Search

The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.

Simple Summary It is generally accepted that fibroblasts represent a heterogeneous population of cells with different functions depending on the cell type. Although numerous reports have stated that cancer-associated fibroblast (CAF) promotes cancer progression, few studies have shown that they inhibit cancer progression. We propose that CAFs derived from some HNSCC patients is less effective in promoting cancer progression than CAFs from other patients and that specific collagen proteins may be involved in this process. Abstract Background: The critical effect of the tumor microenvironment on cancer progression is well recognized. Recent research suggests that the cancer-promoting properties of the tumor stroma may be attributed to fibroblasts. However, the effect of cancer-associated fibroblast (CAF) on the progression of head and neck squamous cell carcinoma (HNSCC) is not well known. Methods: From the immunohistochemical analysis of head and neck squamous cell carcinoma (HNSCC) tissues, we divided CAF into two groups depending on the presence or absence of a well-demarcated boundary between epithelial cancer cells and the surrounding extracellular matrix (ECM). Primary culture of CAF was performed, followed by co-transplantation with HNSCC cells into mice oral mucosa, and the tumorigenesis was compared. The mRNA expression patterns between these two CAF groups were compared using DNA microarray analysis. Results: CAFs from cancer tissues that showed no demarcation between ECM and epithelial cancer cells (CAF-Promote) tended to stimulate Matrigel invasion of HNSCC cells. Conversely, CAFs from cancer tissues that showed a boundary with epithelial cancer cells (CAF-Delay) caused no remarkable increase in Matrigel invasion. Compared with CAF-P, CAF-D is less effective in promoting FaDu tumorigenicity in the mouse model. In DNA microarray analysis, COL3A1 and COL6A6 showed particularly high expression in the CAF-D group. Conclusions: These cancer stroma-derived collagen proteins might delay the HNSCC progression. These findings are expected to provide vital information for predicting HNSCC prognosis and developing drug targets in the future.

In cone-beam computed tomography (CBCT), the minimum threshold of the gray value of segmentation is set to convert the CBCT images to the 3D mesh reconstruction model. This study aimed to assess the accuracy of image registration of optical scans to 3D CBCT reconstructions created by different thresholds of grey values of segmentation in partial edentulous jaw conditions. CBCT of a dentate jaw was reconstructed to 3D mesh models using three different thresholds of gray value (&minus, 500, 500, and 1500), and three partially edentulous models with different numbers of remaining teeth (4, 8, and 12) were made from each 3D reconstruction model. To merge CBCT and optical scan data, optical scan images were registered to respective 3D reconstruction CBCT images using a point-based best-fit algorithm. The accuracy of image registration was assessed by measuring the positional deviation between the matched 3D images. The Kruskal&ndash, Wallis test and a post hoc Mann&ndash, Whitney U test with Bonferroni correction were used to compare the results between groups (&alpha, = 0.05). The correlations between the experimental factors were calculated using the two-way analysis of variance test. The positional deviations were lowest with the threshold of 500, followed by the threshold of 1500, and then &minus, 500. A significant interaction was found between the threshold of gray values and the number of remaining teeth on the registration accuracy. The most significant deviation was observed in the arch model with four teeth reconstructed with a gray-value threshold of &minus, 500. The threshold for the gray value of CBCT segmentation affects the accuracy of image registration of optical scans to the 3D reconstruction model of CBCT. The appropriate gray value that can visualize the anatomical structure should be set, especially when few teeth remain in the dental arch.

Recognition memory, often compromised in psychiatric disorders, is a major component of declarative memory, which permits the realization that an event, object or social subject has been previously encountered. The CA2 region of the dorsal hippocampus (dCA2) is involved in social memory and responds to novel objects, in time and space. However, it remains unclear how these neurons encode either social or inanimate object recognition. Here, we show that in dCA2, encoding of social recognition memory entails suppression of pyramidal neurons’ activity leading to a sparse representation of the familiar conspecific. We discuss the neural coding scheme by which dCA2 pyramidal neurons contribute to social memory.

