We have developed methods to achieve efficient CRISPR-Cas9–mediated gene knockout in ex vivo mouse embryonic salivary epithelial explants. Salivary epithelial explants provide a valuable model for ...characterizing cell signaling, differentiation, and epithelial morphogenesis, but research has been limited by a paucity of efficient gene perturbation methods. Here, we demonstrate highly efficient gene perturbation by transient transduction of guide RNA–expressing lentiviruses into Cas9-expressing salivary epithelial buds isolated from Cas9 transgenic mice. We first show that salivary epithelial explants can be cultured in low-concentration, nonsolidified Matrigel suspensions in 96-well plates, which greatly increases sample throughput compared to conventional cultures embedded in solidified Matrigel. We further show that salivary epithelial explants can grow and branch with FGF7 alone, while supplementing with insulin, transferrin, and selenium (ITS) enhances growth and branching. We then describe an efficient workflow to produce experiment-ready, high-titer lentiviruses within 1 wk after molecular cloning. To track transduced cells, we designed the lentiviral vector to coexpress a nuclear fluorescent reporter with the guide RNA. We routinely achieved 80% transduction efficiency when antibiotic selection was used. Importantly, we detected robust loss of targeted protein products when testing 9 guide RNAs for 3 different genes. Moreover, targeting the β1 integrin gene (Itgb1) inhibited branching morphogenesis, which supports the importance of cell–matrix adhesion in driving branching morphogenesis. In summary, we have established a lentivirus-based method that can efficiently perturb genes of interest in salivary epithelial explants, which will greatly facilitate studies of specific gene functions using this system.
Mice produce various sounds within the ultrasonic range in social contexts. Although these sounds are often used as an index of sociability in biomedical research, their biological significance ...remains poorly understood. We previously showed that mice repeatedly produced calls in a sequence (i.e. calling bout), which can vary in their structure, such as Simple, Complex or Harmonics. In this study, we investigated the use of the three types of calling bouts in different sociosexual interactions, including both same- and opposite-sex contexts. In same-sex contexts, males typically produced a Simple calling bout, whereas females mostly produced a Complex one. By contrast, in the opposite-sex context, they produced all the three types of calling bouts, but the use of each calling type varied according to the progress and mode of sociosexual interaction (e.g. Harmonic calling bout was specifically produced during reproductive behaviour). These results indicate that mice change the structure of calling bout according to sociosexual contexts, suggesting the presence of multiple functional signals in their ultrasonic communication.
We studied the stereoselective propylene polymerization catalyzed by the ion pair (IP) active species of (pyridylamido)hafnium(IV) complex, the catalyst. In particular, we focused on the role of ...the counteranion (CA) for the reaction mechanism of propylene insertion because it had never been investigated from the molecular point of view. First, we searched for a variety of IP configurations by the molecular dynamics (MD) method and quantum chemical calculation, and located the transition states (TSs) of propylene insertion. By comparing the CA-bound catalysts with the isolated ones, it was revealed that the manner of energetically favorable insertion is interestingly altered due to the location of the CA. Next, we showed that the CA is essential to determine the coordination structures of the Hf atom at transition state. Therefore, it was concluded that the role of the CA is critical to accurately control the polymerization mechanism. Finally, we proposed a synthesis experiment which would be advantageous to increase the stereoselectivity of the catalyst on the basis of the present polymerization mechanism.
Summary
Background
There have been no comparative studies of tacrolimus vs. anti‐tumour necrosis factor (anti‐TNF) agents to determine which treatment is safer or more effective in refractory ...ulcerative colitis (UC).
Aim
To compare short‐term safety and efficacy of tacrolimus vs. anti‐TNF agents for active UC.
Methods
One hundred patients with moderate‐to‐severe active UC were studied. Fifty patients were treated with oral tacrolimus (TAC group). The other 50 patients were treated with anti‐TNF agents (anti‐TNF group): 40 with infliximab and 10 with adalimumab. Primary endpoints were clinical response and remission rates, colectomy rate, and the incidence of adverse events during 12 weeks.
Results
The incidence of adverse events was 12% in the TAC vs. 18% in the anti‐TNF groups (P = 0.58). At week 12, clinical remission rate was 40% in the TAC vs. 28% in the anti‐TNF groups (P = 0.29). Clinical response (including remission) rate was 62% in the TAC vs. 64% in the anti‐TNF groups (P > 0.99). Five patients (10%) in the TAC and 8 (16%) in the anti‐TNF groups required colectomy (P = 0.55). In a subgroup analysis restricted to severely active UC, the response rate was 50% in the TAC vs. 25% in the anti‐TNF groups (P = 0.24). In severely active UC, the response rate tended to be higher in patients treated with tacrolimus, albeit not statistically significant.
Conclusions
Both tacrolimus and anti‐TNF agents appeared to be safe and effective in the management of moderate‐to‐severe active UC. However, randomised controlled trials are warranted to confirm the results obtained in this study.
