Rh proteins and H+ transporters involved in ammonia excretion in Amur Ide (Leuciscus waleckii) under high alkali exposure.

High alkaline environment can lead to respiratory alkalosis and ammonia toxification to freshwater fish. However, the Amur ide (Leuciscus waleckii), which inhabits an extremely alkaline lake in China with titratable alkalinity up to 53.57 mM (pH 9.6) has developed special physiological and molecular mechanisms to adapt to such an environment. Nevertheless, how the Amur ide can maintain acid-base balance and perform ammonia detoxification effectively remains unclear. Therefore, this study was designed to study the ammonia excretion rate (Tamm), total nitrogen accumulation in blood and tissues, including identification, expression, and localization of ammonia-related transporters in gills of both the alkali and freshwater forms of the Amur ide. The results showed that the freshwater form Amur ide does not have a perfect ammonia excretion mechanism exposed to high-alkaline condition. Nevertheless, the alkali form of Amur ide was able to excrete ammonia better than freshwater from Amur ide, which was facilitated by the ionocytes transporters (Rhbg, Rhcg1, Na+/H+ exchanger 2 (NHE2), and V-type H+ ATPase (VHA)) in the gills. Converting ammonia into urea served as an ammonia detoxication strategy to reduced endogenous ammonia accumulation under high-alkaline environment.

Iron deficiency and early childhood caries: a systematic review and meta-analysis.

BACKGROUNDS: Previous surveys have found that children with iron deficiency (ID) were likely to suffer from early childhood caries (ECC). We aimed to assess the scientific evidence about whether ID is intrinsically related to ECC. METHODS: The medical subject headings (MeSH) terms and free words were searched on PubMed, Web of Science, Cochrane, China National Knowledge Infrastructure, Wanfang, and the Database for Chinese Technical Periodicals from March 2020 to September 2020. Two researchers independently screened the articles. Data extraction and cross-checking were performed for the studies that met the inclusion criteria. Meta-analysis was performed using the Cochrane Collaboration's Review Manager 5.3 software. RESULTS: After excluding duplication and irrelevant literature, 12 case-control studies were included in the study. The meta-analysis demonstrated that children with ECC were more likely to have ID (odds ratio [OR] = 2.63, 95% confidence interval [CI]: [1.85, 3.73], P < 0.001). There was no statistically significant association found between the level of serum ferritin and ECC (weighted mean difference (WMD) = -5.80, 95% CI: [-11.97, 0.37], P = 0.07). Children with ECC were more likely to have iron-deficiency anemia (OR = 2.74, 95% CI: [2.41,3.11], P < 0.001). The hemoglobin (HGB) levels in the ECC group were significantly lower compared with that in the ECC-free group (WMD = -9.96, 95% CI: [-15.45, -4.46], P = 0.0004). The mean corpuscular volume (MCV) levels in the ECC group were significantly lower compared with that in the ECC-free group (WMD = -3.72, 95% CI: [-6.65, -0.79], P = 0.01). CONCLUSIONS: ID was more prevalent in children with ECC, and the markers of iron status in the ECC group, such as serum ferritin, HGB, and MCV, were relatively lower than the ECC-free group.

Effects of Bicarbonate Stress on Serum Ions and Gill Transporters in Alkali and Freshwater Forms of Amur Ide (Leuciscus waleckii).

The Amur ide (Leuciscus waleckii) is a fish in the Cyprinidae family. Compared with other Amur ide living in freshwater ecosystems, the Amur ide population in Lake Dali Nor of China is famous for its high tolerance to the alkaline conditions of 54 mM (pH 9.6). Yet, surprisingly, the ionoregulatory mechanism responsible for this remarkable alkaline adaptation remains unclear. Therefore, this study sought to investigate how bicarbonate affects the acid-base balancing and ionoregulatory responses of this animal. Here, using a comparative approach, the alkali form of Amur ide and its ancestral freshwater form living in other freshwater basins were each exposed to 50 mM (pH 9.59 ± 0.09), a level close to the alkalinity of Lake Dali Nor, and their physiological (AE1) adjustment of ions and acid-base regulation were investigated. This study highlighted differences in blood pH and serum ions (e.g., Na+, K+, Cl-, and Ca2+), Na+/K+ ATPase (NKA) activity and its mRNA level, and mRNA expression of gill transporters (Na+/H+ exchanger member 2 and/or 3, Na+/ HCO 3 - cotransporter (NBC1), Cl-/ HCO 3 - exchanger, Na+/Cl- cotransporter (NCC), Na+/K+/2Cl- (NKCC1), SLC26A5, and SLC26A6) for alkalinity adaptation between the two forms of Amur ide differing in alkalinity tolerance. Specifically, close relationships among the serum Na+ and mRNA levels of NCC, NKCC1, and NHE, and also NKA and NBC1, in addition to serum Cl- and bicarbonate transporters (e.g., SLC26A5 and SLC26A6), characterized the alkali form of Amur ide. We propose that this ecotype can ensure its transepithelial Cl- and Na+ uptake/base secretions are highly functional, by its basolateral NKA with NBC1 and apical ionic transporters, and especially NCC incorporated with other transporters (e.g., SLC26). This suggests an evolved strong ability to maintain an ion osmotic and acid-base balance for more effectively facilitating its adaptability to the high alkaline environment. This study provides new insights into the physiological responses of the alkaline form of the Amur ide fish for adapting to extreme alkaline conditions. This information could be used as a reference to cultivating alkaline-tolerant fish species in abandoned alkaline waters.

