells after transfecting with miR-16-1 mimics. As a result, miR-16-1 expression level was lower in U251 and U87 cells than normal brain tissues. After miR-16-1 was upregulated in U251 cells, cellular proliferation was notably attenuated but cell apoptosis was not significantly increased. Moreover, overexpression of miR-16-1 attenuated migration and invasion of glioma cells, and U251 cells transfected with miR-16-1 showed significantly lower endogenous mRNA levels of Zyxin than those transfected with nonspecific control miRNA or mock (P < 0.05). In summary, we demonstrated that miR-16-1 expression was markedly decreased in human glioma cell lines, and for the first time, described the roles of miR-16-1 in cellular proliferation, migration, and invasion abilities of high-invasive glioma cells, and suggested that Zyxin may be one of putative target genes of miR-16-1. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

This study analyzes morphometrically 17 skulls of the Mediterranean monk seal Monachus monachus housed in different Italian Museums and collections. We considered several morphometric variables (31 linear, 1 volumetric and 1 surface area measurements). In addition, we identified, measured and compared two nonmorphometric variables, namely, the bone densities of selected areas obtained using a dual-energy X-ray absorptiometry (DXA) device. The high correlation coefficient of all variables indicated continuous growth with the onset of age. The ranking of the hierarchical cluster analysis identified the presence of three main groups containing individuals of similar sizes: lactating pups and yearlings; subadult individuals and adult females; and adult males. Smaller groups were identified within these clusters, and their respective allocations into two subgroups were argued on the basis of skull development and other factors. The discriminant analysis of the three main groups indicated a discriminant diagnostic key, based on condilobasilar length (CBlr-L); maximum mandibular branch height (MB-H); and surface area of the bulla tympanica. The proposed diagnostic key is useful to classify monk seal skulls of unidentified age and sex. The data reported here suggest that in this species certain adult skull growth features (enhanced tympanic bullae surface area extension, occipital bone density) are sexually dimorphic and possibly related to specific anatomical functions. These functions may include an enhanced auditory capacity; an increased development of the cranial musculature capable of supporting a large skull and guaranteeing the mandibular strength necessary for mastication; and male to male social interactions.

A comparative anatomical study was conducted among four mobulid species (Mobula japanica, M. munkiana, M. tarapacana, and M. thurstoni), from the coasts of the Gulf of California. Characters of the skeleton (neurocranium, mandibles, scapular girdle, pelvic girdle, and synarcual) were described. Mobula species had a wide structural variation. In the skeleton, M. tarapacana displayed the most plesiomorphic characters, mainly at the cranial level (cranium longer than wide, reduced internasal width, fontanelle longer than wide), while M. japanica had the most derived characters (cranium wider than longer, fontanelle wider than longer). Based on the observations, the dorsal cephalic musculature were similar in all species; however, oppositely to other batoid species, the epiaxilis is strongly developed, as it extends toward the cephalic part of the neurocranium, at level of the postorbital processes. In the ventral musculature, ventral constrictor muscles are quite reduced, whereas complex mandibular muscles are very developed. Muscles showing the highest variation are the coracohioideus and the coracoarcual.

The morphology of the autonomic cardiac nervous system (ACNS) was examined in 24 sides of 12 New World monkeys (Platyrrhini) of all four families to document the morphology systematically and to study the evolutionary changes of the ACNS in this primate lineage. We report the following: (1) Although several trivial intra- and inter-specific variations are present, a family-dependent morphology of the ACNS does not exist in New World monkeys. (2) The sympathetic ganglia in New World monkeys consist of the superior cervical, the middle cervical, and the cervicothoracic which is composed of the inferior cervical and first and second thoracic, and the thoracic ganglia starting with the third thoracic. The general cardiac nervous system is the sympathetic middle and inferior cardiac nerves and all parasympathetic vagal cardiac branches. (3) The morphology of the ACNS in the New World monkeys is almost consistent regardless of the number of vertebrae, the cardiac position and deviation (axis), and the great arterial branching pattern of the aortic arch, and it is very similar to that in the Old World monkeys, with only one difference: the superior cervical ganglion in the New World monkeys tends to be relatively smaller, higher, and provides a narrower contribution to the spinal nerves than in the Old World monkeys. The ACNS morphology exhibits significant evolutionary changes within the primate lineage from New and Old World monkeys to humans. The comparative morphology within the lineage is concordant with the phylogeny, suggesting that the primate ACNS preserves its evolutionary history in close alignment with phylogeny.

Lizards of the family Gekkonidae display a variety of reproductive patterns, as evidenced by the presence of viviparous and oviparous species. The species Hemidactylus mabouia is oviparous. We examined, in vitellogenic females, oviductal structure by light microscopy after routine histological and histochemical techniques, as well as by scanning and transmission electron microscopy. The oviduct is composed of four different regions: the infundibulum, which opens into the coelomic cavity and receives the oocyte released at the time of ovulation; the uterine tube, where sperm storage takes place; the uterus, which is responsible for the eggshell production; and the vagina, the final portion of the oviduct that leads to the cloaca. The oviductal structure of H. mabouia is similar to that of other oviparous lizard species and can be useful for morphological comparative analysis among reptile species.

The aim of the present study was to investigate the sequence of shell bone formation in the embryos of the Pleurodira, Podocnemis unifilis. Their bones and cartilage were collected and cleared before staining. The shell was also examined by obtaining a series of histological slices. All the bony elements of the plastron have independent ossification centers, which subsequently join together and retain two fontanelles until the period of hatching. This turtle has a mesoplastra, which is characteristic of the Podocnemididae. The carapace begins to form concurrently with the ossification of the ribs at the beginning of stage 20. All the plates, except the suprapygal, initiate ossification during the embryonic period. The main purpose of the histological investigation was to highlight the relationship between the formation of the carapace and ribs with that of the neural plates. The costal and neural plates were found not to independent ossification centers, but to be closely related to components of the endoskeleton, originating as expansions of the perichondral collar of the ribs and the neural arches, respectively. Considering the ribs as an endoskeletal element of the carapace, the carapace and plastron begin ossification at the same stage in P. unifilis. This pattern reveals similarities with other Pleurodira, as well as evident variations, such as the presence of the seven neural bones and the presence of only one ossification center in the nuchal plate.

