In vitro and in vivo suppression of SARS-CoV-2 replication by a modified, short, cell-penetrating peptide targeting the C-terminal domain of the viral spike protein.

Peptides are promising therapeutic agents for COVID-19 because of their specificity, easy synthesis, and ability to be fine-tuned. We previously demonstrated that a cell-permeable peptide corresponding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike C-terminal domain (CD) inhibits the interaction between viral spike and nucleocapsid proteins that results in SARS-CoV-2 replication in vitro. Here, we used docking studies to design R-t-Spike CD(D), a more potent short cell-penetrating peptide composed of all D-form amino acids and evaluated its inhibitory effect against the replication of SARS-CoV-2 S clade and other variants. R-t-Spike CD(D) was internalized into Vero cells and Calu-3 cells and suppressed the replication of SARS-CoV-2 S clade, delta variant, and omicron variant with higher potency than the original peptide. In hemizygous K18-hACE2 mice, intratracheal administration of R-t-Spike CD(D) effectively delivered the peptide to the trachea and lungs, whereas intranasal administration delivered the peptide mostly to the upper respiratory system and stomach, and a small amount to the lungs. Administration by either route reduced viral loads in mouse lungs and turbinates. Furthermore, intranasally administered R-t-Spike CD(D) mitigated pathological change in the lungs and increased the survival of mice after infection with the SARS-CoV-2 S clade or delta variant. Our data suggest that R-t-Spike CD(D) has potential as a therapeutic agent against SARS-CoV-2 infection.

Outbred Mice with Streptozotocin-Induced Diabetes Show Sex Differences in Glucose Metabolism.

Outbred mice (ICR) with different genotypes and phenotypes have been reported to be more suitable for scientific testing than inbred mice because they are more similar to humans. To investigate whether the sex and genetic background of the mice are important factors in the development of hyperglycemia, we used ICR mice and divided them into male, female, and ovariectomized female (FOVX) groups and treated them with streptozotocin (STZ) for five consecutive days to induce diabetes. Our results show that fasting blood glucose and hemoglobin A1c (HbA1c) levels were significantly higher in diabetes-induced males (M-DM) and ovariectomized diabetes-induced females (FOVX-DM) than in diabetes-induced females (F-DM) at 3 and 6 weeks after STZ treatment. Furthermore, the M-DM group showed the most severe glucose tolerance, followed by the FOVX-DM and F-DM groups, suggesting that ovariectomy affects glucose tolerance in female mice. The size of pancreatic islets in the M-DM and FOVX-DM groups was significantly different from that of the F-DM group. The M-DM and FOVX-DM groups had pancreatic beta-cell dysfunction 6 weeks after STZ treatment. Urocortin 3 and somatostatin inhibited insulin secretion in the M-DM and FOVX-DM groups. Overall, our results suggest that glucose metabolism in mice is dependent on sex and/or genetic background.

Age-related hearing loss was accelerated by apoptosis of spiral ganglion and stria vascularis cells in ApoE KO mice with hyperglycemia and hyperlipidemia.

Age-related hearing loss (ARHL) is associated with diabetes and/or dyslipidemia in humans. However, the detailed mechanism for the development of ARHL by diabetes and/or dyslipidemia has not been elucidated. In this study, we investigated the etiology of ARHL in apolipoprotein E (ApoE)-deficient mice with diabetes and dyslipidemia. The atherosclerotic CD-STZ (mice fed with a control diet and received an STZ injection), WD-con (mice fed with a western diet), and WD-STZ (mice fed with a western diet and received an STZ injection) mice showed a 2.4-, 4.9-, and 6.8-fold larger area, respectively, occupied by lesions throughout the aorta compared with the CD-con mice. A significantly larger area under the curve (AUC) was observed in the STZ-treated groups than in the non-treated groups based on the oral glucose tolerance test (OGTT). At 20 weeks of age, HbA1c levels were significantly higher in the CD-STZ and WD-STZ mice than in the CD-con and WD-con mice. In all the groups, the auditory brainstem response (ABR) thresholds of the 16-week-old mice were significantly higher compared with those of the 8-week-old mice. In particular, in the WD-STZ mice, the ABR thresholds of the left and right ears reached the maximum decibel peak equivalent sound pressure levels (130 dBpeSPL), which is a sign of deafness. The apoptotic spiral ganglion neurons (SGNs) of the WD-STZ mice were significantly increased compared with those of the other three groups, indicating that SGN apoptosis resulted in hearing loss in STZ-induced diabetic ApoE KO mice fed with a WD. A significant loss of the stria vascularis cells was observed in the WD-STZ group compared with the CD-con mice. In the organ of Corti, few apoptotic hair cells were found in all the groups; however, no significant difference was observed. Therefore, we consider that the reduced hearing ability in the STZ-treated and WD-fed groups was attributed to the damage to the SGN and stria vascularis in the cochlea. Thus, our results indicated that ototoxicity by diabetes and/or dyslipidemia accelerated ARHL in ApoE KO mice, thereby suggesting the importance of appropriate treatment of patients with diabetes and/or dyslipidemia to prevent ARHL.