Identification and Analysis of Long Non-coding RNAs in Leuciscus waleckii Adapted to Highly Alkaline Conditions.

Leuciscus waleckii is a freshwater fish that is known to inhabit the Dali Nor Lake, Inner Mongolia, China. The water in this lake has an HCO3 -/CO3 2- concentration of 54 mM (pH 9.6) and a salinity of 0.6‰. The physiological mechanisms that allow this fish to tolerate these saline/alkaline conditions have yet to be elucidated. Transcriptional component analysis has shown that the expression levels of a large number of genes involved in the pathways responsible for osmo-ionoregulation and arachidonic acid metabolism pathway expression change significantly (p < 0.05) during the regulation of acid-base balance under high alkaline stress. In this study, we investigated the role of long non-coding RNAs (lncRNAs) during adaptation to high alkaline conditions. Fish were challenged to an NaHCO3-adjusted alkalinity of 0 mM, 30 mM (pH 9.44 ± 0.08), and 50 mM (pH 9.55 ± 0.06) for 20 days in the laboratory. Gill and kidney tissues were then collected for high-throughput sequencing assays. A total of 159 million clean reads were obtained by high-throughput sequencing, and 41,248 lncRNA transcripts were identified. Of these, the mean number of exons and the mean length of the lncRNA transcripts were 4.8 and 2,079 bp, respectively. Based on the analysis of differential lncRNA transcript expression, a total of 5,244 and 6,571 lncRNA transcripts were found to be differentially expressed in the gills and kidneys, respectively. Results derived from Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the coding genes were correlated with the lncRNA expression profiles. GO analysis showed that many lncRNAs were enriched in the following processes: "transporter activity," "response to stimulus," and "binding." KEGG analysis further revealed that metabolic pathways were significantly enriched. A random selection of 16 lncRNA transcripts was tested by RT-qPCR; these results were consistent with our sequencing results. We found that a large number of genes, with the same expression profiles as those with differentially expressed lncRNAs, were associated with the regulation of acid-base balance, ion transport, and the excretion of ammonia and nitrogen. Collectively, our data indicate that lncRNA-regulated gene expression plays an important role in the process of adaptation to high alkaline conditions in L. waleckii.

[Experimental study of a new animal model with trigeminal neuralgia produced by administration of talc to peripheral infraobital nerve in rats].

PURPOSE: To establish a new animal model of trigeminal neuralgia（TN） produced by administration of talc to peripheral infraobital nerve in rats. METHODS: Thirty male Wistar rats were randomly divided into 2 groups. Talcum powder (30％,0.3 mL) was injected into the peripheral infraorbital foramen in one group, the same dose of normal saline was injected by the same method in another group. On 3 day before surgery and 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 and 12 weeks postoperatively, mechanical pain behavior was determined. Statistical analysis of the threshold of pain response was performed and the behavior of pain was observed in the area of infraorbital nerve innervation in rats. Histopathological changes of the peripheral infraorbital nerve tissue in the rats were observed 3 days, 4 weeks, 8 weeks and 12 weeks postoperatively. The expression of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin -1ß (IL-1ß) in the territory of the infraorbital nerve was detected by immunohistochemistry. SPSS16.0 software package was used to analyze the data. RESULTS: The mechanical pain threshold of rats in the infraorbital innervation area 3 days postoperatively in the experimental group was significantly decreased compared with that in the preoperative group and the control group (P<0.01). The rats in the experimental group 3 days postoperatively experienced symptoms of irritability, scratching the face or aggressive behavior. Twelve weeks after operation, the mechanical pain threshold was still significantly decreased. Histopathological examination in the experimental group 3 days postoperatively mainly showed inflammation with a few inflammatory factors（IL-1ß and TNF-α）expression. Inflammation in the experimental group 1week postoperatively was more intense and more inflammatory factors were expressed. Four weeks postoperatively, there was more proliferation of granulation tissue in the area of peripheral infraorbital nerve tissue and expression of inflammatory factors was highest. Four to twelve weeks, the inflammatory response in the experimental group was gradually reduced, increased scar and infraorbital nerve compressing by scar were observed, and the expression of inflammatory factors decreased gradually. CONCLUSIONS: Injection of talc to the peripheral infraorbital foramen can establish a reliable and stable animal model for research of etiology and treatment of TN.