Summary
We analyzed osteoporosis in 20 HME patients. According to the T-score of BMD, 30% and 67.5% of the patients fell in the range of osteopenia in the lumbar spine and femoral neck. Our results ...indicate HME patients have low bone mass. They do not have abnormal bone metabolism.
Introduction
There are few reports of osteoporosis in hereditary multiple exostoses (HME) patients. Therefore, the purpose of this study was to analyze osteoporosis in HME patients.
Methods
This retrospective cohort study included 20 patients diagnosed with HME. Patients underwent bone mineral density (BMD) measurement of the lumbar spine (
n
= 20) and femoral neck (
n
= 40). Bone metabolic parameters, including serum osteocalcin and urinary cross-linked N-telopeptide of type 1 collagen (NTx), were analyzed in all subjects.
EXT1
and
EXT2
genes were sequenced using genomic DNA. We also examined the correlation between genotype and BMD Z-score and T-score.
Results
The mean BMD values of the lumbar spine were 1.085 ± 0.116 g/cm
2
(
n
= 11) in male and 1.108 ± 0.088 g/cm
2
(
n
= 9) in female. The mean BMD values of the femoral neck area were 0.759 ± 0.125 g/cm
2
(
n
= 22) in male and 0.749 ± 0.115 g/cm
2
(
n
= 18) in female. Z-score of most HME patients show < 0, indicating that these patients tend to have low bone mass compared with the age-matched population. According to the T-score of BMD, 30% (6 of 20) and 67.5% (27 of 40) of the patients fell in the range of osteopenia in the lumbar spine and femoral neck areas, respectively. Serum osteocalcin and urinary NTx were in the normal range in most patients. There was no significant correlation between genotypes and Z-score.
Conclusion
HME patients have low bone mass, especially in the femoral neck area. They do not have abnormal bone metabolism, and there was no correlation between genotypes and Z-score.
Exostosin-1 (EXT1) and EXT2 are the major genetic etiologies of multiple hereditary exostoses and are essential for heparan sulfate (HS) biosynthesis. Previous studies investigating HS in several ...mouse models of multiple hereditary exostoses have reported that aberrant bone morphogenetic protein (BMP) signaling promotes osteochondroma formation in Ext1-deficient mice. This study examined the mechanism underlying the effects of HS deficiency on BMP/Smad signaling in articular cartilage in a cartilage-specific Ext−/− mouse model.
We generated mice with a conditional Ext1 knockout in cartilage tissue (Ext1-cKO mice) using Prg4-Cre transgenic mice. Structural cartilage alterations were histologically evaluated and phospho-Smad1/5/9 (pSmad1/5/9) expression in mouse chondrocytes was analyzed. The effect of pharmacological intervention of BMP signaling using a specific inhibitor was assessed in the articular cartilage of Ext1-cKO mice.
Hypertrophic chondrocytes were significantly more abundant (P = 0.021) and cartilage thickness was greater in Ext1-cKO mice at 3 months postnatal than in control littermates (P = 0.036 for femur; and P < 0.001 for tibia). However, osteoarthritis did not spontaneously occur before the 1-year follow-up. matrix metalloproteinase (MMP)-13 and adamalysin-like metalloproteinases with thrombospondin motifs(ADAMTS)-5 were upregulated in hypertrophic chondrocytes of transgenic mice. Immunostaining and western blotting revealed that pSmad1/5/9-positive chondrocytes were more abundant in the articular cartilage of Ext1-cKO mice than in control littermates. Furthermore, the BMP inhibitor significantly decreased the number of hypertrophic chondrocytes in Ext1-cKO mice (P = 0.007).
HS deficiency in articular chondrocytes causes chondrocyte hypertrophy, wherein upregulated BMP/Smad signaling partially contributes to this phenotype. HS might play an important role in maintaining the cartilaginous matrix by regulating BMP signaling.
Aim
To evaluate the efficacy of a prototype root canal dressing containing surface pre‐reacted glass–ionomer (S‐PRG) fillers on repairing induced periapical lesions in a rat model. Calcium hydroxide ...Ca(OH)2 was applied as a comparison in the healing process.
Methodology
The pulp chambers of the maxillary first molars in 64 male Wistar rats aged 16 weeks were opened to induce periapical lesions. After 28 days, the mesial canal of each tooth was prepared, irrigated with 2.5% sodium hypochlorite only (control group: irrigation) or followed by the respective dressing Ca(OH)2 group, irrigation + Ca(OH)2; S‐PRG group, irrigation + S‐PRG and restored with composite resin for 3 or 7 days (10/group). Four rats with healthy molars were used as blank controls. Descriptive analysis of the periapical radiographs, haematoxylin and eosin staining and immunohistochemical observation was performed 3 and 7 days after treatment. The periapical grey value, CD68 macrophages and osteoclasts (cathepsin‐K) were quantified and statistically analysed with Tukey’s honest significant difference test. A significant difference was achieved when P values were <0.05.
