Near-Infrared Photoimmunotherapy Using a Protein Mimetic for EGFR-Positive Salivary Gland Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer therapy based on a monoclonal antibody (mAb) conjugated to a photosensitizer (IR700Dye). The conjugate can be activated by near-infrared light irradiation, causing necrotic cell death with high selectivity. In this study, we investigated NIR-PIT using a small protein mimetic (6-7 kDa, Affibody) which has more rapid clearance and better tissue penetration than mAbs for epidermal growth factor receptor (EGFR)-positive salivary gland cancer (SGC). The level of EGFR expression was examined in vitro using immunocytochemistry and Western blotting. Cell viability was analyzed using the alamarBlue assay. In vivo, the volume of EGFR-positive tumors treated with NIR-PIT using the EGFR Affibody-IR700Dye conjugate was followed for 43 days. It was found that NIR-PIT using the EGFR Affibody-IR700Dye conjugate induced the selective destruction of EGFR-positive SGC cells and restricted the progression of EGFR-positive tumors. We expect that NIR-PIT using the EGFR Affibody-IR700Dye conjugate can efficiently treat EGFR-positive SGC and preserve normal salivary function.

Early revascularization activates quiescent dental pulp stem cells following tooth replantation in mice.

Introduction: The intentional perforation of the pulp chamber floor before tooth replantation promotes pulpal healing by facilitating the revascularization of the pulp cavity. This study aimed to elucidate the effects of this method on the dynamics of quiescent dental pulp stem cells (DPSCs). Methods: The right and left maxillary first molars of Crlj:CD1 mice and TetOP-histone 2B (H2B)-green fluorescent protein (GFP) mice were extracted. The left molars were immediately replanted as the control group (CG), whereas the pulp chamber floor of the right molars were perforated before the tooth was replanted as the experimental group (EG). Immunohistochemistry for Nestin and GFP, and quantitative RT-PCR for Nestin, Opn, CD11c, and Oct3/4 mRNA were performed. Results: The rate of Nestin-positive perimeter along the pulp-dentin border in the EG tended to be higher than that of the CG at days 5 and 7 and was significantly increased between days 3 and 7. The rate of GFP-positive cells in the EG was significantly higher than that of the CG at days 5 and/or 7 in the mesial and middle coronal pulp. CD11c mRNA in the EG at day 5 was significantly higher than that of the CG and tended to be higher than that of the CG during the observation period. Oct3/4 mRNA expression in the EG was significantly higher than that of the CG at day 7. Conclusions: The current experimental model demonstrated the promotion of the survival of DPSCs and their differentiation into odontoblast-like cells (OBLCs). Thus, the use of this model is expected to clarify the crosstalk mechanism between immune cells, including macrophages and dendritic cells, and DPSCs with regards to pulpal healing after tooth replantation. It also provides insight into the differentiation process of DPSCs into OBLCs.

The accuracy of quantifying the degree of hard tissue calcification using an electron probe micro analyzer, micro-focus X-ray computed tomography, and tissue sectioning methods.

OBJECTIVES: Micro-focus X-ray computed tomography (µCT) helps evaluate specimens without destroying it. However, its accuracy of quantifying bone mineral density remains to be fully elucidated. We aimed to verify the accuracy of calcification assessed by µCT, by comparing the images of identical specimens obtained via different methods such as µCT and electron probe micro analyzer (EPMA) analyses. METHODS: The maxillae, mandibles, and tibiae of five-week-old male mice were analyzed. Calcification density was analyzed using µCT. The right sides of the specimens were decalcified and processed for Azan staining. The left side of the specimens underwent elemental mapping for Ca, Mg, and P using EPMA. RESULTS: µCT revealed a significant increase in calcification levels in the following order: enamel, dentin, cortical bone, and trabecular bone. These results reflected the Ca and P levels observed in the EPMA analyses. µCT demonstrated significant differences in the degree of calcification among the enamel tissues or dentin tissues, except for dentin in the maxillary incisors and molars. However, EPMA analysis did not demonstrate significant differences in the Ca and P levels among the same tissue samples. CONCLUSIONS: EPMA elemental analysis can be used to measure Ca and P levels for evaluating the calcification rate of hard tissues. Additionally, the study results validate the evaluation of calcification density via µCT. Furthermore, µCT can evaluate even minute differences in calcification rates compared with EPMA analysis.

The effect of the formalin-fixed paraffin-embedded process on salivary microbiota profiling.