The Amazonian manatee (Trichechus inunguis) is uniparous and has a slow reproduction cycle due to a long gestation period and long interval between births. Even though protected by law, hunting remains one of the main causes hindering the natural population growth of this species in the wild. The histology and reproductive anatomy provide information on the history and reproductive status of the female and offer a tool for the conservation of the species. The present study describes the anatomy of the female reproductive tract in T. inunguis. It is based on materials from three reproductive tracts fixed in 10% buffered formalin. The ovaries, uterine tubes, uterus, vagina, and external genitalia are described. The hymen presents two tiny openings separated by a segment that, upon rupturing during the first copulation, should make up a single vaginal opening. A still intact hymen and the absence of placental scars in the uterus were found in one specimen. Additionally, the presence of a hemorrhagic body and Graafian follicles on the right ovary were observed, as well as whitish scars and among them, possible corpora albicantia. These findings suggest that T. inunguis undergoes infertile estrus cycles before its first gestation. Macroscopically, counting of the whitish scars is hindered by the small diameter of these structures. It is not possible to differentiate between the scars resulting from ruptured (corpora albicantia) and nonruptured follicles (regressed corpora atretica). The presence of whitish scars on both ovaries of the same specimen suggests their bilateral function in T. inunguis.

Cytokines are soluble mediators that are thought to act as communication signals between astroglia and neighboring neural cells. They are both released by, and act on, astroglia. It is hypothesized that it is this effect on astroglia that may be important in widespread phenomena including traumatic brain injury, inflammation, and scar formation. In this article, we examine the effect of mouse recombinant interleukin-1β (IL-1β) on the morphology, organization, and expression of glial fibrillary acidic protein (GFAP) and actin isoforms in cultured mouse astroglia. This study shows that the majority of the astroglia treated with IL-1β acquire long processes. Immunofluorescence staining shows that there are no remarkable changes in the organization of GFAP, F-actin, α-smooth muscle (α-sm) actin, and β-actin isoforms. In fluorescent microplate assay, the short-term treated astroglia (range, 1-2 days) show an increase in the intensity of GFAP and β-actin isoform over the level observed in untreated control, whereas no remarkable changes are observed in the intensity of α-sm actin isoform. In the case of long-term treatment (range, 4-8 days), the intensity of GFAP and α-sm actin isoform progressively decreases below the level of untreated control. In addition, the intensity of β-actin isoform increases above the control level. These results have been confirmed by immunoblotting experiments. The upregulation of β-actin isoform may be important in limiting the noxious effects of an inflammatory reaction. This gives credence to the hypothesis that it might be possible to modulate astroglial effects on neuronal inflammation and scar formation with appropriate therapies.

Dens invaginatus (DI) is a developmental anomaly of teeth. Here we observed the characteristics of DI in 517 permanent teeth from 67 ancient Chinese people using micro-computed tomography (micro-CT) scanning techniques. The individuals were excavated from Shaanxi province of China and identified to be about 2,000 years old. Four DI categories are proposed to distinguish the different types of DI. The invaginated lingual fossa is classified into three classes. The overall prevalence of DI in 67 individuals was 31.34% (21 of 67). DI was found in 25 of 517 teeth (4.83%). All affected teeth were maxillary lateral incisors. The invaginated lingual fossa (Type I DI) occurred most frequently (84%), followed by radicular grooves (Type II; 16%), while Type III and Type IV were not found in the present study. Some of fossae correspond with radicular grooves (8 of 21; 3 located at mesial, 5 were distal). The bilateral incidence of DI was 19.05%. Comparing our results to those of a retrospective survey of DI from 1873 to present, we found a higher rate of DI in the excavated teeth of Chinese individuals 2,000 years ago. The use of the micro-CT technique and ethnic origin might have contributed to the higher incidence of DI. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.

Despite its abundance in Latin America, and its remarkable ability to use tools, there are only a few myological studies on the capuchin monkey, Cebus libidinosus. In the present study, we dissected the forearm extensor muscles of six adult males and two adult females of this species. We describe these muscles and compare them with those of other primates dissected by us and by other authors. The forearm extensor muscles of Cebus monkeys are, in general, more similar to those of other platyrrhines than to distantly related taxa that use tools, such as chimpanzees and modern humans, with three main exceptions: contrary to most other platyrrhines, (1) in Cebus, chimpanzees and modern humans the extensor pollicis longus usually inserts onto Digit I, and not onto Digits I and II; (2) in Cebus the abductor pollicis longus has two separate tendons, as is the case in chimpanzees, and in modern humans (where one of these tendons is associated with a distinct belly, forming the muscle extensor pollicis brevis); (3) in Cebus, and in modern humans and chimpanzees, the extensor pollicis longus is not deeply blended with the extensor indicis. Therefore, the Cebus monkeys provide an illustrative example of how phylogenetic constrains and ecological adaptations have been combined to develop a specific myological configuration that, associated with their sophisticated neurological organization, allow them to easily navigate in their arboreal habitats and, at the same time, to finely manipulate objects in order to search for food and to prepare this food for ingestion.

The present study was designed to define the morphological dimensions of the acetabulum in normal Chinese adults and to statistically compare these data with the available data worldwide. This information is important for the diagnosis of dysplasia and treatment of total hip arthroplasty. In this study, the gender and bilateral differences were evaluated. One-hundred CT scans of patients were retrospectively studied. These individuals showed no signs of developmental disturbances in either of the hip joints. Thirty-five morphometric parameters of the acetabulum were measured. The size of acetabulum was evaluated by the acetabular perimeter, anteroposterior diameter, vertical diameter, the depth and width of fossa ovalis in both transaxial and coronal plane. The parameters of acetabular orientation were the acetabular angle, anterior center edge angle, neck shaft angle, acetabular anteversion, and abduction angle. The coverage of acetabulum was examined as the, acetabular head index, center edge angle, the distance between the femoral head, and acetabulum. Gender and bilateral differences were analyzed for each parameter, and compared with available worldwide data. The results showed statistically significant differences between the Chinese genders and also between the Chinese and other human races in some parameters. In conclusion, gender, bilateral and racial differences exist in the morphology of acetabulum. The data may be helpful for the design of total hip arthroplasty for the Chinese population. Anat Rec, 2014. © 2014 Wiley Periodicals, Inc.

