A high concentrate diet inhibits forkhead box protein A2 expression, and induces oxidative stress, mitochondrial dysfunction and mitochondrial unfolded protein response in the liver of dairy cows.

High-concentrate diet induce subacute ruminal acidosis (SARA) and cause liver damage in ruminants. It has been reported that forkhead box protein A2 (FOXA2) can enhance mitochondrial membrane potential but its function in mitochondrial dysfunction induced by high concentrate diets is still unknown. Therefore, the aim of this study was to elucidate the effect of high-concentrate (HC) diet on hepatic FOXA2 expression, mitochondrial unfolded protein response (UPRmt), mitochondrial dysfunction and oxidative stress. A total of 12 healthy mid-lactation Holstein cows were selected and randomized into 2 groups: the low concentrate (LC) diet group (concentrate:forage = 4:6) and HC diet group (concentrate:forage = 6:4). The trial lasted 21 d. The rumen fluid, blood and liver tissue were collected at the end of the experiment. The results showed that the rumen fluid pH level was reduced in the HC group and the pH was lower than 5.6 for more than 4 h/d, indicating that feeding HC diets successfully induced SARA in dairy cows. Both FOXA2 mRNA and protein abundance were significantly reduced in the liver of the HC group compared with the LC group. The activity of antioxidant enzymes (CAT, G6PDH, T-SOD, Cu/Zn SOD, Mn SOD) and mtDNA copy number in the liver tissue of the HC group decreased, while the level of H2O2 significantly increased, this increase was accompanied by a decrease in oxidative phosphorylation (OXPHOS). The balance of mitochondrial division and fusion was disrupted in the HC group, as evidenced by the decreased mRNA level of OPA1, MFN1, and MFN2 and increased mRNA level of Drp1, Fis1, and MFF. At the same time, HC diet downregulated the expression level of SIRT1, SIRT3, PGC-1α, TFAM, and Nrf 1 to inhibit mitochondrial biogenesis. The HC group induced UPRmt in liver tissue by upregulating the mRNA and protein levels of CLPP, LONP1, CHOP, Hsp10, and Hsp60. In addition, HC diet could increase the protein abundance of Bax, CytoC, Caspase 3 and Cleaved-Caspase 3, while decrease the protein abundance of Bcl-2 and the Bcl-2/Bax ratio. Overall, our study suggests that the decreased expression of FOXA2 may be related to UPRmt, mitochondrial dysfunction, oxidative stress, and apoptosis in the liver of dairy cows fed a high concentrate diet.

tRF3-IleAAT reduced extracellular matrix synthesis in diabetic kidney disease mice by targeting ZNF281 and inhibiting ferroptosis.

It is well established that the synthesis of extracellular matrix (ECM) in mesangial cells is a major determinant of diabetic kidney disease (DKD). Elucidating the major players in ECM synthesis may be helpful to provide promising candidates for protecting against DKD progression. tRF3-IleAAT is a tRNA-derived fragment (tRF) produced by nucleases at tRNA-specific sites, which is differentially expressed in the sera of patients with diabetes mellitus and DKD. In this study we investigated the potential roles of tRFs in DKD. Db/db mice at 12 weeks were adapted as a DKD model. The mice displayed marked renal dysfunction accompanied by significantly reduced expression of tRF3-IleAAT and increased ferroptosis and ECM synthesis in the kidney tissues. The reduced expression of tRF3-IleAAT was also observed in high glucose-treated mouse glomerular mesangial cells. We administered ferrostatin-1 (1 mg/kg, once every two days, i.p.) to the mice from the age of 12 weeks for 8 weeks, and found that inhibition of the onset of ferroptosis significantly improved renal function, attenuated renal fibrosis and reduced collagen deposition. Overexpression of tRF3-IleAAT by a single injection of AAV carrying tRF3-IleAAT via caudal vein significantly inhibited ferroptosis and ECM synthesis in DKD model mice. Furthermore, we found that the expression of zinc finger protein 281 (ZNF281), a downstream target gene of tRF3-IleAAT, was significantly elevated in DKD models but negatively regulated by tRF3-IleAAT. In high glucose-treated mesangial cells, knockdown of ZNF281 exerted an inhibitory effect on ferroptosis and ECM synthesis. We demonstrated the targeted binding of tRF3-IleAAT to the 3'UTR of ZNF281. In conclusion, tRF3-IleAAT inhibits ferroptosis by targeting ZNF281, resulting in the mitigation of ECM synthesis in DKD models, suggesting that tRF3-IleAAT may be an attractive therapeutic target for DKD.