Differential effect of SARS-CoV-2 infection on stress granule formation in Vero and Calu-3 cells.

Stress granule formation is induced by numerous environmental stressors, including sodium arsenite treatment and viral infection. Accordingly, stress granules can inhibit viral propagation and function as part of the antiviral host response to numerous viral infections. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antagonizes stress granule formation, in part, via interaction between SARS-CoV-2 nucleocapsid (N) protein and Ras-GTPase-activating SH3-domain-binding protein 1 (G3BP1). However, it is unclear whether there are differential effects in different cell types. In this study, we assessed interaction between the N protein of SARS-CoV-2 S clade and G3BP1/2 in Vero and Calu-3 cells and investigated the effect of various SARS-CoV-2 strains on sodium arsenite-induced stress granule formation. Our data show that SARS-CoV-2 S clade N protein interacts with both G3BP1 and G3BP2 more strongly in Calu-3 vs. Vero cells. Consistent with this observation, infection with SARS-CoV-2 S clade induces stress granule formation in Vero but not in Calu-3 cells. However, infection with SARS-CoV-2 S clade, as well as other SARS-CoV-2 variants, inhibits sodium arsenite-induced stress granule formation in both cell lines. Taken together, our results show differential effects of SARS-CoV-2 infection on stress granule formation that is dependent on host cell type, rather than virus strain type.

Elucidating the characteristics of Mx1 and resistance to influenza A virus subtype H1N1 in the newly developed KWM/Hym mice.

BACKGROUND: Inbred mice have several advantages, including genetic similarity to humans, a well-established gene manipulation system, and strong tolerance to inbreeding. However, inbred mice derived from a limited genetic pool have a small genetic diversity. Thus, the development of new inbred strains from wild mice is needed to overcome this limitation. Hence, in this study, we used a new strain of inbred mice called KWM/Hym. We sequenced the Mx1 gene to elucidate the genetic diversities of KWM/Hym mice and observed the biological alterations of the Mx1 protein upon influenza A infection. RESULTS: The Mx1 gene in KWM/Hym mice had 2, 4, and 38 nucleotide substitutions compared to those in the Mx1 gene in A2G, CAST/EiJ, and Mus spretus mice, respectively. Moreover, the Mx1 protein in KWM/Hym mice had 2 and 25 amino acid substitutions compared to those in the Mx1 protein in CAST/EiJ and M. spretus mice, respectively. To elucidate the function of the Mx1 protein, we inoculated the influenza A virus (A/WSN/1933) in KWM/Hym mice. Nine days after infection, all infected KWM/Hym mice survived without any weight loss. Four days after infection, the lungs of the infected KWM/Hym mice showed mild alveolitis and loss of bronchiolar epithelium; however, the pulmonary viral titers of the infected KWM/Hym mice were significantly lower than that in the infected BALB/c mice (2.17 × plaque-forming units mL-1). CONCLUSIONS: Our results demonstrate that the KWM/Hym mice are resistant to influenza A virus infection. Further, these mice can be used as a model organism to understand the mechanism of influenza A virus susceptibility.