A yeast artificial chromosome (YAC) transgenic murine model of partial trisomy 21 overexpressing five human genes -- including DYRK1A, which encodes a serine threonine kinase involved in cell cycle control -- has been shown to present an increase in brain weight. We analyzed this new phenotype by measuring total and regional brain volumes at different ages, using a 7 Tesla magnetic resonance imaging volumetric approach. Volumetric measurements showed a total volume increase of 13.6% in adult mice. Changes in brain morphogenesis were already visible at a very early postnatal stage (postnatal days 2-7). Region-specific changes were characterized from postnatal day 15 to 5 months. These results, made it possible to define region-specific effects of DYRK1A overexpression, with the strongest increase seen in the thalamus-hypothalamus area (24%).

A relationship between compromised muscles and other tissues has been demonstrated in mdx mouse, an animal model studied for understanding of Duchenne muscular dystrophy. The hypothesis is that changes in the calcaneal tendon of mdx mice occur previous to the onset of rigorous and most marked episodes of muscle degeneration, which start suddenly after 21 days of life. Thus, this study aimed to identify possible alterations in the calcaneal tendon of mdx mouse at 21 days of age. Control and mdx tendons were submitted to mechanical tensile testing, quantification of hydroxyproline, and staining with toluidine blue and picrosirius red. Hydroxyproline content was similar between mdx and control groups. The control tendon presented higher mechanical strength (load, stress, and elastic modulus) and its morphological analysis showed a larger number of round fibroblasts, nuclei with well-decondensed chromatin, and slightly metachromatic well-stained cytoplasmic material, different from that observed in mdx tendons. The results suggest that the absence of dystrophin in mdx mouse can provoke directly or indirectly alterations in the mechanical properties and morphology of the calcaneal tendon. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.

Fibroblast growth factor-23 (FGF23) is a hormone that modulates circulating phosphate (P(i)) levels by controlling P(i) reabsorption from the kidneys. When FGF23 levels are deficient, as in tumoral calcinosis patients, hyperphosphatemia ensues. We show here in a murine model that Fgf23 ablation disrupted morphology and protein expression within the dentoalveolar complex. Ectopic matrix formation in pulp chambers, odontoblast layer disruption, narrowing of periodontal ligament space, and alteration of cementum structure were observed in histological and electron microscopy sections. Because serum P(i) levels are dramatically elevated in Fgf23(-/-), we assayed for apoptosis and expression of members from the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family, both of which are sensitive to elevated P(i) in vitro. Unlike X-linked hypophosphatemic (Hyp) and wild-type (WT) specimens, numerous apoptotic osteocytes and osteoblasts were detected in Fgf23(-/-) specimens. Further, in comparison to Hyp and WT samples, decreased bone sialoprotein and elevated dentin matrix protein-1 protein levels were observed in cementum of Fgf23(-/-) mice. Additional dentin-associated proteins, such as dentin sialoprotein and dentin phosphoprotein, exhibited altered localization in both Fgf23(-/-) and Hyp samples. Based on these results, we propose that FGF23 and (P(i)) homeostasis play a significant role in maintenance of the dentoalveolar complex.

Gliomas are the most common form of primary brain tumor with the highest mortality rates. Drug resistance is a major cause of treatment failure in patients with glioma. Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) has been demonstrated to play an important role in drug resistance in human cancer cell lines. However, the reversing effect of mda-7/IL-24 on drug resistance of human glioma is not fully clear. Here, we investigated the effects of overexpression of the mda-7/IL-24 gene in human glioma. We established a cisplatin-resistant U87 glioma cell line and found that mda-7/IL-24 was highly correlated with drug resistance. Furthermore, we investigated the apoptotic rate, intracellular accumulation of Rhodamine-123, and expression of glutathione and P-glycoprotein. The over-expression of mda-7/IL-24 enhanced cisplatin cytotoxicity and reversal of drug resistance in glioma cells. The reversing effect of mda-7/IL-24 on drug resistance was induced mainly through the regulation of drug resistance-related genes and efflux drug pumps. Thus, mda-7/IL-24 can be used as a promising predictive biomarker and potential therapeutic target for chemotherapy in glioma. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.

To identify when during fetal development connexins (Cxs) 26 (Cx26) 32 (Cx32), and 36 (Cx36) begin to be expressed, as well as to characterize their spatial distribution, real time polymerase chain reaction and immunolabeling studies were performed. Total RNA from mouse pancreases at 13 and 18 days postcoitum (dpc) and 3 days postpartum (dpp) was analyzed. In addition, pancreatic sections of mouse at 13, 14, 15, 16, 18 dpc and 3 dpp and of rat at term were double labeled with either anti-insulin or anti-α-amylase and anti-Cx26 or -Cx32 or -Cx36 antibodies and studied with confocal microscopy. From day 13 dpc, Cxs 26, 32, and 36 transcripts were identified and their levels increased with age. At 13-14 dpc, Cxs 26 and 32 were localized in few acinar cells, whereas Cx36 was distributed in small beta cell clumps. From day 14 dpc onwards, the number of labeled cells and relative immunofluorescent reactivity of all three Cxs at junctional membranes of the respective cell types increased. Cxs 26 and 32 colocalized in fetal acinar cells. In rat pancreas at term, a similar connexin distribution was found. Relative Cxs levels evaluated by immunoblotting also increased (two-fold) in pancreas homogenates from day 18 dpc to 3 dpp. The early cell specific, wide distribution, and age dependent expression of Cxs 26, 32, and 36 during fetal pancreas ontogeny suggests their possible involvement in pancreas differentiation and prenatal maturation.

The presence of a live cell cohabiting within another cell has fascinated scientists for many decades. Far from being a spurious event, many have attempted to uncover the molecular mechanism underlying this phenomenon. In this study, we observed anchorage-dependent MCF-7 cells internalizing neighboring epithelial cells (entosis) after siRNA-mediated silencing of the Metallothionein-2A (MT-2A) gene. MTs belong to a family of low-molecular weight proteins, which bind metal ions endogenously and its over-expression has been reported in a variety of cancers that include breast, prostate, and colon. We provide microscopic evidence at light and ultrastructural levels of the occurrence of entosis after altering MT expression in a subpopulation of MCF-7 breast cancer cells by silencing the MT-2A gene. Our results demonstrate that adheren junctions may play important roles in the formation of cell-in-cell cytostructure after MT-2A gene downregulation and the entotic process does not appear to involve genes associated with autophagy. Interiorized cells often underwent lysosomal degradation within the cytoplasmic body of the engulfing cell. It would appear that a subset of breast cancer cells could die via entosis after MT-2A gene silencing.