We evaluated potential biomarkers in human whole saliva for the early diagnosis of oral squamous cell carcinoma (OSCC). We selected 30 candidate genes with relevance to cancer from recent reports in PubMed. Saliva samples were obtained from 34 non-tumor control and 33 OSCC patients. Real-time PCR was performed, and mRNA levels were compared. Normalized mRNA levels of six genes (NGFI-A binding protein 2 (NAB2), cytochrome P450, family 27, subfamily A, polypeptide 1 (CYP27A1), nuclear pore complex interacting protein family, member B4 (NPIPB4), monoamine oxidase B (MAOB), sialic acid acetyltransferase (SIAE), and collagen, type III, alpha 1 (COL3A1)) were significantly lower in saliva of OSCC patients. Receiver operating characteristics (ROC) analysis was used to individually evaluate the predictive power of the potential biomarkers for OSCC diagnosis. The area under the curve (AUC) values were evaluated for the OSCC vs. non-tumor groups via univariate ROC analyses, as well as multivariate ROC analyses of combinations of multiple potential biomarkers. The combination of CYP27A1 + SIAE showed a favorable AUC value of 0.84. When we divided saliva samples into two groups according to age using a 60-year cut-off, with OSCC patients and controls evaluated together, the AUC of MAOB&ndash, NAB2 was more predictive of OSCC in the under-60 group (AUC, 0.91, sensitivity, 0.92, and specificity, 0.86) than any other gene combination. These results are expected to aid the early diagnosis of OSCC, especially in patients under 60 years of age. While more studies with larger numbers of patients are necessary, our result suggest that salivary mRNA would be a potent biomarker for early OSCC diagnosis.

Extracellular vesicles (EVs) are evolutionary well-conserved nano-sized membranous vesicles that are secreted by both prokaryotic and eukaryotic cells. Recently, they have gained great attention for their proposed roles in cell-to-cell communication, and as biomarkers for human disease. In particular, small RNAs (sRNAs) contained within EVs have been considered as candidate interspecies-communication molecules, due to their demonstrated capacity to modulate gene expression in multiple cell types and species. While research into this field is in its infancy, elucidating the mechanisms that underlie host–microbe interactions and communications promises to impact many fields of biological research, including human health and medicine. Thus, this review discussed the results of recent studies that have examined the ways in which EVs and sRNAs mediate ‘microbe–host’ and ‘host–microbe’ interspecies communication.

MicroRNAs are small non-coding RNAs that bind to the 3′-untranslated region of target mRNAs and have transcriptional or translational inhibitory function in eukaryotes. Before microRNAs were widely known, bacterial non-coding small RNAs around 50–200 nt in length were discovered whose mechanism of action resembled that of microRNAs. Recently, RNAs that are of similar size to or smaller than microRNAs have been discovered in bacteria and indeed, this class of small RNAs have been found throughout all domains of life. Moreover, recent findings suggest that these tiny RNAs can be released via extracellular vesicles (such as exosomes in eukaryotes and outer membrane vesicles in bacteria), which in turn heralds a new field of research, interkingdom communication. This review discusses two similar classes of small RNAs in evolutionarily distinct eukaryotes and bacteria. In addition to their biogenesis and regulation, we discuss small RNA vehicles and their secretion. Impact statement The possible endogenous functions of small RNAs such as regulatory small RNAs in bacteria and microRNAs in eukaryotes have been extensively studied since they were first discovered. However, their powerful functions should not be seen as limited to their cells of origin. Recently, several papers have demonstrated that small RNAs function as signaling molecules between cells. This is possible because small RNAs can be shuttled around after being incorporated into environmentally protective extracellular vesicles. It is now clearly plausible that secreted small RNAs can regulate other types of cells through biofluids. Given their “common molecule” status, the role of small RNAs in mediating bacteria-human crosstalk is an emerging and competitive area of genetic research. This review provides insight into the function of small RNAs in intercellular and even interkingdom communication.

