Assessment of goat activin receptor type IIB knockdown by short hairpin RNAs in vitro.

BACKGROUND: Targeted knockdown of ACVR2B, a receptor for TGF beta superfamily, has been seen as a potential candidate to enhance the muscle mass through RNAi approach. METHODS: We have evaluated the potential short hairpin RNAs targeting goat ACVR2B in human HEK293T cells and goat myoblasts cells by transient transfection and measured their knockdown efficiency and possible undesired interferon response by quantitative real-time PCR. RESULTS: We observed a significant silencing (64-81%) of ACVR2B in 293T cells with all seven shRNAs (sh1 to sh7) constructs and 16-46% silencing with maximum of 46% by sh6 (p = 0.0318) against endogenous ACVR2B whereas up to 66% (p = 0.0002) silencing by sh6 against exogenously expressed ACVR2B in goat myoblasts cells. Transient knockdown of ACVR2B in goat myoblasts cells by shRNAs did not show significant correlation with the expression of MyoD (r = 0.547; p = 0.102), myogenin (r = 0.517; p = 0.126) and Myf5 (r = 0.262; p = 0.465). As reported earlier, transfection of plasmid DNA induced potent interferon response in 293T and goat myoblasts cells. CONCLUSIONS: The present study demonstrates the targeted knockdown of ACVR2B by shRNAs in HEK293T and goat myoblasts cells in vitro. The transient knockdown of ACVR2B by shRNAs in goat myoblasts did not alter the myogenic gene expression program. However, shRNAs showing significant knockdown efficiency in our study may further be tested for long term and stable knockdown to assess their potential to use for enhancing muscle mass in vivo. As reported earlier, expression of shRNAs through plasmid expression vectors induces potent interferon response raising the concern of safety of its application in vivo.

Comparative study of final visual outcome between open- and closed-globe injuries following surgical treatment of traumatic cataract.

OBJECTIVE: The objective of this work is to compare final visual outcomes in cases of surgically treated traumatic cataract between open-globe and closed-globe groups, as classified by the Birmingham Eye Trauma Terminology system. DESIGN: Observational cohort study. SETTING: Tertiary eye-care center at the trijunction of Gujarat, Madhya Pradesh, and Rajasthan states in central western India. METHODS: We enrolled patients meeting specific inclusion criteria, examined their eyes to review any co-morbidities due to trauma, performed surgery for traumatic cataracts, and implanted lenses. The patients were re-examined 6 weeks postoperatively. We classified the cases of traumatic cataract as either open-globe (group 1) or closed-globe (group 2), according to the Birmingham Eye Trauma Terminology (BETT) system, and compared visual acuity. OUTCOME MEASURES: Visual Acuity. RESULTS: Our cohort of 687 eyes with traumatic cataracts included 496 eyes in group 1 and 191 in group 2. Six weeks postoperatively, the visual acuity was >20/60 in 298 (58%) and 75 (39.1%) operated eyes in groups 1 and 2, respectively (p < 0.001, ANOVA). At follow-up, >20/60 vision was significantly higher in group 1 than in group 2 (OR = 1.61; 95% CI, 0.85-3.02). Overall, 373 eyes (54.3%) regained final visual acuity >20/60. CONCLUSIONS: Open-globe injury has a more favorable prognosis for satisfactory (>20/60) visual recovery after management of traumatic cataracts.

Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector-mediated RNAi.

Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin-signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector-based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic-cell nuclear transfer (SCNT) studies. Sh-RNA positive cells were screened by puromycin selection. Using real-time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down-regulation in sh2 shRNA-treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin-targeting siRNA produced endogenously could efficiently down-regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus-mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals.

Goat activin receptor type IIB knockdown by muscle specific promoter driven artificial microRNAs.

Activin receptor type IIB (ACVR2B) is a transmembrane receptor which mediates signaling of TGF beta superfamily ligands known to function in regulation of muscle mass, embryonic development and reproduction. ACVR2B antagonism has shown to enhance the muscle growth in several disease and transgenic models. Here, we show ACVR2B knockdown by RNA interference using muscle creatine kinase (MCK) promoter driven artificial microRNAs (amiRNAs). Among the various promoter elements tested, the ∼1.26 kb MCK promoter region showed maximum transcriptional activity in goat myoblasts cells. We observed up to 20% silencing in non-myogenic 293T cells and up to 32% silencing in myogenic goat myoblasts by MCK directed amiRNAs by transient transfection. Goat myoblasts stably integrated with MCK directed amiRNAs showed merely 8% silencing in proliferating myoblasts which was increased to 34% upon induction of differentiation at transcript level whereas up to 57% silencing at protein level. Knockdown of ACVR2B by 5'-UTR derived amiRNAs resulted in decreased SMAD2/3 signaling, increased expression of myogenic regulatory factors (MRFs) and enhanced proliferation and differentiation of myoblasts. Unexpectedly, knockdown of ACVR2B by 3'-UTR derived amiRNAs resulted in increased SMAD2/3 signaling, reduced expression of MRFs and suppression of myogenesis. Our study offers muscle specific knockdown of ACVR2B as a potential strategy to enhance muscle mass in the farm animal species.