Iron plays a crucial role in the survival, differentiation, and myelin formation of oligodendrocyte lineages. However, the regulation mechanism of iron homeostasis in oligodendrocytes remains unclear. Recently, much research has focused on Ferroportin 1 (FPN1), an iron exporter protein. First, about 95% pure primary rat O-2A progenitor cells were obtained by shaking methods in our laboratory. The expression of FPN1 mRNA and protein in O-2A progenitor cells were determined by reverse transcription-PCR and western blot. In addition, the localization of FPN1 at the cell membrane, in the cytoplasm and in processes was assayed by double-labeling immunofluorescence. A time-dependent increase of iron efflux from O-2A progenitor cells was confirmed by the calcein-indicated iron efflux assay. However, the same cells treated with FPN1 antibody showed no obvious change in iron release. For further confirmation, overexpression of FPN1 in O-2A progenitor cells was transduced with lentivirus. The release of iron in O-2A progenitor cells was dramatically increased by the overexpressed FPN1 when compared with that of the control group. Both ferritin (Ft) and transferrin receptor (TfR) are routinely used as indicators of labile iron pool. Cells pretreated with FPN1 antibody upregulated Ft and downregulated TfR protein level, while the opposite results occurred in the FPN1 overexpressing cells. Determination of Ft and TfR indirectly indicated that FPN1 might contribute to iron release from O-2A progenitor cells. We suggested that expression of FPN1 in O-2A progenitor cells might play a critical role in iron efflux from these cells. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Quantification of microvascular remodeling as a meaningful discovery tool requires mapping and measurement of site-specific changes within vascular trees and networks. Vessel density and other critical vascular parameters are often modulated by molecular regulators as determined by local vascular architecture. For example, enlargement of vessel diameter by vascular endothelial growth factor (VEGF) is restricted to specific generations of vessel branching (Parsons-Wingerter et al., Microvascular Research72: 91, 2006). The averaging of vessel diameter over many successively smaller generations is therefore not particularly useful. The newly automated, user-interactive software VESsel GENeration Analysis (VESGEN) quantifies major vessel parameters within two-dimensional (2D) vascular trees, networks, and tree-network composites. This report reviews application of VESGEN 2D to angiogenic and lymphangiogenic tissues that includes the human and murine retina, embryonic coronary vessels, and avian chorioallantoic membrane. Software output includes colorized image maps with quantification of local vessel diameter, fractal dimension, tortuosity, and avascular spacing. The density of parameters such as vessel area, length, number, and branch point are quantified according to site-specific generational branching within vascular trees. The sole user input requirement is a binary (black/white) vascular image. Future applications of VESGEN will include analysis of 3D vascular architecture and bioinformatic dimensions such as blood flow and receptor localization. Branching analysis by VESGEN has demonstrated that numerous regulators including VEGF165, basic fibroblast growth factor, transforming growth factor β-1, angiostatin and the clinical steroid triamcinolone acetonide induce 'fingerprint' or 'signature' changes in vascular patterning that provide unique readouts of dominant molecular signaling.

It is now well documented that androgen and estrogen signaling during early development cause a sexual dimorphism in second-to-fourth digit length ratio (2D:4D). It is also well documented that males of mammalian species have a smaller 2D:4D than females. Although there are discrepancies among 2D:4D studies in birds, the consensus is that birds exhibit the opposite pattern with males having a larger 2D:4D than females. The literature currently lacks substantial information regarding the phylogenetic pattern of this trait in amphibians and reptiles. In this study, we examined 2D:4D in two species of frogs (Oophaga pumilio and Craugastor bransfordii) and two species of lizards (Anolis humilis and Anolis limifrons) to determine the existence and the pattern of the sexual dimorphism. Male O. pumilio and C. bransfordii displayed larger 2D:4D than females in at least one of their two forelimbs. Male A. humilis had larger 2D:4D than females in both hindlimbs, but smaller 2D:4D than females in both forelimbs. Male A. limifrons may also have smaller 2D:4D than females in the right forelimb. Finally, digit ratios were sometimes positively related to body length, suggesting allometric growth. Overall, our results support the existence of the 2D:4D sexual dimorphism in amphibians and lizards and add to the knowledge of 2D:4D trait patterning among tetrapods.

Type X collagen (Col-X) deposition is a marker of terminal differentiation during chondrogenesis, in addition to appositional growth and apoptosis. The parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor, or PPR, is a G-Protein coupled receptor (GPCR), which activates several downstream pathways, moderating chondrocyte differentiation, including suppression of Col-X deposition. An Avian sterna model was used to analyze the PPR GPCR downstream kinase role in growth rate and extracellular matrix (ECM) including Col-II, IX, and X. Phosphatidylinositol kinase (PI3K), mitogen activating protein kinase (MAPK) and protein kinase A (PKA) were inhibited with specific established inhibitors LY294002, PD98059, and H89, respectively to test the hypothesis that they could reverse/inhibit the PTH/PTHrP pathway. Excised E14 chick sterna were PTH treated with or without an inhibitor and compared to controls. Sternal length was measured every 24 hr. Cultured sterna were immuno-stained using specific antibodies for Col-II, IX, or X and examined via confocal microscopy. Increased growth in PTH-treated sterna was MAPK, PI3K, and PKA dose dependent, suggesting growth was regulated through multiple pathways. Col-X deposition was rescued in PTH-treated sterna in the presence of PI3K or MAPK inhibitors, but not with the PKA inhibitor. All three inhibitors moderately disrupted Col-II and Col-IX deposition. These results suggest that PTH can activate multiple pathways during chondrocyte differentiation.