Silk Fibroin Promotes the Regeneration of Pancreatic ß-Cells in the C57BL/KsJ-Leprdb/db Mouse.

Diabetes mellitus is a chronic metabolic disease, and its progression leads to serious complications. Although various novel therapeutic approaches for diabetes mellitus have developed in the last three decades, its prevalence has been rising more rapidly worldwide. Silk-related materials have been used as anti-diabetic remedies in Oriental medicine and many studies have shown the effects of silk fibroin (SF) in both in vitro and in vivo models. In our previous works, we reported that hydrolyzed SF improved the survival of HIT-T15 cells under high glucose conditions and ameliorated diabetic dyslipidemia in a mouse model. However, we could not provide a precise molecular mechanism. To further evaluate the functions of hydrolyzed SF on the pancreatic ß-cell, we investigated the effects of hydrolyzed SF on the pancreatic ß-cell proliferation and regeneration in the mouse model. Hydrolyzed SF induced the expression of the proliferating cell nuclear antigen (PCNA) and reduced the apoptotic cell population in the pancreatic islets. Hydrolyzed SF treatment not only induced the expression of transcription factors involved in the pancreatic ß-cell regeneration in RT-PCR results but also increased neurogenin3 and Neuro D protein levels in the pancreas of those in the group treated with hydrolyzed SF. In line with this, hydrolyzed SF treatment generated insulin mRNA expressing small cell colonies in the pancreas. Therefore, our results suggest that the administration of hydrolyzed SF increases the pancreatic ß-cell proliferation and regeneration in C57BL/KsJ-Leprdb/db mice.

Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet.

Age-related hearing loss (ARHL) is a neurodegenerative disease associated with an aged population. ARHL is influenced by biological factors such as aging, sex difference, and atherosclerosis. The mechanisms of ARHL caused by atherosclerosis have not been previously determined in apolipoprotein E knockout (ApoE KO) male mice. To investigate the onset and cause of the hearing loss, ApoE KO male mice were treated with a western diet (ApoE KO-WD) for 16 weeks. The lipid profile, atherosclerotic plaques throughout the aorta, and auditory brainstem response (ABR) thresholds were measured in the ApoE KO-WD male mice. The expression of S100 calcium-binding protein B (S100B), a neuronal damage biomarker, was also observed. Reactive oxygen species (ROS) and apoptosis rates were detected in the cochlea of the ApoE KO male mice. Atherosclerotic plaques on the aorta and ABR thresholds were significantly increased in the ApoE KO-WD male mice at 24 weeks of age. ABR thresholds had a statistically significant positive correlation with the area of atherosclerotic plaques (r = 0.783, p = 0.013) in male mice at 24 weeks of age. S100B protein expression and the dihydroethidium (DHE) reaction to ROS in the cochlear spiral ganglion neurons (SGNs) were significantly increased in the ApoE KO and ApoE KO-WD male mice. Cells positive for active caspase-3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) in the SGNs were significantly increased in ApoE KO-WD male mice indicating an increased rate of cellular apoptosis. In conclusion, ROS in the SGNs were activated by increased S100B expression in ApoE KO-WD male mice, and this resulted in an increased apoptosis rate. Thus, hearing loss began at 16 weeks in ApoE KO-WD male mice. Our results suggest that the ApoE KO-WD male mice are a suitable animal model for studying ARHL associated with exacerbated atherosclerosis.

Metagenomic Analysis of the Gut Microbiota of Wild Mice, a Newly Identified Reservoir of Campylobacter.