[Preliminary report of botulinum toxin type A injection at trigger point for treatment of trigeminal neuralgia: experiences of 16 cases].

PURPOSE: To investigate the effects of injection of botulium toxin type A at trigger point for treatment of patients with primary trigeminal neuralgia. METHODS: Sixteen patients with primary Trigeminal Neuralgia were treated with injection of botulium toxin type A. Visual analog scores(VAS) at 1 week, 2 weeks, 1 month, 3 months and 6 months after treatment and Barrow Neurological Institute (BNI) pain evaluation criteria were utilized to measure the degree of pain. The data was analyzed with SPSS 10.0 software package. RESULTS: The VAS score was 9.12±0.65 before botulium toxin type A injection while the scores were 2.8±1.36, 2.2±1.26, 1.3±1.45, 1.3±1.45 and 1.2±2.52 at 1 week, 2 weeks, 1 month,3 months and 6 months after treatment. There was significant difference in VAS compared with before treatment. VAS score was lower and stable at 1 month, 3 months and 6 months after treatment, but no significant difference was found at 1-week and 2-week after treatment. BNI evaluation results showed good therapeutic effect 1 week after treatment, while the best therapeutic effect was noted 1-3 months after treatment. 6 months later, 1 patient had recurrence and 11 patients had complete relief of pain. CONCLUSIONS: Botulium toxin type A injection is an effective way for treatment of patients with primary trigeminal neuralgia.

A preliminary study on the application of bone marrow stromal cell sheet on the formation of functional tissue-engineered bone in dogs.

PURPOSE: The aim of this study was to construct functional tissue-engineered bone in dogs using cell sheet engineering, a new technique to gain and transfer seed cells. MATERIALS AND METHODS: Demineralized bone matrixes, prepared from homologous bone, were coated with recombinant human bone morphogenetic protein-2. Bone marrow stromal cells (BMSCs) were isolated and subcultured. Osteogenic-induced BMSCs were incubated in a temperature-responsive culture dish to form the BMSC sheet. The complex of demineralized bone matrix, recombinant human bone morphogenetic protein-2, and BMSCs wrapped with BMSC sheets was implanted around the blood vessels of the latissimus dorsi muscle in the experimental side, and the same complex without BMSC sheets was implanted around the blood vessels of the latissimus dorsi muscle on the other side as a control. At 4, 8, 12, and 16 weeks after implantation, the implants were removed for radiographic evaluation, descriptive histologic observation, and histologic quantitative analysis. RESULTS: Radiographic analysis showed that the optical density of the tissue-engineered bone on the 2 sides increased with time. However, the optical density of the experimental side was significantly greater than that of the control side at the same points. Sixteen weeks after implantation, mature lamellar bone was formed in the experimental side, with red bone marrow in the bone marrow cavity. In contrast, the control side exhibited significantly less lamellar bone. Histologic quantitative analysis showed that the experimental side exhibited significantly more bone per area compared with the control side. CONCLUSION: BMSC sheet engineering may be useful to construct functional tissue-engineered bone.

[Establishment of palatal organ culture model of C57BL/6J mouse embryos in vitro].

PURPOSE: To establish palatal organ culture model of C57BL/6J mouse embryos in vitro and provide platform for study of embryo palatal development. METHODS: The mouse palatal shelves were harvested under sterilization from a female mouse of gestation day(GD) 13.5 by stereoscopic microscope and cultured in vitro. Totally 36 pairs of palatal shelves were divided into three groups equally and cultured 6 h, 24 h and 48 h, respectively. Finally, all palatal shelves were embedded and stained by Hematoxylin and Eosin (HE) and subjected to scanning electron microscope (SEM) analysis. RESULTS: The results of HE dyeing showed that the palatal shelves did not fuse on 6 h group, and began to fuse on 24 h group, but still had some medial edge epithelial (MEE) cells remained. The palatal shelves completely fused and all the MEE cells disappeared on 48 h group. The results of SEM showed that there was a gap between palatal shelves on 6 h group. The palatal shelves began to contact and form the medial epithelial seam (MES) on 12 h group. Finally, palatal shelves completely fused and MES disappeared on 48 h group. CONCLUSION: This method provides an effective way for investigating the etiology of cleft palate in vitro.