With age, alpha-synuclein (α-SYNC) misfolds and forms insoluble deposits of protein in the myenteric plexus, leading presumably to dystrophy and degeneration in the circuitry controlling gastrointestinal (GI) function. The present experiment examined aggregates of α-SYNC in the aging small intestine and investigated how macrophages in the wall of the GI tract respond to these aberrant deposits. Groups of adult and aged Fisher 344 rats were studied. Whole mounts of duodenal, jejunal, and ileal smooth muscle wall, including the myenteric plexus, were prepared. Double labeling immunohistochemistry was used to stain α-SYNC protein and the phenotypic macrophage antigens CD163 and MHCII. Alpha-synuclein accumulated in dense aggregates in axons of both postganglionic and preganglionic neurons throughout the small intestine. Staining patterns suggested that deposits of protein occur initially in axonal terminals and then spread retrogradely toward the somata. Macrophages that were adjacent to dystrophic terminal processes were swollen and contained vacuoles filled with insoluble α-SYNC, and these macrophages commonly had the phenotype of alternatively activated phagocytes. The present results suggest that macrophages play an active phagocytotic role in removing α-SYNC aggregates that accumulate with age in the neural circuitry of the gut. Our observations further indicate that this housekeeping response does not clear the protein sufficiently to eliminate all synucleinopathies or their precursor aggregates from the healthy aging GI tract. Thus, accumulating deposits of insoluble α-SYNC in the wall of the GI tract may contribute, especially when compounded by disease or inflammation, to the age-associated neuropathies in the gut that compromise GI function. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.

Nuclear speckles, which are sites of pre-mRNA splicing and/or assembly components, are diffusely distributed throughout the nucleoplasm. They are composed of splicing factors (SFs), including SC-35, which are nuclear proteins that remove introns (noncoding sequences in the genes) from precursor mRNA molecules, to form mature RNA, which will be transported to the cytoplasm, site of protein synthesis and activation. In light of such evidences, here we report that hypoxia modulates in vivo SC-35 SF phosphorylation via protein kinase C (PKC) delta in young rat heart. Trichrome Mallory staining and TUNEL analysis along with immunohistochemistry and Western blotting have been performed on left ventricles excised from young and old rats exposed to intermittent hypoxia. Although young hypoxic myocardial cells appear smaller than normoxic cells, connective and endothelial components increase, SC-35 phosphorylation is particularly evident in the endothelium and paralleled by an increased expression of vascular endothelial growth factor (VEGF). In addition, SC-35 and PKC delta coimmunoprecipitation occurs, suggesting that SC-35 phosphorylation could be PKC delta-mediated and that hypoxic young heart needs to counteract the damage through a process of neoangiogenesis involving such SF. Even though the levels of SC-35 and PKC delta are high, the similar response disclosed by normoxic and hypoxic old rat hearts (both showing a fibrotic organization, similar endothelial components and VEGF levels) could be due to the existence of an impaired oxygen sensing mechanism and thus to a low rate of angiogenesis.

Hepatocellular carcinoma (HCC) is one of the most frequent malignant neoplasms worldwide and is the second leading cause of cancer death in China. We have previously demonstrated that LAPTM4B-35, encoded by lysosomal protein transmembrane 4 beta gene, is overexpressed in over 80% of HCCs and is a novel-independent prognostic factor for metastasis, recurrence, and postoperative survival in HCC. In this study, we investigated the role of LAPTM4B-35 in malignant transformation and tumorigenesis using L02 cells, a cell line originated from human normal liver cells. Our data show that replication-deficient adenovirus vector-mediated upregulation of LAPTM4B-35 promotes anchorage-independent proliferation and resistance to adriamycin-induced apoptosis. Study of the underlying mechanisms demonstrated alterations of molecular events involved in these processes, which included the activation of phosphoinositide 3-kinases (PI3K)/serine/threonine protein kinase B (PKB/AKT)/bcl-xL/bcl-2-associated death promoter homolog (Bad) signaling pathway, inhibition of caspase-3 activation, upregulation of Bcl-2, and downregulation of Bax. In addition, upregulation of LAPTM4B-35 in L02 cells resulted in tumorigenesis in 100% (6/6) of inoculated nude mice and accelerated the death of mice with xenografts in vivo. In conclusion, LAPTM4B-35 promotes malignant transformation and tumorigenesis in human liver L02 cell line through promotion of deregulated proliferation and inhibition of apoptosis. These findings suggest that overexpression of LAPTM4B-35 may play a critical role in hepatocarcinogenesis and therefore, may be a therapeutic target for HCC.

We developed the Case Cryo-imaging system that provides information rich, very high-resolution, color brightfield, and molecular fluorescence images of a whole mouse using a section-and-image block-face imaging technology. The system consists of a mouse-sized, motorized cryo-microtome with special features for imaging, a modified, brightfield/fluorescence microscope, and a robotic xyz imaging system positioner, all of which is fully automated by a control system. Using the robotic system, we acquired microscopic tiled images at a pixel size of 15.6 microm over the block face of a whole mouse sectioned at 40 microm, with a total data volume of 55 GB. Viewing 2D images at multiple resolutions, we identified small structures such as cardiac vessels, muscle layers, villi of the small intestine, the optic nerve, and layers of the eye. Cryo-imaging was also suitable for imaging embryo mutants in 3D. A mouse, in which enhanced green fluorescent protein was expressed under gamma actin promoter in smooth muscle cells, gave clear 3D views of smooth muscle in the urogenital and gastrointestinal tracts. With cryo-imaging, we could obtain 3D vasculature down to 10 microm, over very large regions of mouse brain. Software is fully automated with fully programmable imaging/sectioning protocols, email notifications, and automatic volume visualization. With a unique combination of field-of-view, depth of field, contrast, and resolution, the Case Cryo-imaging system fills the gap between whole animal in vivo imaging and histology.

Realistic airway geometry is required for accurate biomechanical modeling, particle deposition predictions and ultimately risk assessment and inhaled drug delivery protocols. Morphometric studies to date provide data for specific anatomical locations or for more generational average data for the entire lung. In an attempt to provide a realistic geometry representative of a typical human, the National Institute of Health (NIH) Visible Human (VH) female data set was reconstructed and compared to available morphometric data from the literature. The reconstructed NIH VH female airway model extended from just distal to the larynx down through the fifth generation of bronchial passageways. Casting and scanning techniques were used to create the upper airway geometries so that the model could be used realistically for oral exposure. Each reconstruction stage was examined to show the loss of data during segmentation, decimation, and smoothing processes. The resulting dimensions of the complete female model were consistent with morphometric data from the literature, indicating that the model is a reasonable representation of an adult female that could be used for biomechanical modeling.