Myostatin knockdown and its effect on myogenic gene expression program in stably transfected goat myoblasts.

Myostatin, a negative regulator of skeletal muscle mass, is a proven candidate to modulate skeletal muscle mass through targeted gene knockdown approach. Here, we report myostatin (MSTN) knockdown in goat myoblasts stably expressing small hairpin RNA (shRNAs) against MSTN gene through lentivirus vector-mediated integration. We observed 72% (p = 0.003) and 54% (p = 0.022) downregulation of MSTN expression with sh2 shRNA compared to empty vector control and untransduced myoblasts, respectively. The knockdown of MSTN expression was accompanied with concomitant downregulation of myogenic regulatory factor MYOD (77%, p = 0.001), MYOG (94%, p = 0.000), and MYF5 (36%, p = 0.000), cell cycle regulator p21 (62%, p = 0.000), MSTN receptor ACVR2B (23%, p = 0.061), MSTN antagonist follistatin (81%, p = 0.000), and downstream signaling mediators SMAD2 (20%, p = 0.060) and SMAD3 (49%, p = 0.006). However, the expression of MYF6 was upregulated by 14% compared to control lentivirus-transduced myoblasts (p = 0.354) and 79% compared to untransduced myoblasts (p = 0.018) in sh2 shRNA-transduced goat myoblasts cells. Although, MSTN knockdown led to sustained cell proliferation of myoblasts, the myoblasts fusion was suppressed in both MSTN knocked down and control lentivirus-transduced myoblasts. The expression of interferon response gene OAS1 was significantly upregulated in control lentivirus (10.86-fold; p = 0.000)- and sh2 (1.71-fold; p = 0.002)-integrated myoblasts compared to untransduced myoblasts. Our study demonstrates stable knockdown of MSTN in goat myoblasts cells and its potential for use in generation of transgenic goat by somatic cell nuclear transfer.

Effect of interval between time of injury and timing of intervention on final visual outcome in cases of traumatic cataract.

PURPOSE: There are no clear guidelines to treat traumatic cataract. This study was conducted to provide evidence-based care to patients with traumatic cataracts and to examine the effect of the time interval between injury and the first intervention on the final visual outcome. METHODS: In a prospective cohort study, all patients presenting to our hospital with traumatic cataracts between January 2003 and December 2009 were enrolled. Information regarding demographics and ocular trauma was collected on the pretested World Eye Trauma Registry form for both the first and follow-up visits. In particular, we collected specific information on the time interval between the injury and intervention. The relationship between this time interval and the final visual outcome was analyzed. The study was conducted at a tertiary eye care center, in Dahod, at the junction of Gujarat, Madhya Pradesh, and Rajasthan states, in central western India. RESULTS: The time interval between the injury and first intervention had a significant effect on the final visual outcome (p = 0.02, chi2 test). CONCLUSIONS: The morphology of traumatic cataracts plays an important role in determining the appropriate surgical technique and the final visual outcome.

Morphology of traumatic cataract: does it play a role in final visual outcome?

Aim To study the morphology of traumatic cataract as an important predictor for final visual outcome after treatment of traumatic cataracts. Setting Tertiary eye care centre in Dahod at the trijunction of Gujarat, Madhya Pradesh, and Rajasthan states in central western India. Methods This was a prospective observational cohort study among all patients presenting at the hospital with traumatic cataracts between January 2003 and December 2009. All information regarding demographic and ocular trauma was collected on a pretested World Eye Trauma Registry form for both the first visit and follow-up. In particular, the authors collected specific information about the morphology of traumatic cataracts; the surgical technique was determined accordingly. Data were entered and analysed with regard to the relationship between type of trauma and resulting injury, results achieved with particular surgical techniques, and the relationship between morphology and final visual outcome. Outcome measures Final visual outcome. Results Traumatic cataracts of different morphologies showed significant differences in the final visual outcome (χ(2) test, p=0.014). Conclusion The morphology of traumatic cataract plays an important role in the final visual outcome.