Background: DJ-1 (PARK7) was reported as an oncogene in a Ras-dependent manner. Recent studies have shown that DJ-1 stimulates cell proliferation, cell invasion, and cancer metastasis. However, the molecular mehchanism by which DJ-1 induces cancer cell invasion and metastasis remains unclear. Methods: Breast cancer cells were transfected with DJ-1 siRNA or DJ-1 overexpression to investigate the effect of DJ-1 on KLF17 expression. ID-1 luciferase promoter assay was performed to evaluate DJ-1-dependent KLF17 expression changes. In addition, Epistasis analysis of DJ-1 and KLF17 was performed to evaluate their regulatory interactions. Ras inhibitors were pretreated to determine whether DJ-1 regulates cell invasion in a Ras-dependent manner. Results: In the present study, we found increased DJ-1 expression in highly invasive breast cancer cells as compared with non-metastatic cells. Furthermore, DJ-1 promoted breast cancer cell invasion by downregulating E-cadherin and increasing Snail expression. Interestingly, exogenous DJ-1 overexpression markedly decreased mRNA and protein expression of KLF17, the EMT negative regulator. These data were confirmed by ID-1 promoter activity, which is directly regulated by DJ-1-dependent KLF17 transcription factor. Epistasis analysis showed that KLF17 overexpression overcomes increased cell invasion by DJ-1, suggesting that KLF17 might be one of the downstream signalling molecules of DJ-1. Acceleration of cell invasion by DJ-1 was alleviated by Ras inhibitors, suggesting that DJ-1 cooperates with Ras to increase cell invasion. Conclusion: Altogether, these data suggest for the first time that DJ-1 acts as an EMT-positive regulator in breast cancer cells via regulation of the KLF17/ID-1 pathway.

During microRNA (miRNA) biogenesis, one strand of a 21-23 nucleotide RNA duplex is preferentially selected for entry into an RNA-induced silencing complex (RISC). The other strand, known as the miRNA* species, is typically thought to be degraded. Previous studies have provided miRNA* selection models, but it remains unclear how the dominance of one arm arises during the biogenesis of miRNA. Using miRNA sponge-like methods, we cloned four tandem target sequences (artificial target) of miR-7b* and then measured miR-7b* expression levels after transfection of the artificial target. miR-7b* levels were found to significantly increase after transfection of the artificial target. We postulate that the abundance of target transcripts drives miRNA arm selection.

Oxytocin (Oxt), produced in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) for transport to and release from the posterior pituitary, was originally discovered through its role in lactation and parturition. Oxt also plays important roles in the central nervous system by influencing various behaviors. MicroRNAs (miRNAs), endogenous regulators of many genes, are a class of small noncoding RNAs that mediate post-transcriptional gene silencing. We performed miRNA expression profiling of the mouse hypothalamus by deep sequencing. Among the sequenced and cross-mapped small RNAs, expression of known miRNAs and unknown miRNAs candidates were analyzed. We investigated in detail one miRNA, miR-24, and found that it is a novel regulator of Oxt and controls both transcript and peptide levels of Oxt. These results provide insights into potential neurohypophysial hormone regulation mediated by miRNAs.

Estrogen uses two mechanisms to exert its effect on the skeleton: it inhibits bone resorption by osteoclasts and, at higher doses, can stimulate bone formation. Although the antiresorptive action of estrogen arises from the inhibition of the MAPK JNK, the mechanism of its effect on the osteoblast remains unclear. Here, we report that the anabolic action of estrogen in mice occurs, at least in part, through oxytocin (OT) produced by osteoblasts in bone marrow. We show that the absence of OT receptors (OTRs) in OTR(-/-) osteoblasts or attenuation of OTR expression in silenced cells inhibits estrogen-induced osteoblast differentiation, transcription factor up-regulation, and/or OT production in vitro. In vivo, OTR(-/-) mice, known to have a bone formation defect, fail to display increases in trabecular bone volume, cortical thickness, and bone formation in response to estrogen. Furthermore, osteoblast-specific Col2.3-Cre(+)/OTR(fl/fl) mice, but not TRAP-Cre(+)/OTR(fl/fl) mice, mimic the OTR(-/-) phenotype and also fail to respond to estrogen. These data attribute the phenotype of OTR deficiency to an osteoblastic rather than an osteoclastic defect. Physiologically, feed-forward OT release in bone marrow by a rising estrogen concentration may facilitate rapid skeletal recovery during the latter phases of lactation.

A wealth of studies has implicated oxytocin (Oxt) and its receptors (Oxtr) in the mediation of social behaviors and social memory in rodents. It has been suggested that failures in this system contribute to deficits in social interaction that characterize autism spectrum disorders (ASD). In the current analyses, we investigated the expression of autism-related behaviors in mice that lack the ability to synthesize the oxytocin receptor itself, Oxtr knockout (KO) mice, as compared to their wild-type (WT) littermates. In the visible burrow system, Oxtr KO mice showed robust reductions in frontal approach, huddling, allo-grooming, and flight, with more time spent alone, and in self-grooming, as compared to WT. These results were corroborated in the three-chambered test: unlike WT, Oxtr KO mice failed to spend more time in the side of the test box containing an unfamiliar CD-1 mouse. In the social proximity test, Oxtr KO mice showed clear reductions in nose to nose and anogenital sniff behaviors oriented to an unfamiliar C57BL/6J (B6) mouse. In addition, our study revealed no differences between Oxtr WT and KO genotypes in the occurrence of motor and cognitive stereotyped behaviors. A significant genotype effect was found in the scent marking analysis, with Oxtr KO mice showing a decreased number of scent marks, as compared to WT. Overall, the present data indicate that the profile for Oxtr KO mice, including consistent social deficits, and reduced levels of communication, models multiple components of the ASD phenotype. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.