Reconstruction of fractured, distorted, or missing parts in human skeleton presents an equal challenge in the fields of paleoanthropology, bioarcheology, forensics, and medicine. This is particularly important within the disciplines such as orthodontics and surgery, when dealing with mandibular defects due to tumors, developmental abnormalities, or trauma. In such cases, proper restorations of both form (for esthetic purposes) and function (restoration of articulation, occlusion, and mastication) are required. Several digital approaches based on three-dimensional (3D) digital modeling, computer-aided design (CAD)/computer-aided manufacturing techniques, and more recently geometric morphometric methods have been used to solve this problem. Nevertheless, comparisons among their outcomes are rarely provided. In this contribution, three methods for hemimandibular body reconstruction have been tested. Two bone defects were virtually simulated in a 3D digital model of a human hemimandible. Accordingly, 3D digital scaffolds were obtained using the mirror copy of the unaffected hemimandible (Method 1), the thin plate spline (TPS) interpolation (Method 2), and the combination between TPS and CAD techniques (Method 3). The mirror copy of the unaffected hemimandible does not provide a suitable solution for bone restoration. The combination between TPS interpolation and CAD techniques (Method 3) produces an almost perfect-fitting 3D digital model that can be used for biocompatible custom-made scaffolds generated by rapid prototyping technologies.

Looking in microscopic detail at the 3D organization of initiating teeth within the embryonic jaw has long-proved technologically challenging because of the radio-translucency of these tiny un-mineralized oral tissues. Yet 3D image data showing changes in the physical relationships among developing tooth and jaw tissues are vital to understand the coordinated morphogenesis of vertebrate teeth and jaws as an animal grows and as species evolve. Here, we present a new synchrotron-based scanning solution to image odontogenesis in 3D and in histological detail using a silver-based contrast agent. We stained fixed, intact wild-type mice aged embryonic (E) day 10 to birth with 1% Protargol-S at 37°C for 12-32 hr. Specimens were scanned at 4-10 µm pixel size at 28 keV, just above the silver K-edge, using micro-computed tomography (µCT) at the Canadian Light Source synchrotron. Synchrotron µCT scans of silver-stained embryos showed even the earliest visible stages of tooth initiation, as well as many other tissue types and structures, in histological detail. Silver stain penetration was optimal for imaging structures in intact embryos E15 and younger. This silver stain method offers a powerful yet straightforward approach to visualize at high-resolution and in 3D the earliest stages of odontogenesis in situ, and demonstrates the important of studying the tooth organ in all three planes of view. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.

Pronosupination is a component of the hominoid orthograde corporal plane that enables primates to execute efficient and sure locomotion in their habitat and is an essential movement for the development of manipulative capacities. We analyze human variability in the rotational efficiency of the pronator teres muscle by applying the biomechanical model created by Galtés et al. (Am J Phys Anthropol 2008; 135:293-300; Am J Phys Anthropol 2009a; 140:589-594) to skeletal remains of a human sample (N = 29) and three nonhuman hominoid specimens (chimpanzee, gorilla, and orangutan) by means of 3D technology. We aim to examine whether there is a distinctive human pattern of rotational efficiency and determine which structural features of the upper-limb bones have the greatest influence on the determination of rotational efficiency. Our results show that the human pattern differs from efficiencies observed in nonhuman hominoids, which may be interpreted in the light of morphofunctional adaptations. We identify medial epicondylar form as the key structure of the upper-limb bones for the determination of the rotational efficiency of the forearm. Results indicate that the more medially projected epicondyle of nonhuman hominoids relative to humans leads to higher values of maximum rotational efficiency. Moreover, the orientation of the medial epicondyle determines the pronounced differences in the position of the maximum efficiencies in the pronosupination range between humans and the studied nonhuman hominoids. Proximodistal orientation of the medial epicondyle is suggested to be a more appropriate feature for distinguishing between humans and nonhuman hominoids than anteroposterior orientation and, therefore, for inferring behavioral aspects from skeletal remains and fossils of primate upper-limb bones.

Previous studies showed that in modern humans the basicranium is formed of two modules: the midline cranial base and the lateral basicranium which are integrated with the face in very different ways. The study of the relationship between these structures is of prime interest in the context of hominids craniofacial evolutionary history. In this study, we aim to test if the relationship between the midline cranial base and the face on one hand and the lateral basicranium and the face on the other hand are qualitatively and quantitatively different in modern humans and chimpanzees: two phylogenetically close but morphologically different hominids. This work is performed using three-dimensional (3D) landmarks to take into account the face and basicranium 3D shape. Modern humans and chimpanzees both exhibit a significant relationship between lateral basicranium and face, and a nonsignificant relationship between midline cranial base and face. However, the patterns of integration are different for the two species. These results underscore the essential role of the lateral basicranial shape in the setting of the facial morphology in modern humans and chimpanzees. The important differences in the patterns of integration may be related to the genetic, developmental, and functional requirements of each taxon, acquired along their respective evolution. From a common, tight, relationship between lateral basicranium, and face, each taxon may develop different patterns of integration in order to adapt to particular functions and morphologies. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.

The aim of the present study was to develop a method for three-dimensional (3D) reconstruction of the brachial plexus to study its morphology and to calculate strain and displacement in relation to changed nerve position. The brachial plexus was finely dissected and injected with contrast medium and leaden markers were implanted into the nerves at predefined places. A reverse shoulder prosthesis was inserted in a cadaveric specimen what induced positional change in the upper limb nerves. Computed tomography (CT) was performed before and after this surgical intervention. The computer assisted image processing package Mimics(R) was used to reconstruct the pre- and postoperative brachial plexus in 3D. The results show that the current interactive model is a realistic and detailed representation of the specimen used, which allows 3D study of the brachial plexus in different configurations. The model estimated strains up to 15.3% and 19.3% for the lateral and the medial root of the median nerve as a consequence of placing a reverse shoulder prosthesis. Furthermore, the model succeeded in calculating the displacement of the brachial plexus by tracking each implanted lead marker. The presented brachial plexus 3D model currently can be used in vitro for cadaver biomechanical analyses of nerve movement to improve diagnosis and treatment of peripheral neuropathies. The model can also be applied to study the exact location of the plexus in unusual upper limb positions like during axillary radiation therapy and it is a potential tool to optimize the approaches of brachial plexus anesthetic blocks.