Satellite cell-derived exosome-mediated delivery of microRNA-23a/27a/26a cluster ameliorates the renal tubulointerstitial fibrosis in mouse diabetic nephropathy.

Renal tubulointerstitial fibrosis (TIF) is considered as the final convergent pathway of diabetic nephropathy (DN) without effective therapies currently. MiRNAs play a key role in fibrotic diseases and become promising therapeutic targets for kidney diseases, while miRNA clusters, formed by the cluster arrangement of miRNAs on chromosomes, can regulate diverse biological functions alone or synergistically. In this study, we developed clustered miR-23a/27a/26a-loaded skeletal muscle satellite cells-derived exosomes (Exos) engineered with RVG peptide, and investigated their therapeutic efficacy in a murine model of DN. Firstly, we showed that miR-23a-3p, miR-26a-5p and miR-27a-3p were markedly decreased in serum samples of DN patients using miRNA sequencing. Meanwhile, we confirmed that miR-23a-3p, miR-26a-5p and miR-27a-3p were primarily located in proximal renal tubules and highly negatively correlated with TIF in db/db mice at 20 weeks of age. We then engineered RVG-miR-23a/27a/26a cluster loaded Exos derived from muscle satellite cells, which not only enhanced the stability of miR-23a/27a/26a cluster, but also efficiently delivered more miR-23a/27a/26a cluster homing to the injured kidney. More importantly, administration of RVG-miR-23a/27a/26a-Exos (100 µg, i.v., once a week for 8 weeks) significantly ameliorated tubular injury and TIF in db/db mice at 20 weeks of age. We revealed that miR-23a/27a/26a-Exos enhanced antifibrotic effects by repressing miRNA cluster-targeting Lpp simultaneously, as well as miR-27a-3p-targeting Zbtb20 and miR-26a-5p-targeting Klhl42, respectively. Knockdown of Lpp by injection of AAV-Lpp-RNAi effectively ameliorated the progression of TIF in DN mice. Taken together, we established a novel kidney-targeting Exo-based delivery system by manipulating the miRNA-23a/27a/26a cluster to ameliorate TIF in DN, thus providing a promising therapeutic strategy for DN.

Effects of Plant Growth-Promoting Rhizobacteria on the Growth and Soil Microbial Community of Carya illinoinensis.

Plant growth-promoting rhizobacteria (PGPR) are important members of soil microbial communities. In this study, the effects of several PGPR on the growth of Carya illinoinensis plants, the microbial community composition and soil nutrients were investigated by inoculation tests to identify excellent PGPR strains. The experiment showed that after PGPR application, the plant height, ground diameter, and dry weight of C. illinoinensis were significantly increased compared with those of the control group, and Bacillus velezensis YH20 had the most significant effect in promoting growth (p < 0.05). In addition, all the PGPRs used for inoculation promoted plant root growth, and the Brevibacillus reuszeri MPT17 strain had the most significant promoting effect on plant root growth (p < 0.05). The application of PGPRs also affected the nutrient levels in plants and plant rhizosphere soil. For example, compared with the control, the levels of available phosphorus and potassium in rhizosphere soil and the total potassium content in plant roots were significantly increased under Br. reuszeri MPT17 treatment (p < 0.05). The experiment showed that the relative abundance of Mortierella, Dictyophora, and Bacillus in the rhizosphere soil increased significantly after the application of PGPR (p < 0.05). These genera could effectively improve the rate of soil nutrient use, antagonize plant pathogenic bacteria, and promote plant growth. This study provides basic reference data regarding the use of PGPR to improve the microecological environment and promote the growth and development of C. illinoinensis plants.

Osteochondroma of the mandibular condyle: a classification system based on computed tomographic appearances.

