Proteinase activated receptor 2: Role of extracellular loop 2 for ligand-mediated activation

1. Rat proteinase-activated receptor-2 (PAR2) variants were stably expressed in rat KNRK cells: (a) wild-type (wt) - PAR2; (b) PAR2PRR, with the extracellular loop 2 (EL-2) sequence P231E232E233mutated to PRR and (c) PAR2NET, with the EL-2 sequence, PEEV changed to NETL. Cell lines were evaluated for their sensitivity (calcium signalling) towards trypsin and the receptor-activating peptides, SLIGRL-NH2, SLIGEL-NH2, trans-cinnamoyl(tc)-LIGRLO-NH2, and SFLLR-NH2. 2. SLIGEL-NH2 exhibited low potency (1 : 200 relative to SLIGRL-NH2) in wild-type PAR2. Its activity was increased 5 fold in PAR2PRR, but it was inactive in PAR2NET. 3. In PAR2PRR, the potencies of SLIGRL-NH2, tc-LIGRLO-NH2, and SFLLR-NH2 were decreased by 80 - 100 fold. But, the potency of trypsin was decreased by only 7 fold. 4. In PAR2NET, highly homologous in EL-2 with proteinase-activated receptor-1 (PAR1), the potency of the PAR1-derived peptide, SFLLR-NH2, was reduced by 100 fold compared with wt-PAR2, whereas the potency of the PAR2-derived AP, SLIGRL-NH2 was reduced 10 fold. In contrast, the potency of trypsin in PAR2NET was almost the same as in wt-PAR2. 5. We conclude that the acidic EL-2 tripeptide, PEE, in PAR2 plays an important role in governing agonist activity. 6. The data obtained with the PEEV-->NETL mutation suggested: (a) that SLIGRL-NH2 and SFLLR-NH2 interact in a distinct manner with PAR2 and (b) that SFLLR-NH2 may interact differently with PAR2 than it does with PAR1. 7 The differential reductions in the potencies of SLIGRL-NH2, compared with trypsin in the PAR2PRR and PAR2NET cell lines point to differences between the interactions of the trypsin-revealed tethered ligand and the free receptor-activating peptide with PAR2.     

Various aspects of hearing loss in newborns: A narrative review

Hearing loss is considered the most common birth defect. The estimated prevalence of moderate and severe hearing loss in a normal newborn is 0.1%-0.3%, while the prevalence is 2%-4% in newborns admitted to the newborn intensive care unit. Neonatal hearing loss can be congenital (syndromic or non-syndromic) or acquired such as ototoxicity. In addition, the types of hearing loss can be conductive, sensorineural, or mixed. Hearing is vital for the acquisition of language and learning. Therefore, early detection and prompt treatment are of utmost importance in preventing the unwanted sequel of hearing loss. The hearing screening program is mandatory in many nations, especially for high-risk newborns. An automated auditory brainstem response test is used as a screening tool in newborns admitted to the newborn intensive care unit. Moreover, genetic testing and screening for cytomegalovirus in newborns are essential in identifying the cause of hearing loss, particularly, mild and delayed onset types of hearing loss. We aimed to update the knowledge on the various aspects of hearing loss in newborns with regard to the epidemiology, risk factors, causes, screening program, investigations, and different modalities of treatment.     

Is Use of BMP-2 Associated with Tumor Growth and Osteoblastic Differentiation in Murine Models of Osteosarcoma?