Campylobacter, the most common etiologic agent of zoonotic gastroenteritis in humans, is present in many reservoirs including livestock animals, wildlife, soil, and water. Previously, we reported a novel Campylobacter jejuni strain SCJK02 (MLST ST-8388) from the gut of wild mice (Micromys minutus) using culture-dependent methods. However, due to fastidious growth conditions and the presence of viable but non-culturable Campylobacter spp., it is unclear whether M. minutus is a Campylobacter reservoir. This study aimed to: 1) determine the distribution and proportion of Campylobacter spp. in the gut microbiota of wild mice using culture-independent methods and 2) investigate the gut microbiota of wild mice and the relationship of Campylobacter spp. with other gut microbes. The gut microbiota of 38 wild mice captured from perilla fields in Korea and without any clinical symptoms (18 M. minutus and 20 Mus musculus) were analyzed. Metagenomic analysis showed that 77.8% (14 of 18) of the captured M. minutus harbored Campylobacter spp. (0.24-32.92%) in the gut metagenome, whereas none of the captured M. musculus carried Campylobacter spp. in their guts. Notably, 75% (6 of 8) of M. minutus determined to be Campylobacter-negative using culture-dependent methods showed a high proportion of Campylobacter through metagenome analysis. The results of metagenome analysis and the absence of clinical symptoms suggest that Campylobacter may be a component of the normal gut flora of wild M. minutus. Furthermore, linear discriminant analysis (LDA) showed that Campylobacter was the most enriched genus in the gut microbiota of M. minutus (LDA score, 5.37), whereas Lactobacillus was the most enriched genus in M. musculus (LDA score, -5.96). The differences in the presence of Campylobacter between the two species of wild mice may be attributed to the differential abundance of Campylobacter and Lactobacillus in their respective gut microbiota. In conclusion, the results indicate that wild M. minutus may serve as a potential Campylobacter reservoir. This study presents the first metagenomics analysis of the M. minutus gut microbiota to explore its possible role as an environmental Campylobacter reservoir and provides a basis for future studies using culture-independent methods to determine the role of environmental reservoirs in Campylobacter transmission.

Comparing the Superficial Vasculature of the Central Nervous System in Six Laboratory Animals: A Hypothesis About the Role of the "Circle of Willis".

We provide images of the entire central nervous system vasculature, and compare the anatomical findings in six different laboratory animals. A detailed understanding of the specific anatomy for each is important in the design of experimental modeling and for understanding the specific function of each target organ. Six different types of animals, the Korean wild mouse, C57BL/6J mouse, F344 rat, mongolian gerbil, Syrian hamsters, and guinea pigs, were included. To stain the blood vessels in each of the animals, Alcian blue reagent was used to perfuse each species. The bifurcation and anastomotic patterns of the anterior cerebral arteries differed in each species. The vascular supply to the olfactory nerve was visualized as a single artery supplying both olfactory nerves, and arteries supplying the lateral portion of the olfactory nerves originating from the olfactory bulb area. The posterior communicating arteries of the six animals demonstrated unique morphologies. The shape of the hypophyseal portal system varied by species. Most animals used in this study had a hexagonal Circle of Willis, except for the Korean wild mouse. Using this approach, we successfully mapped the brain vascular system in six different species of animals. This information and the images created can guide other researchers as they design research studies and create experimental models for new surgical procedures and approaches. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc. Anat Rec, 302:2049-2061, 2019. © 2019 American Association for Anatomy.

Applicability of vital staining and tissue clearing to vascular anatomy and melanocytes' evaluation of temporal bone in six laboratory species.

The purpose of the present study was to define the applicability of tissue clearing to the field of otology. We combined tissue clearing with vital staining perfusion via a pumping system to examine the vascular anatomy of temporal bones in laboratory animals. We used six different types of species including Korean wild mouse, mouse, Mongolian gerbil, hamsters and Guinea pigs. A mixture of Alcian blue reagent and 4% paraformaldehyde was circulated throughout the entire circulatory system of the animal via a perfusion pump system. Transparency images were obtained from the temporal bones according to the protocol of the SunHyun 3D Imaging Kit. In examining the inner surface of the tympanic membrane, flaccid part (pars flaccida) was positioned along the entire marginal area in Guinea pig. In the Guinea pig, unlike the other species, the cortical bone of the mastoid (bullae) was easily removed using cold instruments, allowing a direct approach to the enclosed structures. The distribution and pattern of cochlea melanocytes were compared among the species. "Mobius strip"-like accumulated melanocytes in vestibules were shown in both the Korean wild mouse and mouse. The collateral blood supply to the cochlea in six different species was checked in various pattern. Combining dye infusion with tissue-clearing techniques, we documented the middle ear and transparent inner ear structures in six different species. The information and associated images will help other researchers to develop hypotheses and design experimental investigations.

