CRISPR/Cas tool designs for multiplex genome editing and its applications in developing biotic and abiotic stress-resistant crop plants.

Crop plants are prone to several yield-reducing biotic and abiotic stresses. The crop yield reductions due to these stresses need addressing to maintain an adequate balance between the increasing world population and food production to avoid food scarcities in the future. It is impossible to increase the area under food crops proportionately to meet the rising food demand. In such an adverse scenario overcoming the biotic and abiotic stresses through biotechnological interventions may serve as a boon to help meet the globe's food requirements. Under the current genomic era, the wide availability of genomic resources and genome editing technologies such as Transcription Activator-Like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and Clustered-Regularly Interspaced Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas) has widened the scope of overcoming these stresses for several food crops. These techniques have made gene editing more manageable and accessible with changes at the embryo level by adding or deleting DNA sequences of the target gene(s) from the genome. The CRISPR construct consists of a single guide RNA having complementarity with the nucleotide fragments of the target gene sequence, accompanied by a protospacer adjacent motif. The target sequence in the organism's genome is then cleaved by the Cas9 endonuclease for obtaining a desired trait of interest. The current review describes the components, mechanisms, and types of CRISPR/Cas techniques and how this technology has helped to functionally characterize genes associated with various biotic and abiotic stresses in a target organism. This review also summarizes the application of CRISPR/Cas technology targeting these stresses in crops through knocking down/out of associated genes.

Identification and validation of stage-specific reference genes for gene expression analysis in Callosobruchus maculatus (Coleoptera: Bruchidae).

In light of a number of recent studies highlighting the increasing research interest in bruchids, it is crucial to validate suitable reference genes that could be used in quantitative gene expression studies. Callosobruchus maculatus is a serious pest of stored grains and field legumes in which reference genes have not been assessed and validated to date. The present study aimed to identify and validate reference genes in different developmental stages of C. maculatus shortlisted from commonly used reference genes such as VATPase, TRIP12, TBP, TF11D, ACTIN, GST, ANNEXIN, PTCD3, RPL32, and ß -Tub in various insects. Dedicated algorithms like GeNorm, NormFinder, and BestKeeper were used to analyze the stability of these candidate genes, which revealed GST for third instar, ANNEXIN and PTCD3 for the fourth instar, TF11D and VATPase for male pupa, RPL32 and ß-tub for female pupa, ß-tub and TBP for adult male and VATPase and GST for adult females as suitable reference genes for expression studies in C. maculatus. The final comprehensive ranking using RefFinder identified GST and TBP as the best reference genes for all the developmental stages of C. maculatus. To the best of our knowledge, this is the first report which evaluates and validates stable reference genes in C. maculatus. The information of stage-specific gene expression, generated in this study will be useful for future molecular, physiological, and biochemical studies on C. maculatus and other closely related bruchids.

Scheimpflug imaging of pediatric posterior capsule rupture.

We report a case of an 11-year-old boy who presented two days after blunt trauma to the left eye with a slingshot. On examination his best corrected visual acuity (BCVA) was 20/20 in the right eye and 20/400 in the left eye. Slit-lamp examination of the left eye revealed a Vossius ring, traumatic cataract, traumatic posterior capsule tear (PCT). The contour of the posterior capsule bulge corresponded to the edges of the PCT. Rotating Scheimpflug imaging (Pentacam 70700:Oculus, Wetzlar Germany) confirmed the traumatic cataract in the region of the PCT visualized as increased lens density at the cortex-vitreous interface. The extent of the PCT in the greatest and least dimensions was documented before and after intraocular lens (IOL) implantation. Intra-operatively, the PCT was evident and phaco-emulsification with an IOL implant was performed. Postoperatively, his BCVA improved to 20/20 in the left eye with a well-centered in-the-bag IOL as found on slit-lamp and Scheimpflug images.

Pentacam tomograms: a novel method for quantification of posterior capsule opacification.

PURPOSE: To develop and validate a method to quantify posterior capsule opacification (PCO) in eyes after cataract surgery and intraocular lens implantation using Scheimpflug Pentacam tomograms and compare its validity with slit lamp retroillumination image analysis. METHODS: One hundred twenty-four pseudophakic eyes of 124 patients were divided into two groups. Group 1 consisted of 40 eyes with visually significant PCO, and group 2 consisted of 84 eyes without visually significant PCO. Pentacam Imaging was performed after full mydriasis using the 50-scan acquisition protocol, and high-resolution tomograms were reconstructed and analyzed using ImageJ freeware. Retroillumination photographs were captured for group 1 eyes using the Topcon digital slit lamp, and these were analyzed using POCOman software to calculate an aggregate severity grade and percentage PCO value. Correlation coefficients were calculated for PCO values obtained using POCOman and ImageJ. RESULTS: Mean PCO percentage value obtained using POCOman software was 23.34 +/- 6.25 U, mean aggregate PCO severity grade was 0.46 +/- 0.28 U, and mean pixel-intensity value using ImageJ was 31.071 +/- 8.26 U. There was a significant positive correlation between the percentage PCO (P = 0.000; r = 0.864) and PCO severity grade (P = 0.001; r = 0.490) obtained for group 1 eyes using slit lamp retroillumination images and PCO pixel intensity obtained using Pentacam tomograms. CONCLUSIONS: Retroillumination photographs are the standard for quantifying PCO. Pentacam tomograms are easier to obtain and are free of flash reflections, and they allow for a more objective analysis. The correlation between the two methods demonstrates that ImageJ analysis of Pentacam tomograms is a viable tool for PCO analysis.