OBJECTIVE: The objectives of this study were to introduce the classification of osteochondroma of the mandibular condyle based on computed tomographic images and to present our treatment experiences. MATERIALS AND METHODS: From January 2002 and December 2012, a total of 61 patients with condylar osteochondroma were treated in our division. Both clinical and radiologic aspects were reviewed. The average follow-up period was 24.3 months with a range of 6 to 120 months. RESULTS: Two types of condylar osteochondroma were presented: type 1 (protruding expansion) in 50 patients (82.0%) and type 2 (globular expansion) in 11 patients (18.0%). Type 1 condylar osteochondroma presented 5 forms: anterior/anteromedial (58%), posterior/posteromedial (6%), medial (16%), lateral (6%), and gigantic (14%). Local resection was performed on patients with type 1 condylar osteochondroma. Subtotal condylectomy/total condylectomy using costochondral graft reconstruction with/without orthognathic surgeries was performed on patients with type 2 condylar osteochondroma. During the follow-up period, tumor reformation, condyle absorption, and new deformity were not detected. The patients almost reattained facial symmetry. CONCLUSIONS: Preoperative classification based on computed tomographic images will help surgeons to choose the suitable surgical procedure to treat the condylar osteochondroma.

Current status of common cold-related knowledge, attitude, and practices among nursing students as well as relevant factors in China.

To understand the current status of cold-related knowledge, attitude, and practices (KAP) among nursing students as well as relevant factors, and provide a scientific basis for offering effective health education and promoting health care levels among nursing students. We conducted a survey of 668 nursing students using a self-designed "General Condition Questionnaire" and "Survey of common cold-related knowledge, attitude, and practices among students majoring in nursing." We used SPSS22.0 for data sorting and analysis; mean ±â€…standard deviation was used for statistical description of the questionnaire scores, and t-test and ANOVA (analysis of variance) were used for differences comparison between binary variables and polytomous variables of the relevant factors. The test level α was 0.05, and the difference was considered statistically significant when P < .05. The total KAP score of the nursing students was 128.47 ±â€…13.91 points, which is a good level. There were significant differences in the KAP scores based on gender, educational background, grade, whether relevant knowledge had been acquired, extracurricular activities, weekly exercise frequency, exercise time per session, coping method after catching a cold, and medicine purchase methods (P < .05). The KAP level for cold among nursing students is at a good level. It is recommended that the relevant departments of nursing schools strengthen the education of nursing students on the knowledge about cold and physical exercise.

Validity of inorganic nanosheets as an efficient planar substituent to enhance the enantioselectivity of transition-metal-catalyzed asymmetric synthesis.

Effectively enhancing the enantioselectivity is a persistent challenge in heterogeneous asymmetric catalysis. Here, the validity of a layered double hydroxides (LDH) nanosheet as an efficient planar substituent to enhance the enantioselectivity has been investigated theoretically; first in vanadium-catalyzed asymmetric epoxidation of allylic alcohols, and then in zinc-catalyzed direct asymmetric aldol addition. The computational predication is further confirmed experimentally in zinc-catalyzed direct asymmetric aldol addition by controlling the location of catalytic sites.

A novel protocol for three-dimensional reconstruction of pharyngeal airway on computed tomography.

Three-dimensional measurement of the pharyngeal airway has been widely used, but the three-dimensional reconstruction of pharyngeal airway has been performed in various ways, especially during the anterior boundary demarcation of the nasopharyngeal airway and oropharyngeal airway. This would inevitably affect the measurement and lead to noncomparison between different studies. Our study provided a novel method for anterior boundary demarcation of pharyngeal airway that defined the anterior boundary of nasopharyngeal airway as the "choana" according to the anatomical definition and defined the anterior boundary of oropharyngeal airway as a plane perpendicular to the long axis of soft palate and through the intersections of the lateral space and inferior space to soft palate according to the physiologic characteristics of soft palate. By 2-step segmentation, a three-dimensional image of pharyngeal airway was eventually reconstructed.Ten computed tomographic scans of pharyngeal airway were included for the anterior boundary demarcation and three-dimensional reconstruction by a medical imaging software (Surgicase 5.0; Materialise Interactive Medical Image Control System, Leuven, Belgium), with the volume and surface area being calculated. By using intraclass correlation coefficient, the reliability between intra- and interobservers of this method was well tested.The method established in this study for anterior boundary demarcation and three-dimensional reconstruction of pharyngeal airway is highly reliable and could more veritably reflect the intrinsic anatomical characteristics of the pharyngeal airway.