BackgroundThe putative benefit of rhBMP-2 is in the setting of limb reconstruction using structural allografts, whether it be allograft-prosthetic composites, osteoarticular allografts, or intercalary segmental grafts. There are also potential advantages in augmenting osseointegration of uncemented endoprosthetics and in reducing infection. Recombinant human BMP-2 might mitigate nonunion in structural allograft augmented osteosarcoma limb salvage surgery; however, its use is limited because of concerns about the prooncogenic effects of the agent.Questions/purposes(1) To assess if BMP-2 signaling influences osteosarcoma cell line growth. (2) To characterize degree of osteosarcoma cell line osteoblastic differentiation in response to BMP-2. (3) To assess if BMP-2 signaling has a consistent effect on local or systemic tumor burden in various orthotopic murine models of osteosarcoma.MethodsIn this study, 143b, SaOS-2 and DLM8-M1 osteosarcoma cell lines were transfected with BMP-2 cDNA controlled by a constitutive promoter (experimental) or an empty vector (control) using a PiggyBac transposon system. Cellular proliferation was assessed using a quantitative MTT colorimetric assay. Osteoblastic differentiation was compared between control and experimental cell lines using quantitative real-time polymerase chain reaction of the osteoblastic markers connective tissue growth factor, Runx-2, Osterix, alkaline phosphatase and osteocalcin. Experimental and control cell lines were injected into the proximal tibia of either NOD-SCID (143b and SaOS-2 xenograft model), or C3H (DLM8-M1 syngeneic model) mice. Local tumor burden was quantitatively assessed using tumor volume caliper measurements and bioluminescence, and qualitatively assessed using post-mortem ex vivo microCT. Lung metastasis was qualitatively assessed by the presence of bioluminescence, and incidence was confirmed using histology. rhBMP-2 soaked absorbable collagen sponges (experimental) and sterile-H₂O soaked absorbable collagen sponges (control) were implanted adjacent to 143b proximal tibial cell line injections to compare the effects of exogenous BMP-2 application with endogenous upregulation.ResultsConstitutive expression of BMP-2 increased the in vitro proliferation of 143b cells (absorbance values 1.2 ± 0.1 versus 0.89 ± 0.1, mean difference 0.36 [95% CI 0.12 to 0.6]; p = 0.01), but had no effect on SaOS-2 and DLM8-M1 cell proliferation. In response to constitutive BMP-2 expression, 143b cells had no differences in osteoblastic differentiation, while DLM8-M1 cells downregulated the early marker connective tissue growth factor (mean ΔCt 0.2 ± 0.1 versus 0.6 ± 0.1; p = 0.002) and upregulated the early-mid range marker Runx-2 (mean ΔCt -0.8 ± 0.1 versus -1.1 ± 0.1; p = 0.002), and SaOS-2 cells upregulated the mid-range marker Osterix (mean ΔCt -2.1 ± 0.6 versus -3.9 ± 0.6; p = 0.002). Constitutive expression of BMP-2 resulted in greater 143b and DLM8-M1 local tumor volume (143b: 307.2 ± 106.8 mm³ versus 1316 ± 387.4 mm³, mean difference 1009 mm³ [95% CI 674.5 to 1343]; p < 0.001, DLM8-M1 week four: 0 mm³ versus 326.1 ± 72.8 mm³, mean difference 326.1 mm³ [95% CI 121.2 to 531]; p = 0.009), but modestly reduced local tumor growth in SaOS-2 (9.5 x 10⁸ ± 8.3x10⁸ photons/s versus 9.3 x 10⁷ ± 1.5 x 10⁸ photons/s, mean difference 8.6 x 10⁸ photons/s [95% CI 5.1 x 10⁸ to 1.2 x 10⁹]; p < 0.001). Application of exogenous rhBMP-2 also increased 143b local tumor volume (495 ± 91.9 mm³ versus 1335 ± 102.7 mm³, mean difference 840.3 mm³ [95% CI 671.7 to 1009]; p < 0.001). Incidence of lung metastases was not different between experimental or control groups for all experimental conditions.ConclusionsAs demonstrated by others, ectopic BMP-2 signaling has unpredictable effects on local tumor proliferation in murine models of osteosarcoma and does not consistently result in osteosarcoma cell line differentiation. Further investigations into other methods of safe bone and soft tissue healing augmentation and the use of differentiation therapies is warranted.Clinical RelevanceOur results indicate that BMP-2 has the potential to stimulate the growth of osteosarcoma cells that are poorly responsive to BMP-2 mediated osteoblastic differentiation. As this differentiation potential is unpredictable in the clinical setting, BMP-2 may promote the growth of microscopic residual tumor burden after resection. Our study provides further support for the recommendation to avoid the use of BMP-2 after limb-salvage surgery in patients with osteosarcoma.     

骨髓干细胞分化为肝细胞的多种移植途径

近年来许多研究证实,人类和啮齿类动物的骨髓细胞可分化为多种细胞类型,包括骨骼肌细胞、心肌细胞、神经细胞和肝细胞等,这些骨髓干细胞的可塑性研究,为肝细胞移植提供了新的供体来源．骨髓干细胞替代肝细胞进行移植具有来源丰富,费用相对低廉,对患者损伤小等优点,且自体骨髓干细胞移植可以完全避免移植排斥反应,同时,骨髓干细胞只有 5-15 μm,移植后不会发生栓塞等并发症．因此骨髓干细胞移植在治疗肝病以及解决供体肝脏来源短缺方面具有广泛的应用前景．本文就骨髓干细胞的移植途径做一综述．     