In recent years, bacterial DNA in formalin-fixed paraffin-embedded (FFPE) samples has been recognized as a valuable bioresource for microbiota studies. This study aimed to examine the effect of the FFPE process on microbiota profiling to evaluate whether FFPE samples could serve as an alternative bioresource to fresh samples in oral microbiota studies. Fresh saliva was collected from nine subjects. The pellets obtained by centrifuging the collected saliva were fixed in formalin, then dehydrated and embedded in paraffin to prepare FFPE samples. The abundance of the hypervariable regions V1-9, V1-2, and V3-4 of the 16S rRNA gene in fresh and FFPE samples was relatively compared. In addition, microbiota profiling was performed to compare the results between the two sample types. The results showed that the FFPE process resulted in a certain degree of fragmentation of the 16S rRNA gene. However, the V1-2 region was relatively well-preserved compared to the V1-9 and V3-4 regions, suggesting that short regions are suitable targets for oral microbiota analysis. Importantly, there were no significant differences in alpha and beta diversity of microbiota between fresh and FFPE samples, and microbiota profiles were similar between the two sample types, suggesting that FFPE samples could be a valuable bioresource for oral microbiota studies.

The effect of intentionally perforating the floor of the pulp chamber on pulpal healing after tooth replantation in mice.

OBJECTIVES: Shortening the root of a mouse molar prior to tooth replantation results in early revascularization in the pulp cavity and activation of the dental pulp quiescent stem cells. This study aimed to validate the effects of pulp chamber floor perforation on pulpal healing after tooth replantation as a strategy to promote early revascularization into the pulp. METHODS: The maxillary first molars of three-week-old Crlj:CD1 mice were extracted and repositioned into the original socket: the left teeth were immediately replanted (control group: CG), whereas the floor of the pulp chamber of the right teeth was perforated with a tungsten carbide bur before tooth replantation (experimental group: EG). The samples were collected from three days to eight weeks postoperatively. In addition to the TUNEL assay, immunohistochemistry for Nestin, CK14, and Ki-67 was conducted. RESULTS: In the EG, early revascularization occurred with a decrease in apoptosis and an increase in cell proliferation, facilitating pulpal healing, compared with the CG. The rate of Nestin-positive perimeter in the distal root significantly increased on days 5 and 14 and the amount of Nestin-positive hard tissue increased on day 14. However, on day 7, the number of epithelial cell rests of Malassez in the EG significantly decreased, making the EG susceptible to ankylosis at the floor. CONCLUSIONS: Intentionally perforating the floor of the pulp chamber provides a route for early revascularization, resulting in better pulpal healing after tooth replantation.

Profiling of the microbiota of breast milk before and after feeding with an artificial nipple.

OBJECTIVES: Breast milk is a valuable and useful source of nutrition; however, surplus milk is routinely discarded for hygiene reasons despite an unclear scientific basis. Here, we profiled the microbiota of expressed breast milk before and after feeding with an artificial nipple and examined the bacterial survival in breast milk stored at 4 °C. METHODS: Eleven mother-baby pairs were included in the study. Samples of expressed breast milk were collected before and after feeding with an artificial nipple and examined both immediately (0 h) and after storage for 3 and 12 h at 4 °C. Each sample was inoculated onto a blood agar plate and incubated anaerobically and aerobically at 37 °C. Genomic DNA was extracted from individual bacterial colonies, which were identified by 16S rRNA gene sequencing. RESULTS: Before feeding, the bacterial counts at 0 and 12 h were (1.4 ± 1.6) × 105 colony-forming units (CFU)/mL and (1.4 ± 0.6) × 105 CFU/mL, respectively. Staphylococcus (47.7% and 41.9%, respectively), Cutibacterium (20.7% and 36.0%, respectively), and Streptococcus (16.1% and 6.6%, respectively) were identified among the samples. In contrast, after feeding, the bacterial counts at 0 and 12 h were (2.7 ± 1.7) × 105 CFU/mL and (2.1 ± 2.5) × 105 CFU/mL, respectively. Staphylococcus (30.1% and 37.4%, respectively), Cutibacterium (11.7% and 31.7%, respectively), and Streptococcus (41.5% and 25.2%, respectively), were identified among the samples. CONCLUSIONS: Bacteria were present in the breast milk before feeding. Although the main component of the microbiota shifted from Staphylococcus to Streptococcus species after feeding, these results suggest that surplus expressed breast milk may be preserved safely in a refrigerator for at least 12 h after feeding with an artificial nipple.

Microbiota profiles on the surface of non-woven fabric masks after wearing.