Nanosheet-enhanced asymmetric induction of chiral α-amino acids in catalytic aldol reaction.

An efficient ligand design strategy towards boosting asymmetric induction was proposed, which simply employed inorganic nanosheets to modify α-amino acids and has been demonstrated to be effective in vanadium-catalyzed epoxidation of allylic alcohols. Here, the strategy was first extended to zinc-catalyzed asymmetric aldol reaction, a versatile bottom-up route to make complex functional compounds. Zinc, the second-most abundant transition metal in humans, is an environment-friendly catalytic center. The strategy was then further proved valid for organocatalyzed metal-free asymmetric catalysis, that is, α-amino acid catalyzed asymmetric aldol reaction. Visible improvement of enantioselectivity was experimentally achieved irrespective of whether the nanosheet-attached α-amino acids were applied as chiral ligands together with catalytic Zn(II) centers or as chiral catalysts alone. The layered double hydroxide nanosheet was clearly found by theoretical calculations to boost ee through both steric and H-bonding effects; this resembles the role of a huge and rigid substituent.

Nanosheet-enhanced enantioselectivity in the vanadium-catalyzed asymmetric epoxidation of allylic alcohols.

The use of suitable chiral ligands is an efficient means of producing highly enantioselective transition-metal catalysts. Herein, we report a facile, economic, and effective strategy for the design of chiral ligands that demonstrate enhanced enantioselectivity and catalytic efficacy. Our simple strategy employs naturally occurring or synthetic inorganic nanosheets as huge and rigid planar substituents for, but not limited to, naturally available α-amino-acid ligands; these ligands were successfully used in the vanadium-catalyzed asymmetric epoxidation of allylic alcohols. The crucial role of the inorganic nanosheets as planar substituents in improving the enantioselectivity of the reaction was clearly revealed by relating the observed enantiomeric excess with the distribution of the catalytic centers and the accessibility of the substrate molecules to the catalytic sites. DFT calculations indicated that the LDH layer improved the enantioselectivity by influencing the formation and stability of the catalytic transition states, both in terms of steric resistance and H-bonding interactions.

The Endophytic Strain ZS-3 Enhances Salt Tolerance in Arabidopsis thaliana by Regulating Photosynthesis, Osmotic Stress, and Ion Homeostasis and Inducing Systemic Tolerance.

Soil salinity is one of the main factors limiting agricultural development worldwide and has an adverse effect on plant growth and yield. To date, plant growth-promoting rhizobacteria (PGPR) are considered to be one of the most promising eco-friendly strategies for improving saline soils. The bacterium Bacillus megaterium ZS-3 is an excellent PGPR strain that induces growth promotion as well as biotic stress resistance and tolerance to abiotic stress in a broad range of host plants. In this study, the potential mechanisms of protection against salinity stress by B. megaterium ZS-3 in Arabidopsis thaliana were explored. Regulation by ZS-3 improved growth in A. thaliana under severe saline conditions. The results showed that ZS-3 treatment significantly increased the biomass, chlorophyll content and carotenoid content of A. thaliana. Compared to the control, the leaf area and total fresh weight of plants inoculated with ZS-3 increased by 245% and 271%, respectively; the chlorophyll a, chlorophyll b, and carotenoid contents increased by 335%, 146%, and 372%, respectively, under salt stress. Physiological and biochemical tests showed that ZS-3 regulated the content of osmotic substances in plants under salt stress. Compared to the control, the soluble sugar content of the ZS-3-treated group was significantly increased by 288%, while the proline content was significantly reduced by 41.43%. Quantification of Na+ and K+ contents showed that ZS-3 treatment significantly reduced Na+ accumulation and increased the K+/Na+ ratio in plants. ZS-3 also isolated Na+ in vesicles by upregulating NHX1 and AVP1 expression while limiting Na+ uptake by downregulating HKT1, which protected against Na+ toxicity. Higher levels of peroxidase and catalase activity and reduced glutathione were detected in plants inoculated with ZS-3 compared to those in uninoculated plants. In addition, it was revealed that ZS-3 activates salicylic acid (NPR1 and PR1) and jasmonic acid/ethylene (AOS, LOX2, PDF1.2, and ERF1) signaling pathways to induce systemic tolerance, thereby inducing salt tolerance in plants. In conclusion, the results of this study indicate that ZS-3 has the potential to act as an environmentally friendly salt tolerance inducer that can promote plant growth in salt-stressed environments.