三维重建射频损伤区域观察猪舌根射频损伤体积与射频能量及时间的关系

目的：目前临床进行隧道法舌根射频治疗时,其作用参数的设置仍缺乏统一的标准,故通过计算机三维重建射频损伤区域,分析猪舌根射频损伤体积与射频能量、时间的关系,从中得出应用舌根隧道法射频治疗的最佳作用能级和作用时间。 方法：实验于2006-06/2007-05在上海交通大学耳鼻喉科研究所完成。将36只实验用猪以射频作用能级1,2,3,4,5,6随机分成6组,每组6头猪,各个猪舌的作用时间分别设置为2,5,10,15,20,25s。用Coblation射频发生仪及Reflex55刀头进行猪舌根射频操作。射频作用后的舌根组织行连续冰冻切片,苏木精-伊红染色后,进行序列组织切片的全貌二维图像采集,对拟重建的结构进行边界提取和图像分割。将提取分割图像导入Image-Pro Solution图像处理软件,利用3D Constructor插件进行三维重建,并根据设定参数进行体积计算。用SPSS10.0统计学软件对所测数据进行统计学分析。 结果：①作用能级固定时,舌根组织射频损伤体积随时间延长而增大,符合Logarithmic回归曲线。②作用时间固定时,舌根组织射频损伤体积随能级增大而增大,符合直线回归。③射频损伤体积随能量增大而增加亦符合Logarithmic回归曲线。④Coblation射频治疗系统在能级6时,在作用10s之前,损伤体积随作用时间增加而迅速增加,其后变化趋势平缓,超过20s后损伤体积无显著增加。 结论：①舌根区域射频治疗时,舌根组织射频损伤体积与时间或能量呈Logarithmic曲线相关,与能级呈直线相关。②Coblation射频治疗系统在能级6时,最佳作用时间范围为10-20s。     

Prediction of Caesarean Section from Ultrasound and Clinical Assessment of Fetal Size

EDITORIAL COMMENT: We accepted this paper for publication because the authors have explored the possible value of ultrasound versus clinical assessment of fetal size to see whether they can predict the need for Caesarean section when the baby is large. In this study both the ultrasound and the clinical assessment were impressively accurate and fetal femur length was the most accurate of the ultrasound parameters in assessing fetal weight in predicting the need for Caesarean section. However, as the authors indicate, none of these methods of assessment of fetal size are recommended as an absolute indication for Caesarean section. Every obstetrician uses clinical assessment of the fetus when evaluating management of the patient but the need for clinical judgment remains.
Summary: One hundred and five women with singleton pregnancies and cephalic presentation were assessed. Fundal height and a clinical estimate of fetal weight were recorded. The fetal biparietal diameter, abdominal circumference and femur length were measured with ultrasound. Ultrasound estimated fetal weight was calculated using 3 different formulas (Shepard, Campbell and CUHK). The liquor volume was assessed using the amniotic fluid index.
Ultrasound was able to predict Caesarean section with more reliability than clinical assessment of fetal size or weight. The biparietal diameter, fundal height and amniotic fluid index were poor predictors of mode of delivery. The measurements which best predicted the mode of delivery were the fetal femur length and abdominal circumference. Femur length, but not abdominal circumference, was a statistically better predictor of Caesarean section than clinical estimation of fetal weight. There was no improvement in prediction using ultrasound estimated fetal weight.     

Suppression by protease-activated receptor-2 activation of gastric acid secretion in rats

Activation of protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, induces neurally mediated gastric mucus secretion accompanied by mucosal cytoprotection. In the present study, we investigated whether PAR-2 could modulate gastric acid secretion in rats. Messenger RNAs for PAR-2 and PAR-1 were detected in the gastric mucosa and smooth muscle. The PAR-2-activating peptide SLIGRL-NH(2), but not the inactive control peptide, when administered i.v., strongly suppressed gastric acid secretion in response to carbachol, pentagastrin or 2-deoxy-D-glucose in the rats with a pylorus ligation. The PAR-2-mediated suppression of acid secretion was resistant to cyclooxygenase inhibition or ablation of sensory neurons by capsaicin. Our results provide novel evidence that in addition to stimulating neurally mediated mucus secretion, activation of PAR-2 suppresses gastric acid secretion independently of prostanoid production or sensory neurons. These dual actions of PAR-2 would result in gastric mucosal cytoprotection.