This study aimed to characterize commensal microbiota on the skin before and after wearing masks, and to characterize the microbiota on the surface of used masks after 1 week of drying. From the 13 human subjects (age range, 19-26 years), mean bacterial concentrations of (6.1 ± 11.0) × 105 and (1.0 ± 1.4) × 106 colony-forming units (CFU)/mL were recovered from the skin of the buccal areas wiped with a sterile cotton swab before and after wearing non-woven fabric masks for 8 h, respectively. Furthermore (3.4 ± 4.9) × 104 CFU/mL of bacteria were recovered from the mask surfaces. The bacteria contained in the masks, which consisted mainly of Cutibacterium acnes and Staphylococcus epidermidis/aureus, virtually disappeared after drying the masks indoors for 1 week.

Exploration of the role of the subodontoblastic layer in odontoblast-like cell differentiation after tooth drilling using Nestin-enhanced green fluorescent protein transgenic mice.

OBJECTIVES: Original odontoblasts and regenerated odontoblast-like cells (OBLCs) may differently regulate Nestin expression. This study aimed to investigate the role of the subodontoblastic layer (SOBL) using green fluorescent protein (GFP) reactivity in the process of OBLC differentiation after tooth drilling in Nestin-enhanced GFP transgenic mice. METHODS: A groove-shaped cavity was prepared on the mesial surface of the maxillary first molars of 5- or 6-week-old mice under deep anesthesia. Immunohistochemical staining for Nestin and GFP and Nestin in situ hybridization were conducted on the sections obtained at 1-14 days postoperative. RESULTS: Odontoblasts showed intense endogenous Nestin protein and mRNA expression, whereas the coronal SOBL cells showed a Nestin-GFP-positive reaction in the control groups. The injured odontoblasts had significantly decreased Nestin immunoreactivity as well as decreased expression of Nestin mRNA 1-2 days after the injury; subsequently, newly differentiated OBLCs were arranged along the pulp-dentin border, with significantly increased Nestin expression as well as increased expression of Nestin mRNA on days 3-5 to form reparative dentin. Nestin-GFP-positive cells at the pulp-dentin border significantly increased in number on days 1 and 2. GFP(+)/Nestin(+) and GFP(-)/Nestin(+) cells were intermingled in the newly differentiated OBLCs. CONCLUSIONS: The commitment of Nestin-GFP-positive cells into Nestin-positive OBLCs suggests that the restriction of endogenous Nestin protein and mRNA expression in the static SOBL cells was removed by exogenous stimuli, resulting in their migration along the pulp-dentin border and their differentiation into OBLCs.

Profiling of Microbiota at the Mouth of Bottles and in Remaining Tea after Drinking Directly from Plastic Bottles of Tea.

It has been speculated that oral bacteria can be transferred to tea in plastic bottles when it is drunk directly from the bottles, and that the bacteria can then multiply in the bottles. The transfer of oral bacteria to the mouth of bottles and bacterial survival in the remaining tea after drinking directly from bottles were examined immediately after drinking and after storage at 37 °C for 24 h. Twelve healthy subjects (19 to 23 years of age) were asked to drink approximately 50 mL of unsweetened tea from a plastic bottle. The mouths of the bottles were swabbed with sterile cotton, and the swabs and the remaining tea in the bottles were analyzed by anaerobic culture and 16S rRNA gene sequencing. Metagenomic analysis of the 16S rRNA gene was also performed. The mean amounts of bacteria were (1.8 ± 1.7) × 104 colony-forming units (CFU)/mL and (1.4 ± 1.5) × 104 CFU/mL at the mouth of the bottles immediately after and 24 h after drinking, respectively. In contrast, (0.8 ± 1.6) × 104 CFU/mL and (2.5 ± 2.6) × 106 CFU/mL were recovered from the remaining tea immediately after and 24 h after drinking, respectively. Streptococcus (59.9%) were predominant at the mouth of the bottles immediately after drinking, followed by Schaalia (5.5%), Gemella (5.5%), Actinomyces (4.9%), Cutibacterium (4.9%), and Veillonella (3.6%); the culture and metagenomic analyses showed similar findings for the major species of detected bacteria, including Streptococcus (59.9%, and 10.711%), Neisseria (1.6%, and 24.245%), Haemophilus (0.6%, and 15.658%), Gemella (5.5%, and 0.381%), Cutibacterium (4.9%, and 0.041%), Rothia (2.6%, and 4.170%), Veillonella (3.6%, and 1.130%), Actinomyces (4.9%, and 0.406%), Prevotella (1.6%, and 0.442%), Fusobacterium (1.0%, and 0.461%), Capnocytophaga (0.3%, and 0.028%), and Porphyromonas (1.0%, and 0.060%), respectively. Furthermore, Streptococcus were the most commonly detected bacteria 24 h after drinking. These findings demonstrated that oral bacteria were present at the mouth of the bottles and in the remaining tea after drinking.