Case Report: Clinical characteristics and genetic analysis of two patients with hereditary hemorrhagic telangiectasia.

Objective: To analyze the clinical features and genetic characteristics of two patients with hereditary hemorrhagic telangiectasia (HHT) and to review the relevant literature. Methods: The clinical data of two HHT patients admitted to the author's hospital between April 2019 and February 2022 were retrospectively analyzed. Meanwhile, the genetic analysis was performed with their consent. Results: The first patient was a 62-year-old woman who had been complaining of shortness of breath and fever for 20 days. Her previous medical history included brain abscess drainage and video-assisted thoracoscopic surgery for a pulmonary hemangioma. A right heart catheterization revealed no pulmonary arterial hypertension, and an abdominal enhanced magnetic resonance imaging revealed multiple arteriovenous malformations in the liver. Her ACVRL1 heterozygous variants were discovered through whole-exon gene testing. The second case involved a 47-year-old woman who had been experiencing chest tightness for the past 2 years. Several years ago, she underwent brain abscess drainage and embolization of a pulmonary arteriovenous fistula. Ultrasound revealed generalized hepatic vascular dilation, and enhanced computed tomography revealed numerous pulmonary venous fistulas scattered in both lungs as well as multiple arteriovenous malformations in the liver. Her whole-exon gene testing revealed that she, like her son, had heterozygous ENG variants. Conclusion: HHT patients may experience infection, bleeding, dyspnea, and other symptoms. Imaging is important in disease diagnosis and management because early detection and treatment can prevent major complications and disability or even death.

[Shanghai municipal expert consensus on standardized prevention and control of COVID-19 during procedures of oral radiology].

The announcement of National Health Commission on January 20, 2020 (No.1 of 2020) has included novel coronavirus pneumonia into the B class infectious diseases according to the law of the People's Republic of China on the prevention and control of infectious diseases, and has been managed as A class infectious diseases. People's governments at all levels and health administration departments have been paying high attention to it. With the alleviation of COVID-19 nationwide, dental clinics gradually resume to work. The main transmission routes of COVID-19 are respiratory droplets and contact transmission, hence oral radiological examination is kind of a high-risk operation. Standardized radiologic process is of great significance to reduce the risk of COVID-19 transmission. In accordance with the national and Shanghai epidemic prevention requirements, and in combination with the actual situation of various medical institutions, Oral and Maxillofacial Radiology Committee of Shanghai Stomatological Association formulated the expert consensus on standardized prevention and control of COVID-19 for clinical reference. This recommendation will be updated according to the situation of epidemic prevention and control in China and the new relevant diagnosis and treatment plans.

B-scan ultrasound and cytology of the vitreous in primary central nervous system lymphoma with vitreoretinal involvement.

AIM: To evaluate the diagnostic value of B-scan ultrasound and explore the cytological characteristics of patients with vitreoretinal lymphoma (VRL) and primary central nervous system lymphoma (PCNSL). METHODS: The clinical data and pathologic specimens from patients with VRL diagnosed at the North Huashan Hospital from 2016 to 2017 were retrospectively reviewed. The patients were diagnosed by slit lamp ophthalmoscopy, B-scan ultrasound, cytology of the vitreous, which was obtained by vitrectomy, and cytokine measurements of interleukin (IL)-10 and IL-6. RESULTS: Twenty-six eyes (19.4%) out of 134 eyes of 67 patients (47 men and 20 women) with PCNSL were diagnosed with VRL by B-scan ultrasound, and 14 eyes (10.4%) were diagnosed by slit lamp ophthalmoscopy. Twenty-four eyes (17.9%) of 17 patients were confirmed as having VRL with cytology. No difference in the association between intracranial lesion location and ocular involvement was found. VRL patients had higher levels of vitreous IL-10 and IL-10/IL-6 when compared with macular hole cases, but the difference was not statistically significant. CONCLUSION: A total of 25.4% of the PCNSL patients had VRL, B-scan ultrasound examination had characteristic features and is recommended over slit lamp ophthalmoscopy for the screening diagnosis of PCNSL with intraocular involvement. Moreover, the cytological and immunohistochemical analyses performed after 25-gauge diagnostic vitrectomy were accurate diagnostic techniques.