Results
S‐PRG and Ca(OH)2 dressings were associated with increased periapical grey values and inhibited osteoclast activity at 3 and 7 days; a significant difference in radiographic results and the number of osteoclasts was obtained at 3 and 7 days compared with the control group (P < 0.05). Reparative tissue was observed histologically in the space of the periapical resorbed necrotic area after S‐PRG and Ca(OH)2 treatment for 3 and 7 days. The number of macrophages was significantly decreased at 3 and 7 days in the S‐PRG and Ca(OH)2 specimens when compared with the controls (P < 0.05).
Conclusions
In a rat experimental model, the S‐PRG root canal dressing was comparable to Ca(OH)2 in promoting the healing of experimentally induced periapical lesions. S‐PRG paste has the potential to be used as an alternative intracanal dressing in teeth with apical periodontitis.
Summary
Background
No studies have monitored the levels of faecal calprotectin (FC) during mesalazine suppository therapy for proctitis in ulcerative colitis (UC).
Aims
To evaluate the value of ...consecutive monitoring of FC in patients with UC during mesalazine suppository therapy.
Methods
One hundred and sixty patients with active inflammation limited to the rectum were treated with mesalazine 1 g suppository once daily for 8 weeks. Patients who achieved clinical remission were advised to maintain the treatment, and were followed up for further 40 weeks. FC levels were measured every 8 weeks during the study.
Results
At week 8, 118 patients (74%) went into clinical remission, of whom 88 achieved endoscopic healing. The median FC level significantly decreased in patients with clinical and endoscopic remission (both P < 0.0001), while it did not change significantly in those without remission. Eighty (68%) of the 118 patients with remission continued the treatment. Twenty‐four patients (30%) relapsed during the 40‐week follow‐up. In patients with clinical relapse, the median FC level elevated already 8 weeks before the diagnosis of relapse. In contrast, in patients who maintained remission it remained at a low level and did not significantly change during the follow‐up. Elevated FC level (≥55 μg/g) was useful for the early diagnosis of relapse (88% sensitivity and 80% specificity).
Conclusions
Faecal calprotectin may represent a useful biomarker for the assessment of disease activity in UC patients treated with mesalazine suppositories. Serial monitoring of faecal calprotectin appears to be valuable for the prediction and early diagnosis of relapse during maintenance therapy.
Although it is known that human leukocyte antigen (HLA)-DPB1 disparity has a strong impact on outcomes in unrelated hematopoietic transplantation with induction of acute graft-versus-host disease ...(GVHD) and a graft-versus-leukemia (GVL) effect, its role in unrelated umbilical cord blood transplantation (UR-CBT) has yet to be fully clarified. Our current study is being conducted to elucidate the impact of HLA-DPB1 mismatch, along with the effect of other HLA loci mismatches at the allele level. HLA six loci alleles were retrospectively typed in 1157 Japanese donors and patients with leukemia or myelodysplastic syndrome who underwent transplantation with a single unit of cord blood. HLA-DPB1 mismatch was associated with a significant reduction in leukemia relapse (hazard ratio 0.61, P<0.001), whereas the other HLA loci allele-level mismatches did not. No significant effect of HLA-DPB1 mismatch was observed in the risk of acute GVHD, engraftment or mortality. This HLA-DPB1 GVL effect without induction of severe acute GVHD or deterioration of survival rate has not been reported in unrelated bone marrow or peripheral blood stem cell transplantations, suggesting apparent advantages of UR-CBT. Accordingly, selection of an HLA-DPB1 mismatch cord blood might be the preferable choice for single-unit UR-CBT.
Abstract
This paper presents the experimental results of 4.2 K cooling power of a Gifford-McMahon (G-M) cryocooler. To improve the cooling power, an original regenerator structure named coaxial pipe ...regenerator was applied to the second stage regenerator. This structure is that a stainless steel pipe is inserted in the coaxial direction of the second stage regenerator. An effect of the coaxial pipe is considered to rectify the helium flow in the regenerator that leads to an increase in the mass flow rate to the expansion space. Then, an improvement in the cooling power is expected. The second stage regenerator is comprised of three-layer of Pb (warm side), HoCu
2
(middle) and Gd
2
O
2
S (cold side) spheres of which the volume filling rate is 50, 20 and 30%, respectively. The coaxial pipe is applied to each material layer independently to survey an improvement effect of the cooling power at 4.2 K. These coaxial pipe regenerators were tested with a G-M cryocooler of one-watt model (RDK-408D2, SHI) and a scroll-type compressor (SSC-3700, SUZUKISHOKAN). The three-layer regenerator (without coaxial pipe) obtained the cooling power of 1.70 W. When the coaxial pipe was applied to the Pb layer, the cooling power of 1.79 W was achieved with the electrical input of compressor of 7.06 kW. In contrast, the coaxial pipe of HoCu
2
layer and Gd
2
O
2
S layer showed no conspicuous improvement. These results prove that the flow condition of helium in the Pb layer significantly affects the 4 K cooling power.
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