[Role of p38 mitogen activated protein kinase on cyclic stretch in human facial hypertrophic scar fibroblasts differentiation into myofibroblasts].

OBJECTIVE: To explore the signal transduction mechanism of p38 mitogen activated protein kinase (p38MAPK) in human facial hypertrophic scar fibroblast (FB) differentiation into myofibroblasts (MFB). METHODS: Fibroblasts of primary culture were simple randomly assigned into two groups: cyclic stretch (control group) and cyclic stretch pre-treated with SB203580(experimental group). Expression of P-p38MAPK and α-smooth muscle actin (α-SMA) protein were examined using Western blotting and expression of transforming growth factor ß1 (TGF-ß1) mRNA and α-SMA mRNA were examined using reverse transcription PCR (RT-PCR). RESULTS: In control group, the expressions of α-SMA, p38MAPK, TGF-ß1 mRNA and α-SMA mRNA (0 h: 0.134 ± 0.011, 0.239 ± 0.015, 0.214 ± 0.018, 0.252 ± 0.010; 6 h: 0.152 ± 0.014, 0.287 ± 0.016, 0.288 ± 0.011, 0.277 ± 0.013; 12 h: 0.172 ± 0.017, 0.320 ± 0.017, 0.335 ± 0.013, 0.297 ± 0.006) , were significantly increased with loading time (6 h>0 h; 12 h>0 and 6 h). In experimental group (pre-treated with SB203580), the expressions of α-SMA, p38MAPK, TGF-ß1 mRNA,α-SMA mRNA (6 h: 0.116 ± 0.017,0.128 ± 0.016,0.134 ± 0.014,0.163 ± 0.009; 12 h: 0.149 ± 0.013,0.136 ± 0.018,0.144 ± 0.013,0.187 ± 0.010) on corresponding time decreased sharply compared with those in control groups (6, 12 h). CONCLUSIONS: The human facial hypertrophic scar fibroblasts differentiation in response to cyclic stretch was mediated by p38MAPK phosporylation.

[Treatment of zygomatio- orbital fractures with small local incisions: report of 32 cases].

PURPOSE: To evaluate the surgical result of small local incisions in the treatment of zygomatio-orbital fractures. METHODS: 32 patients with zygomatio-orbital fractures were operated on with two or more kinds of incisions, including subciliary incision, lateral eyebrow incision, transverse incision along the superior margin of zygomatic arch, oral vestibular incision, maxillary sinus incision, the original wound according to the area and type of the fractures. The fracture line was exposed via the small local incisions and fixed with micro-titanium plates. The therapeutic effectiveness was evaluated 3 to 6 months after operation. RESULTS: All patients had primary healing without apparent scars in the face,except that one patient developed mild ectropion and two had temporary paralysis of the temporal branches of the facial nerve. The function of the eyes recovered completely, while the jaw movement improved significantly. The facial appearance recovered to normal height and width and the bilateral face became symmetrical. CONCLUSION: Treatment of zygomatic-orbital fractures with small local incisions is minimally invasive and the facial appearance and function can be recovered significantly with few complications.

[The influence of botulinum toxin on the masticatory muscles of the temporomandibular joint osteoarthritis in rabbit].

PURPOSE: To study the influence of botulinum toxin on masticatory muscle activity in temporomandibular joint model osteoarthritis(TMJOA). METHODS: 30 adult rabbits were examined with EMG, and TMJOA was established by collagenase injection, then divided into experimental group and control group. EMG of masticatory muscles was recorded during postural position and clenching at the end of 4 weeks, 8 weeks and 12 weeks. Botulinum toxin was injected into the masseter muscle and temporal muscle in the experimental group. Those of the control group were only with TMJOA. All data were analyzed with group t test in SPSS 11.0. RESULTS: During postural position, EMG activity of masticatory muscles in the control group was significantly higher than that of the normal at each examination(P<0.05). EMG activity in th experimental group was lower than normal at the end of 4 weeks and 8 weeks(P<0.05), but at the end of 12 weeks, the two groups showed no difference(P>0.05). During clenching, EMG activity in the control group was lower than normal at each measurement(P<0.05), EMG activity in the experimental group was lower than normal at the end of 4 weeks and 8 weeks(P<0.05), but showed no difference at the end of 12 weeks. CONCLUSION: Botulinum toxin can change and improve masticatory muscle function of TMJOA in rabbit.