[HPLC fingerprint chromatogram analysis of some Taraxacum in Henan province].

OBJECTIVE: To analyze the HPLC fingerprint chromatogram of some Taraxacum in Henan. METHODS: Samples of different species, producing areas, harvest seasons and medicinal parts were determined by RP-HPLC. The chromatogram was evaluated by software of evaluating similarity. RESULTS: The components of different species in Taraxacum were the same and could be substituted for each other. The contents of coffeic acid and chlorogenic acid in different producing areas were very different,which in fecund soil was better. The period of flowering and fruiting in Spring was the best gather period, and the components in different parts were different. CONCLUSION: The quality of medicinal materal within Taraxacum should be controlled better by this method.

[Diagnostic value of diffusion-weighted magnetic resonance imaging for solid tumors affecting the base of tongue and glossopharynx].

PURPOSE: The objective of the present study was to evaluate the potential application value of diffusion-weighted magnetic resonance imaging (DW-MRI) to differentiate solid benign from malignant tumors affecting the base of tongue and glossopharynx. METHODS: Sixty-one patients who presented with solid tumors affecting the base of tongue and glossopharynx underwent conventional MRI and DW-MRI before pathologic verification. Based on pathologic findings, the tumors were classified into 4 groups: Group 1, solid benign tumor (n=10); Group 2, epithelial carcinoma (n=35); Group 3: non- epithelial malignant tumor (n=16); and Group 4: malignant tumor (n=51, a combination of Group 2 and 3). The mean apparent diffusion coefficients (ADCs) were computed from DW-MRI scans obtained with b factors of 0 and 1000 s/mm2. SAS 9.1 software package was used for Wilcoxon test. RESULTS: The mean ADCs were significantly different (P<0.05) between group 1 and the other 3 groups, and between group 2 and group 3, respectively. Among all kinds of pathological types of lesions, non-Hodgkin lymphoma had a lowest mean ADC value, and schwannoma had a highest mean ADC value. In addition, the mean ADCs among epithelial carcinoma subtypes were not significantly different(P>0.05). CONCLUSIONS: DW-MRI has differential diagnostic value of solid tumors affecting the base of tongue and glossopharynx, especially in distinguishing between benign solid tumors and malignant tumors, and between epithelial carcinomas and non-epithelial malignant tumors.

CCL28 promotes breast cancer growth and metastasis through MAPK-mediated cellular anti-apoptosis and pro-metastasis.

Breast cancer is one of the most commonly diagnosed cancers worldwide and the second leading cause of cancer-related deaths among females. CCL28 (mucosa-associated epithelial chemokine, MEC), a CC subfamily chemokine, has been well studied in the process of inflammation, and recently increasing evidence indicates that CCL28 is related to tumor progression. However, little is known concerning its function in breast cancer. In the present study, we generated a CCL28-overexpressing breast cancer cell line MDA-MB-231HM/CCL28 from parental MDA-MB­231HM cells. We found that overexpression of CCL28 promoted cell proliferation and tumor formation, and also enhanced migration, invasion and metastasis both in vitro and in vivo. Mechanistic studies revealed that CCL28 mediated intracellular activation of the mitogen-activated protein kinase (MAPK) signaling pathway to promote breast cancer cell proliferation and metastasis by upregulating anti-apoptotic protein Bcl-2 and suppressing cell adhesion protein ß-catenin. However, overexpression of CCL28 did not influence the expression of metastasis­related protein matrix metalloproteinase MMP2 and MMP9 and VEGF. Tissue sample analysis from animal models also indicated that overexpression of CCL28 was associated with enhanced pERK expression and reduced ß-catenin expression in breast carcinomas. Thus, our results show for the first time that CCL28 contributes to breast cancer progression through the ERK/MAPK­mediated anti-apoptotic and metastatic signaling pathway. Antagonists of CCL28 and the MAPK signaling pathway may be used synergistically to treat breast cancer patients.