Profiling system of oral microbiota utilizing polymerase chain reaction-restriction fragment length polymorphism analysis.

OBJECTIVES: Profiling of oral microbiota has traditionally been performed using conventional methods. These methods are relatively time-consuming and labor-intensive. Metagenomic analysis of oral microbiota using high-speed next-generation sequencing is a highly promising technology. However, it is expensive. This study sought to develop a simple and cost-effective profiling method for oral microbiota using 16S rRNA gene polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of PCR-amplified 16S ribosomal RNA genes. METHODS: Oral isolates of 59 bacterial species from human saliva, including Streptococcus, Actinomyces, and Veillonella, were cultured anaerobically on CDC Anaerobe 5% sheep blood agar plates. Genomic DNA was extracted from single colonies and 16S rRNA genes were PCR-amplified using the 27F and 1492R universal primers. The PCR products were purified and characterized by single digestion with HpaII restriction endonuclease. 16S rRNA gene sequences were obtained from the GenBank database, and the expected restriction profiles were compared with the RFLP patterns obtained from agarose gel electrophoresis. RESULTS: Sixty-five RFLP patterns were obtained from 27 genera and 59 species. The expected fragment sizes of these species were calculated based on GenBank 16S rRNA gene sequences. Fifty-nine patterns were obtained from the analysis of GenBank sequences. The RFLP patterns produced with HpaII distinguished many oral bacterial species. RFLP patterns enabling identification of oral bacteria were generated. The 16S rRNA gene PCR-RFLP analysis did not require expensive equipment and reagents and was cost-effective. CONCLUSION: PCR-RFLP analysis based on 16S rRNA genes could be an alternative method for oral microbiota analysis in smaller laboratories.

The effects of reducing the root length by apicoectomy on dental pulp revascularization following tooth replantation in mice.

BACKGROUND/AIM: Root length is a critical factor for dental pulp regeneration following tooth replantation. The aim of this study was to analyze the effects of reducing the root length by apicoectomy on the pulp healing process using a model for tooth replantation. MATERIAL AND METHODS: After extraction of the upper first molars (M1) of 3-week-old mice, the roots from the experimental group (EG) were shortened to half to two-thirds of their length before replantation, whereas in the control group (CG) the extracted teeth were immediately repositioned into their alveolar sockets. To determine the effects of root resection on the survival of inherent pulp cells, this study included tooth transplantation with root resection using wild-type (WT) and green fluorescent protein (GFP) transgenic mice. The M1 of GFP transgenic mice were transplanted into the alveolar socket of the M1 of WT mice. The roots of the right M1 were shortened (EG), whereas the left M1 remained untreated (CG). RESULTS: Apoptotic cells in the EG significantly decreased in number compared with the CG at day 3. Cell proliferative activity in the EG was significantly higher than that in the CG in the root pulp during days 3-5, and nestin-positive odontoblast-like cells began to arrange themselves along the pulp-dentin border in the cusp area at day 5 in the EG but not in the CG. At week 2, tertiary dentin had formed throughout the pulp in the EG, whereas the combined tissue of dentin and bone occupied the pulp space in 60% of the CG. Root resection also positively affected the survival of inherent pulp cells to differentiate into odontoblast-like cells as demonstrated by transplantation using GFP transgenic mice. CONCLUSIONS: Reducing the root length accelerated pulp regeneration following tooth replantation due to the better environment for revascularization.

Bacterial concentration and composition in liquid baby formula and a baby drink consumed with an artificial nipple.

OBJECTIVES: To clarify the characteristics and growth of bacteria that may infiltrate liquid baby formula during feeding and after storage for more than 3 h, the transfer of oral bacteria through artificial nipples, and bacterial survival in liquid baby formula and a baby drink were examined immediately after drinking and after storage at 4 °C for 12 h and 24 h. METHODS: Thirteen human subjects (aged 19-24 years) were asked to drink approximately 50 mL of liquid baby formula and a baby drink, via the artificial nipple of a baby bottle. Samples of the remaining liquid after storage at 4 °C for 12 h and 24 h were inoculated onto blood agar plates and incubated anaerobically at 37 °C for 7 days. Genomic DNA was extracted from individual colonies, and the bacterial species were identified by 16S rRNA gene sequencing. RESULTS: The mean concentrations of bacteria in the liquid baby formula were (2.6 ± 2.8) × 104 and (4.1 ± 6.6) × 104 colony-forming unit/mL after storage at 4 °C for 12 h and 24 h, respectively. Streptococcus (43.2%), Veillonella (9.3%), and Schaalia (8.2%) species were recovered from the remaining liquid baby formula after storage at 4 °C for 12 h. In contrast, no bacteria were detected in the remaining baby drink after storage at 37 °C for 24 h. CONCLUSIONS: The levels of bacteria immediately after drinking and after storage at 4 °C for 12 h or 24 h were similar, suggesting that remaining liquid baby formula may be preserved safely in a refrigerator for more than 3 h.