Interest in the lateralization of the human brain is evident through a multidisciplinary number of scientific studies. Understanding volumetric brain asymmetries allows the distinction between normal development stages and behavior, as well as brain diseases. We aimed to evaluate volumetric asymmetries in order to select the best gyri able to classify right- versus left cerebral hemispheres. A cross-sectional study performed in 47 right-handed young-adults healthy volunteers. SPM-based software performed brain segmentation, automatic labeling and volumetric analyses for 54 regions involving the cerebral lobes, basal ganglia and cerebellum from each cerebral hemisphere. Multivariate discriminant analysis (DA) allowed the assembling of a predictive model. DA revealed one discriminant function that significantly differentiated left vs. right cerebral hemispheres: Wilks' λ = 0.008, χ(2) (9) = 238.837, p < .001. The model explained 99.20% of the variation in the grouping variable and depicted an overall predictive accuracy of 98.8%. With the influence of gender; the selected gyri able to discriminate between hemispheres were: middle orbital frontal gyrus (g.), angular g., supramarginal g., middle cingulum g., inferior orbital frontal g., calcarine g., inferior parietal lobule and the pars triangularis inferior frontal g. Specific brain gyri are able to accurately classify left vs. right cerebral hemispheres by using a multivariate approach; the selected regions correspond to key brain areas involved in attention, internal thought, vision and language; our findings favored the concept that lateralization has been evolutionary favored by mental processes increasing cognitive efficiency and brain capacity. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.

Introduction: Macroanatomical right-left hemispheric differences in the brain are termed asymmetries, although there is no clear information on the global influence of gender and brain-regions. The aim of this study was to evaluate the main effects and interactions of these variables on the measurement of volumetric asymmetry indices (VAIs). Materials and methods: Forty-seven healthy young-adult volunteers (23 males, 24 females) agreed to undergo brain magnetic resonance imaging in a 3T scanner. Image post processing using voxel-based volumetry allowed the calculation of 54 VAIs from the frontal, temporal, parietal and occipital lobes, limbic system, basal ganglia, and cerebellum for each cerebral hemisphere. Multivariate ANCOVA analysis calculated the main effects and interactions on VAIs of gender and brain regions controlling the effect of age. Results: The only significant finding was the main effect of brain regions (F (6, 9373.605) 44.369, P < .001; partial η2 = .101, and power of 1.0), with no significant interaction between gender and brain regions (F (6, 50.517) .239, P = .964). Conclusion: Volumetric asymmetries are present across all brain regions, with larger values found in the limbic system and parietal lobe. The absence of a significant influence of gender and age in the evaluation of the numerous measurements generated by multivariate analyses in this study should not discourage researchers to report and interpret similar results, as this topic still deserves further assessment.

The previous studies have shown that amyloid-β peptide (Aβ) was mainly found in neurons of neurodegenerative diseases, such as Alzheimer's disease (AD) and glaucoma and little is known about its expression in normal nerve cells. The aim of the present study was to investigate the expression of amyloid-β peptide 42 (Aβ-42) in retinal ganglion cells of the postnatal rats. Rats were divided into seven experimental groups: 3, 6, 13, 15, 25, 60, and 90 days postnatal groups. Rats from 15 and 25 days postnatal groups were further divided into light-exposure and non light-exposure group. Cryosections or flat-mounted retinas of rat eyes were used for testing Aβ-42 by immunocytochemistry staining. Aβ-42 expression was not observed in rats within 13 days after birth, but was easily detectable in all groups of rats over 15 days after birth. In addition, the expression of Aβ-42 in retina was increasing as the rats got older, reached to highest level in 60 days after birth. Furthermore, the expression of Aβ-42 was not detected in rats kept under dark indicating that light is required for the expression of Aβ-42 in retina. This is the first report showing that normal retinal ganglion cells express Aβ-42, and that the expression of Aβ-42 in retinal ganglion cells requires the exposure to light. These data suggest that Aβ-42 may play a important role in vision development.

Gap junction expression has been studied in the atrioventricular junction (AVJ) of many species, however, their distribution in the human AVJ is unknown. The AVJ expression of the gap junction protein connexin 43 (Cx43) is species dependent; therefore we investigated its distribution in the human AVJ. Using Masson trichrome histology, we reconstructed the AVJ of three normal human hearts and one with dilated cardiomyopathy in three dimensions. Cx43 was immunolabeled with vimentin and alpha-actinin to determine the cellular origin of Cx43 and was quantified in the following structures: interatrial septum (IAS), His bundle, compact node (CN), lower nodal bundle (LNB), leftward and rightward nodal extensions (LE and RE), and inferior, endocardial, and left-sided transitional cells. Histology revealed two nodal extensions in three of four hearts. Cx43 was found in the myocytes, but not fibroblasts, of the AVJ. LE and CN Cx43 was lower than the IAS (P < 0.05) and the RE, LNB, and His all expressed Cx43 similarly, with approximately half of IAS expression (RE: 44 +/- 36%; LNB: 50 +/- 26%; His: 48 +/- 12%, P = NS compared with IAS). Cx43 levels in transitional cells were similar to the IAS (P = not significant). Cx43 was found in myocytes of the human AVJ, and its expression pattern delineates two separate continuous structures: one consists of the LE and CN with little Cx43, and the other consists of the His, LNB, and RE expressing approximately half the Cx43 of the IAS. The differential Cx43 expression may provide each structure with unique conduction properties, contributing to arrhythmias arising from the AVJ.