Overexpression of ALK5 Induces Human Tenon's Capsule Fibroblasts Transdifferentiation and Fibrosis In Vitro.

PURPOSE: To investigate the involvement of activin receptor-like kinase 5 (ALK5) in human Tenon's capsule fibroblasts (HTFs) transdifferentiation and fibrosis. METHODS: (1) Cultured HTFs were treated with transforming growth factor beta 1 (TGF-ß1) at different concentrations for different durations, mRNA expression of ALK5 and plasminogen activator inhibitor-1 (PAI-1) was measured by quantitative polymerase chain reaction (PCR) while protein expression of ALK5, α-smooth muscle actin (α-SMA), and extracellular matrix deposition including fibronectin (FN) and collagen I (Col1) was assessed by western blot. HTFs with or without TGF-ß1 were also treated with an ALK5 activity inhibitor, SB-431542, and fibrosis-related genes were assessed. (2) HTFs were transduced with ALK5 lentivirus (ALK5-OE group) or empty lentivirus (NC-OE) with or without the treatment of SB-431542. Protein expression of ALK5, α-SMA, FN, and Col1 was evaluated. (3) HTFs in the ALK5-OE group and NC-OE group were subjected to a scratch-wound assay and their migratory activities assessed. RESULTS: (1) TGF-ß1, in a concentration-dependent manner, upregulated ALK5 and PAI-1 expressions in the HTFs, which peaked between 24 and 36 h. These changes were associated with increases in protein levels of FN, Col1, and α-SMA. These TGF-ß1 effects were blocked by the ALK5 inhibitor SB-431542. (2) Similarly, overexpression of ALK5 by lentiviral vector significantly increased protein expression of α-SMA, FN, and Col1. Addition of TGF-ß1 to the ALK5-OE cells did not produce additional expression of any of the marker proteins. The upregulation of extracellular matrix and α-SMA can be reduced by SB-431542. (3) In ALK5-OE group, HTFs migration was significantly increased compared with normal control and TGF-ß1 could still promote ALK5-OE cells migration. CONCLUSIONS: Our findings suggest that ALK5 is an important mediator of HTFs fibrosis. ALK5 is a potential therapeutic target to suppress scar formation after filtration surgery.

Outflow facility efficacy of five drugs in enucleated porcine eyes by a method of constant-pressure perfusion.

This study aimed to characterize a technique that assesses the outflow facility (C) efficacy of five kinds of IOP-lowering drugs commonly used clinically in enucleated porcine Eyes. Eyes were perfused at 15 mmHg with GPBS first to establish the baseline outflow facility (C0). Then the anterior chamber contents were exchanged for GPBS with corresponding concentration eye drops (4.9×10(3) nM Brimonidine, 41.1 nM Latanoprost, 3.4×10(3) nM Levobunolol, 3.0×10(3) nM Brinzolamide, 8.3×10(3) nM Pilocarpine) in five groups (n = 6 each), while 6 eyes received GPBS alone as control. The mean stable facility obtained after drug administration (C1) was continuously recorded. The changes between C0 and C1 (ΔC = C1-C0) were analyzed. Finally, for drugs among the five experiment groups with statistical significance, the concentration was reduced 3 times, otherwise the drugs' concentration was increased to 10 times to confirm its effectiveness further using the same methods (n = 6 each). We found that the average baseline outflow facility was 0.24±0.01 µl·min(-1)·mmHg(-1). C increased significantly in Brimonidine and Latanoprost groups, even the concentration of Brimonidine and Latanoprost was decreased 3 times (P < 0.05). However, there was no significantly increase in Levobunolol, Brinzolamide, Pilocarpine and control group (P > 0.05), but when drugs' concentration was increased to 10 times, the C value of Pilocarpine decreased significantly (P = 0.04). No significant washout effects in porcine eyes were observed. To conclude, outflow facility efficacy of five drugs in enucleated porcine eyes may provide a reference for clinical medicine. A constant-pressure perfusion technique should be useful to evaluate effect of pharmacologic agents or surgical manipulations on aqueous humor dynamics.