The Wild Mouse (Micromys minutus): Reservoir of a Novel Campylobacter jejuni Strain.

Campylobacter jejuni is one of the most common zoonotic pathogens worldwide. Although the main sources of human C. jejuni infection are livestock, wildlife can also affect C. jejuni transmission in humans. However, it remains unclear whether wild mice harbor C. jejuni and are involved in the "environment-wildlife-livestock-human" transmission cycle of C. jejuni in humans. Here, we characterized C. jejuni from wild mice and identified genetic traces of wild mouse-derived C. jejuni in other hosts using a traditional approach, along with comparative genomics. We captured 115 wild mice (49 Mus musculus and 66 Micromys minutus) without any clinical symptoms from 22 sesame fields in Korea over 2 years. Among them, M. minutus were typically caught in remote areas of human houses and C. jejuni was solely isolated from M. minutus (42/66, 63.6%). We identified a single clone (MLST ST-8388) in all 42 C. jejuni isolates, which had not been previously reported, and all isolates had the same virulence/survival-factor profile, except for the plasmid-mediated virB11 gene. No isolates exhibited antibiotic resistance, either in phenotypic and genetic terms. Comparative-genomic analysis and MST revealed that C. jejuni derived from M. minutus (strain SCJK2) was not genetically related to those derived from other sources (registered in the NCBI genome database and PubMLST database). Therefore, we hypothesize that C. jejuni from M. minutus is a normal component of the gut flora following adaptation to the gastro-intestinal tract. Furthermore, M. minutus-derived C. jejuni had different ancestral lineages from those derived from other sources, and there was a low chance of C. jejuni transmission from M. minutus to humans/livestock because of their habitat. In conclusion, M. minutus may be a potential reservoir for a novel C. jejuni, which is genetically different from those of other sources, but may not be involved in the transmission of C. jejuni to other hosts, including humans and livestock. This study could form the basis for further studies focused on understanding the transmission cycle of C. jejuni, as well as other zoonotic pathogens originating from wild mice.

Aged black garlic extract regulates lipid metabolism by inhibiting lipogenesis and promoting lipolysis in mature 3T3-L1 adipocytes.

As the mechanism of aged black garlic (ABG) extract affecting lipid metabolism in adipocytes remains unclear, this study evaluated the effect of ABG extract on lipid metabolism and the expression of related proteins in mature 3T3-L1 adipocytes. ABG extract treatments at 0, 0.625, 1.25, and 2.5, and 5 mg/mL had no effect on cell morphology or viability in adipocytes. ABG extract suppressed lipogenesis and induced lipolysis in a dose-dependent manner compared to control. Furthermore, ABG extract at 2.5 and 5 mg/mL significantly reduced protein expression of proliferator activated receptor γ (PPARγ) and perilipin in mature 3T3-L1 adipocytes. The hormone-sensitive lipase (HSL) and Ser563-pHSL levels were also significantly reduced by treatment with 5 mg/mL of ABG extract. Taken together, these results suggest that ABG extract has anti-lipogenic and lipolytic effects in mature 3T3-L1 adipocytes, indicating a potential in anti-obesity therapies.

Clusterin is highly expressed in tubular complexes during spontaneous pancreatitis of spontaneous hypertensive rats.

Pancreatitis is an inflammatory disorder of pancreas which leads to varying degrees of pancreatic endocrine and exocrine dysfunction and manifests in either acute or chronic forms. Spontaneous pancreatitis in experimental animals has rarely been reported. Here, we found acute to chronic courses of spontaneous pancreatitis in spontaneously hypertensive rats (SHRs), showing the formation of tubular complexes (TCs) and enhanced islet regeneration. We investigated the expression pattern of clusterin in the pancreas of SHRs based on immunohistochemistry (IHC). IHC analysis revealed the strong expression of clusterin in dedifferentiated duct-like cells and regenerative islets of TCs. These results imply that clusterin might be involved in the formation of TCs and parenchymal regeneration during rat pancreatitis.