We have used the histochemical and immunohistochemical staining methods and maps of gene expression to analyze the structure of the inferior olive of the C57BL mouse. As in other mammals, the inferior olive of the C57BL mouse contains three major nuclei, the medial nucleus, the principal nucleus, and the dorsal nucleus. The medial nucleus can be divided into a rostral medial nucleus and a more complex caudal part, which is formed by subnuclei C, B, A, the cap of Kooy, and the beta subnucleus. The principal nucleus includes the major principal nucleus and the arcuate subnucleus. Most of the inferior olive neurons are small to medium size, the smallest of which are found in the arcuate subnucleus. Calbindin and the vesicular glutamate transporter 2 gene are expressed in nearly all inferior olive neurons, but acetlycholinesterase, glutamate decarboxylase 1 gene, cocaine- and amphetamine-regulated transcript protein prepropeptide gene, galanin gene, and calretinin are selectively expressed within different subnuclei. These findings are consistent with a pattern of extensive functional differentiation among the neuron groups of the inferior olive. Anat Rec, 297:289-300, 2014. © 2014 Wiley Periodicals, Inc.

Staphylococcal nuclease domain containing 1 (SND1) is a ubiquitously expressed multifunctional protein involved in transcriptional regulation, RNA splicing and RNA metabolism. Ectopic expression of SND1 has been observed in various tumors including colon cancer, breast cancer, prostate cancer and hepatocellular carcinoma (HCC), indicating a positive role of SND1 in tumor initiation and progression. However, the exact role of SND1 in cancers has not been thoroughly investigated. In the present study, we investigated the role of SND1 in HCC. Immunohistochemistry analysis revealed that the expression level of SND1 was higher in HCC tissues than in adjacent nontumor tissues. Stable knock-down of SND1, performed on the HCC cell line SMMC-7721 using shRNA lentiviral expression system, led to reduced cell proliferation, clone formation and tumor formation in nude mice. The insulin-like growth factor (IGF) signaling pathway was frequently dysregulated in HCC, which could facilitate tumor progression. Screening of gene expression levels of the IGF pathway, using real-time PCR, revealed that a decrease in SND1 expression could increase the expression of IGF-binding protein 3 (IGFBP3), which can negatively regulate activation of the IGF pathway by restricting interactions between IGF and IGF receptors. Results from previous studies showed that the downregulation of IGFBP3 expression is a common feature in HCC, and the upregulation of IGFBP3 expression could suppress HCC cells proliferation. We further confirmed that stable knock-down of IGFBP3 could promote SMMC-7721 cells proliferation. Therefore, we concluded that SND1 could affect SMMC-7721 cells proliferation by regulating IGFBP3 expression and IGF signaling pathway. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.

Annexin A1 (ANXA1) is a multifunctional molecule, which mediates various important physiologic processes depending on its subcelluar localization. The purpose of this study was to investigate the expression of ANXA1 level and its subcellular localization in paired clinical samples of gastric adenocarcinoma and adjacent normal counterpart. The study also assesses the clinical significance of ANXA1 subcelluar localization in gastric adenocarcinoma. A total of 104 paired resected gastric adenocarcinoma and corresponding normal specimens were collected in this study. Expression of ANXA1 was examined by immunohistochemical staining. Both cytoplasmic and nuclear ANXA1 expression levels and their correlation with clinicopathological parameters were assessed. ANXA1 protein expression was positive in 72 of 104 (69.2%) normal tissues and 47 of 104 (45.2%) gastric adenocarcinoma tissues. ANXA1 staining was predominantly localized in the cytoplasm in all 72 ANXA1-positive normal specimens, whereas 12 ANXA1-positive gastric adenocarcinoma specimens showed positive nuclear staining. The positive nuclear staining correlated well with serosal invasion, peritoneal dissemination and TNM stage. Cases with positive nuclear staining presented more peritoneal dissemination (41.7%, 5/12) than those with negative nuclear staining (8.7%, 8/92; P = 0.007). A logistic regression model revealed that positive ANXA1 nuclear staining had an independent association with peritoneal dissemination (P = 0.039; hazards ratio, 9.499; 95% confidence interval, 1.159-77.815). These results indicated that ANXA1 is expressed in both gastric adenocarcinoma and normal tissues. In gastric adenocarcinoma tissues ANXA1 is expressed both in cytoplasm and nucleus and its nuclear localization correlates with advanced disease stage and peritoneal dissemination.

Several Eph receptors, prominently EphA4 and EphA7, and their corresponding ligands are known to influence neocortical development, including topographic sorting of thalamocortical axons within primary somatosensory cortex (SI). This study investigated postnatal expression of a ligand that can bind to these receptors, ephrin-A2. Quantitative methods revealed that expression of ephrin-A2 mRNA in SI reached maximum levels on postnatal day (P) 4 and dropped thereafter to background by P18. Ephrin-A2 mRNA expression assessed by in situ hybridization qualitatively revealed a similar time course and localized the expression pattern primarily in two broad laminae in SI, comprising the supragranular and infragranular layers, and with additional expression in the subplate. This expression pattern was investigated in greater detail using immunohistochemistry for ephrin-A2 protein. Immunoreactivity generally showed the same laminar distribution as seen with in situ hybridization, except that it persisted longer, lasting to approximately P14. Expression in the cortical plate was low or absent within presumptive layer IV, and it remained so as cortical lamination progressed. Double-labeling immunohistochemistry with confocal microscopy revealed that cortical neurons were the principal elements expressing ephrin-A2 protein. These findings are consistent with possible involvement of ephrin-A2, in concert with one or more Eph receptors, in influencing arbor development of thalamocortical axons at cortical layer IV boundaries.

Survivin has been implicated in tumor genesis, progression, and resistance to anticancer agents. However, the precise regulatory mechanism for survivin expression is not thoroughly defined. In this study, we showed that Sp1 was co-overexpressed with survivin in adenocarcinoma of lung cells A549, but not in differentiated human bronchial epithelial cells 4F0439 or small airway epithelial cells 3F1584. Subsequently, transfection experiments demonstrated that the inhibition of Sp1 signaling suppressed survivin expression in A549 cells, whereas Sp1 overexpression increased the level of survivin protein as well as its mRNA. We also found that Sp1 could decrease capase-9 activity, which is shown to be suppressed by survivin during apoptosis inhibition. Finally, Luciferase activity and ChIP assays revealed that Sp1 activated survivin promoter by direct interaction with it. Taken together, our data suggest Sp1 plays a potent role in the upregulation of survivin expression in lung cancer cells at the transcriptional level.