Objective: To examine the effect of garlic extract on the biofilm formation by Streptococcus mutans on orthodontic wire and on glucosyltransferase gene expression. Materials and Methods: Growth inhibition of oral bacteria was tested after 50 µL of garlic extract was placed on an agar plate. The minimum inhibitory concentration (MIC) of garlic extract on S mutans growth was first determined. After cultivating streptococci in biofilm medium (BM)-sucrose with garlic extract and orthodontic wire, adenosine triphosphate (ATP) measurement and viable cell counting was performed from the bacteria attached on the wire. Scanning electron microscopy (SEM) analysis of morphology was observed on bacterial cells attached to orthodontic wire. The effect of garlic extract on gene expression was evaluated using quantitative real-time polymerase chain reaction (PCR) of glucosyltransferase. Results: Though garlic extract had a clear antibacterial effect on all microorganisms, it also enhanced S mutans attachment on orthodontic wire. Low concentration of garlic extract also increased glucosyltransferase gene expression of S mutans. Conclusions: Despite its antibacterial function, garlic extract increases biofilm formation by S mutans to orthodontic wire, likely through upregulation of glucosyltransferase expression. Garlic extract may thus play an important role in increased bacterial attachment to orthodontic wires.

Oxytocin (Oxt) is a nonapeptide hormone best known for its role in lactation and parturition. Since 1906 when its uterine-contracting properties were described until 50 years later when its sequence was elucidated, research has focused on its peripheral roles in reproduction. Only over the past several decades have researchers focused on what functions Oxt might have in the brain, the subject of this review. Immunohistochemical studies revealed that magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei are the neurons of origin for the Oxt released from the posterior pituitary. Smaller cells in various parts of the brain, as well as release from magnocellular dendrites, provide the Oxt responsible for modulating various behaviors at its only identified receptor. Although Oxt is implicated in a variety of "non-social" behaviors, such as learning, anxiety, feeding and pain perception, it is Oxt's roles in various social behaviors that have come to the fore recently. Oxt is important for social memory and attachment, sexual and maternal behavior, and aggression. Recent work implicates Oxt in human bonding and trust as well. Human disorders characterized by aberrant social interactions, such as autism and schizophrenia, may also involve Oxt expression. Many, if not most, of Oxt's functions, from social interactions (affiliation, aggression) and sexual behavior to eventual parturition, lactation and maternal behavior, may be viewed as specifically facilitating species propagation.

The Sox genes define a family of transcription factors that play a key role in the determination of cell fate during development. The preferential expression of the Sox15 in the myogenic precursor cells led us to suggest that the Sox15 is involved in the specification of myogenic cell lineages or in the regulation of the fusion of myoblasts to form myotubes during the development and regeneration of skeletal muscle. To identify the physiological function of Sox15 in mice, we disrupted the Sox15 by homologous recombination in mice. Sox15-deficient mice were born at expected ratios, were healthy and fertile, and displayed normal long-term survival rates. Histological analysis revealed the normal ultrastructure of myofibers and the presence of comparable amounts of satellite cells in the skeletal muscles of Sox15(-/-) animals compared to wild-type animals. These results exclude the role of Sox15 in the development of satellite cells. However, cultured Sox15(-/-) myoblasts displayed a marked delay in differentiation potential in vitro. Moreover, skeletal muscle regeneration in Sox15(-/-) mice was attenuated after application of a crush injury. These results suggest a requirement for Sox15 in the myogenic program. Expression analyses of the early myogenic regulated factors MyoD and Myf5 showed the downregulation of the MyoD and upregulation of the Myf5 in Sox15(-/-) myoblasts. These results show an increased proportion of the Myf5-positive cells and suggest a role for Sox15 in determining the early myogenic cell lineages during skeletal muscle development.