Genetic and morphometric characteristics of Korean wild mice (KWM/Hym) captured at Chuncheon, South Korea.

Laboratory inbred mice are used widely and commonly in biomedical research, but inbred mice do not have a big enough gene pool for the research. In this study, genetic and morphometric analyses were performed to obtain data on the characteristics of a newly developing inbred strain (KWM/Hym) captured from Chuncheon, Korea. All of five Korean wild male mice have the zinc-finger Y (ZfY) gene. Also, all of 19 Korean wild mice used in this analysis have the AKV-type murine leukemia virus gene, indicating that Korean wild mice might be Mus musculus musculus. To identify the genetic polymorphism in KWM/Hym, SNP analysis was performed. In a comparison with 28 SNP markers, there was a considerable difference between KWM/Hym and several inbred strains. The homogeneity between KWM/Hym and the inbred strains was as follows: C57BL/6J (39.3%), BALB/c AJic (42.9%), and DBA/2J (50%). KWM/Hym is most similar to the PWK/PhJ inbred strain (96.4%) derived from wild mice (Czech Republic). To identify the morphometric characteristics of KWM/Hym, the external morphology was measured. The tail ratio of male and female was 79.60±3.09 and 73.55±6.14%, respectively. KWM/Hym has short and agouti-colored hairs and its belly is white with golden hair. Taking these results together, KWM/Hym, a newly developing inbred mouse originated from wild mouse, might be use as new genetic resources to overcome the limitations of the current laboratory mice.

Improving glycemic control in model mice with type 2 diabetes by increasing superoxide dismutase (SOD) activity using silk fibroin hydrolysate (SFH).

Islet cell dysfunction in type 2 diabetes is primarily attributed to the increased apoptosis of pancreatic beta cells. Silk fibroin hydrolysate (SFH) has an effect on blood in type 2 diabetes model mice (C57BL/KsJ-db/db). However, its exact mechanism is unknown. The type 2 diabetes model mice were randomly divided into non-diabetic mice (ND), diabetic mice (DB), and diabetic mice treated with silk fibroin hydrolysate (DB-SFH). The results showed that SFH significantly decreased fasting blood glucose and hemoglobin A1c (HbA1c). The oral glucose tolerance and insulin tolerance were significantly improved in the DB-SFH group. The DB-SFH group exhibited increased superoxide dismutase (SOD) activity in the plasma, as well as increased Mn-SOD and CuZn-SOD activities in the pancreatic islets. Furthermore, the pancreatic islet cells' death was decreased in the DB-SFH group. In the DB-SFH group, the protein expression of caspase-3 was significantly decreased compared with the DB group. The expression of the Nkx6.1 and Pdx1 proteins were increased in the DB-SFH group. The results suggest that SFH prevents the degeneration of pancreatic islets via increasing SOD while hyperglycemia is alleviated by maintaining beta cell mass in type 2 diabetes model mice.

Comparing the Organs and Vasculature of the Head and Neck in Five Murine Species.

BACKGROUND/AIM: The purpose of the present study was to delineate the cervical and facial vascular and associated anatomy in five murine species, and compare them for optimal use in research studies focused on understanding the pathology and treatment of diseases in humans. MATERIALS AND METHODS: The specific adult male animals examined were mice (C57BL/6J), rats (F344), mongolian gerbils (Merionesunguiculatus), hamsters (Syrian), and guinea pigs (Hartley). To stain the vasculature and organs, of the face and neck, each animal was systemically perfused using the vital stain, Trypan Blue. Following this step, the detailed anatomy of the head and neck could be easily visualized in all species. RESULTS: Unique morphological characteristics were demonstrated by comparing the five species, including symmetry of the common carotid origin bilaterally in the Mongolian Gerbil, a large submandibular gland in the hamster and an enlarged buccal branch in the Guinea Pig. In reviewing the anatomical details, this staining technique proves superior for direct surgical visualization and identification. CONCLUSION: The anatomical details provided through these five species atlas will help experimental researchers in the future to select the most appropriate animal model for specific laboratory studies aimed to improve our understanding and treatment of diseases in patients.

Over-expression of myosin7A in cochlear hair cells of circling mice.