Profiling of microbiota in liquid baby formula consumed with an artificial nipple.

It is suspected that oral bacteria are transferred to the liquid baby formula through the artificial nipple and multiply in the bottle after feeding. In the present study, in order to understand the influence of bacteria on liquid baby formula after feeding, the transfer of oral bacteria through artificial nipples and their survival in liquid baby formula were examined immediately after drinking as well as after storage at 4°C for 3 h. Four healthy human subjects (20-23 years old) were asked to drink liquid baby formula (Aptamil®, ca. 50 mL) from baby bottles using artificial nipples. Samples of the liquid baby formula (immediately after drinking and 3 h later) were inoculated onto blood agar plates and incubated anaerobically at 37°C for 7 days. Salivary samples from each subject and 6 newborn infants were also cultured. Genomic DNA was extracted from individual colonies, and bacterial species were identified by 16S rRNA gene sequencing. The mean amounts of bacteria (CFU/mL) were (3.2 ± 3.0) ×104 and (3.4 ± 3.3) ×104 immediately after drinking and 3 h later, respectively. Streptococcus (41.6 and 40.5%), Actinomyces (24.3 and 21.5%) and Veillonella (16.2 and 11.0%) were recovered from the samples immediately after drinking and 3 h later, respectively. On the other hand, Streptococcus (38.9%), Actinomyces (17.1%), Neisseria (9.1%), Prevotella (6.9%), Rothia (6.9%) and Gemella (5.1%) were predominant in the saliva of adult subjects, and Streptococcus (65.2%), Staphylococcus (18.5%), Gemella (8.2%) and Rothia (5.4%) were predominant in the saliva of infant subjects. From these findings, oral bacteria, e.g., Streptococcus, Gemella and Rothia, were found to transfer into the liquid baby formula through artificial nipples, and the bacterial composition in the remaining liquid baby formula was found to resemble that of human saliva. The bacterial levels were similar between immediately after drinking and when stored at 4°C for 3 h, suggesting that the remaining liquid baby formula may be preserved in a refrigerator for a specified amount of time.

10-Mesityl-1,8-diphenylanthracene Dimer: Synthesis, Structure, and Properties.

Macrocyclic 10-mesityl-1,8-diphenylanthracene dimer 4 was synthesized by using the electron-transfer oxidation of Lipshutz cuprate derived from 1,8-bis(4-bromophenyl)-10-mesityl-anthracene 7 in moderate yield. This dimer 4 is a considerably fluorescent molecule (ΦF 0.40) with high thermal, photo, and air stability. The X-ray analysis of 4 revealed a unique structure with a small inner cavity which can incorporate a small molecule or atom. 1H NMR spectra in solution and emission spectra of 4 in the solid state showed that copper(I) ion was incorporated to form a 1:1 complex 4·CuOTf, whereas silver(I) ion only weakly interacted with 4 under similar conditions.

Effects of forced movements on learning: Findings from a choice reaction time task in rats.

To investigate how motor sensation facilitates learning, we used a sensory-motor association task to determine whether the sensation induced by forced movements contributes to performance improvements in rats. The rats were trained to respond to a tactile stimulus (an air puff) by releasing a lever pressed by the stimulated (compatible condition) or nonstimulated (incompatible condition) forepaw. When error rates fell below 15%, the compatibility condition was changed (reversal learning). An error trial was followed by a lever activation trial in which a lever on the correct or the incorrect response side was automatically elevated at a preset time of 120, 220, 320, or 420 ms after tactile stimulation. This lever activation induced forepaw movement similar to that in a voluntary lever release response, and also induced body movement that occasionally caused elevation of the other forepaw. The effects of lever activation may have produced a sensation similar to that of voluntary lever release by the forepaw on the nonactivated lever. We found that the performance improvement rate was increased by the lever activation procedure on the incorrect response side (i.e., with the nonactivated lever on the correct response side). Furthermore, the performance improvement rate changed depending on the timing of lever activation: Facilitative effects were largest with lever activation on the incorrect response side at 320 ms after tactile stimulation, whereas hindering effects were largest for lever activation on the correct response side at 220 ms after tactile stimulation. These findings suggest that forced movements, which provide tactile and proprioceptive stimulation, affect sensory-motor associative learning in a time-dependent manner.