Circling mouse (C57BL/6J-cir/cir) deleted the transmembrane inner ear (Tmie) gene is an animal model for human non-syndromic recessive deafness, DFNB6. In circling mouse, hair cells in the cochlea have degenerated and hair bundles have become irregularity as time goes on. Tmie protein carries out a function of the mechanoelectrical transduction channel in cochlear hair cells. Myosin7a (MYO7A) protein has key roles in development of the cochlear hair bundles as well as in the function of cochlear hair cells. To find whether Tmie protein interacts with MYO7A proteins in the cochlea postnatal developmental stage, we investigated expression of the MYO7A proteins in the cochlear hair cells of circling mice by western blot analysis and whole mount immunofluorescence at postnatal day 5 (P5). The expression of MYO7A showed statistically significant increase in the cochlea of C57BL/6J-+/cir and C57BL/6J-cir/cir mice than that of C57BL/6J-+/+ mice. The MYO7A intensity of the cochlear hair cells also increased in C57BL/6J-+/cir and C57BL/6J-cir/cir mice compared with those of C57BL/6J-+/+ mice. Taken together, the results indicate that Tmie protein may have an important role with MYO7A protein in the development and maintenance of the stereociliary bundles during postnatal developmental stage of the cochlea.

Fine tuning of Rac1 and RhoA alters cuspal shapes by remolding the cellular geometry.

The anatomic and functional combinations of cusps and lophs (ridges) define the tooth shape of rodent molars, which distinguishes species. The species-specific cusp patterns result from the spatiotemporal induction of enamel knots (EKs), which require precisely controlled cellular behavior to control the epithelial invagination. Despite the well-defined roles of EK in cusp patterning, the determinants of the ultimate cuspal shapes and involvement of epithelial cellular geometry are unknown. Using two typical tooth patterns, the lophodont in gerbils and the bunodont in mice, we showed that the cuspal shape is determined by the dental epithelium at the cap stage, whereas the cellular geometry in the inner dental epithelium (IDE) is correlated with the cuspal shape. Intriguingly, fine tuning Rac1 and RhoA interconvert cuspal shapes between two species by remolding the cellular geometry. Either inhibition of Rac1 or ectopic expression of RhoA could region-distinctively change the columnar shape of IDE cells in gerbils to drive invagination to produce cusps. Conversely, RhoA reduction in mice inhibited invagination and developed lophs. Furthermore, we found that Rac1 and RhoA modulate the choices of cuspal shape by coordinating adhesion junctions, actin distribution, and fibronectin localization to drive IDE invagination.

Reduction of dynamin 1 in the hippocampus of aged mice is associated with the decline in hippocampal­dependent memory.

Dynamin 1 is a known synaptic protein, which has is key in the presynaptic regulation of endocytosis. The present study investigated the association between age and the observed changes in Morris water maze performance, and immunoreactivity and protein levels of dynamin 1 in the mouse hippocampal formation. In addition, the effects of dynasore, an inhibitor of dynamin 1, on the hippocampal dependent memory were determined to elucidate the correlation between dynamin 1 and memory. In the training phase of the Morris water maze task, the mean escape latency of the aged group (24 months old) was significantly longer, compared with that of the adult group (4 months old), although the average swimming speed and the total distance traveled during the probe trial were similar in the two groups. In the aged group, the time spent locating the target platform was significantly longer and the time spent in the correct quadrant was significantly shorter, compared with those in the adult group. In the adult group, a moderate level of dynamin 1 was detected in the hippocampal CA1 and CA3 regions, and in the dentate gyrus. In the aged group, the immunoreactivity of dynamin 1 was almost eliminated in the CA3 region and the dentate gyrus. In addition, the protein levels of dynamin 1 in the brain were significantly lower in the aged group, compared with those in the adult group. The direct infusion of dynasore, significantly reduced the contextual memory, compared with that of animals in the vehicle­treated group. These results suggested that dynamin 1 was susceptible to the aging process, and that a reduction in dynamin 1 may result in hippocampal­dependent memory deficits by disrupting endocytosis and the release of neurotransmitters.