Visual outcome and posterior capsular opacification following implantation of silicone and acrylic refractive multifocal intraocular lenses.

We studied visual outcome and posterior capsular opacification (PCO) following implantation of hydrophilic acrylic and silicone multifocal lenses in 29 eyes who underwent uneventful phacoemulsification. Both the multifocal IOLs provide satisfactory distant and near vision in the majority of the patients, however tripod acrylic hydrophobic IOL has higher rate of PCO, jeopardizing visual performance.

Unilateral electric cataract: Scheimpflug imaging and review of the literature.

A 22-year-old man presented with diminution of vision in the left eye 1 month after sustaining an injury from a high-voltage electric current to the left side of his head. Slitlamp examination and Scheimpflug imaging confirmed the typical anterior subcapsular lenticular opacities in the left eye and also documented early anterior subcapsular vacuoles in the right eye. Based on history, the electric shock wound, and the typical appearance of the opacities, an electric cataract was diagnosed. The difference in lens density between the 2 eyes was also documented. The best corrected visual acuity (BCVA) was 20/20 in the right eye and counting fingers at 1 foot in the left eye. Phacoaspiration and posterior chamber intraocular lens implantation were performed in the left eye. Postoperatively, the BCVA improved to 20/20. The developmental mechanisms of these opacities after exposure to an electric current and the role of Scheimpflug imaging in assessing the earliest subclinical manifestations of electric cataract injuries are discussed.

Primary intraocular lens implantation in the first two years of life: safety profile and visual results.

PURPOSE: To study the safety profile of primary intraocular lens (IOL) implantation in the first two years of life. MATERIALS AND METHODS: A prospective nonrandomized, interventional study was done at the tertiary care center. Forty-five eyes of 27 children (aged three weeks to two years) with congenital cataract who underwent phacoaspiration with continuous curvilinear capsulorrhexis combined with primary posterior capsulotomy and anterior vitrectomy with in the bag placement of IOL at primary surgery were included. Outcome measures were clarity of visual axis, postoperative inflammation, fixation pattern and retinoscopy. RESULTS: The mean IOL power was 23.95 +/- 0.87 diopter (D) (range 22D to 27D). Follow-up ranged from 12 months to 48 months (mean 18 +/- 9.13 months). In infants, hypermetropia decreased from 6.60D +/- 2.64D at one week postoperatively to 3.03D +/- 2.53D at one year following surgery and 4.78D +/- 1.93D to 2.56D +/- 1.5D in children older than one year at the time of surgery. Re-opacification of the central visual axis was observed in six eyes (13.3%). Significant posterior synechiae due to irido-capsular adhesion was seen in seven eyes (15.6%). Five of these seven eyes also had significant posterior capsular opacification. CONCLUSIONS: Meticulously performed primary IOL implantation and primary posterior capsulorrhexis with anterior vitrectomy in the first two years of life is a safe and effective method of aphakic correction.

A case of spontaneously resolved primary congenital glaucoma.

Primary congenital glaucoma usually presents as enlarged and hazy cornea at birth or early childhood. The diagnosis is based on a thorough clinical examination under anesthesia. Most cases require surgical intervention as the definitive treatment. In very rare instances, primary congenital glaucoma may arrest and resolve spontaneously. We describe a case of spontaneously arrested and resolved primary congenital glaucoma in a 37-year-old male presenting with large cornea, Haab's striae, and normal intraocular pressure in one eye. Such a case has not been previously described from the Indian subcontinent.

Phacoemulsification in patients with Fuchs' heterochromic uveitis.

PURPOSE: To evaluate the results of phacoemulsification with capsular bag fixation of a poly(methyl methacrylate) (PMMA), acrylic, or silicone intraocular lens (IOL) in patients with complicated cataract resulting from Fuchs' heterochromic uveitis (FHU). SETTING: Department of Ophthalmology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. METHODS: This study evaluated 20 eyes of 19 patients with FHU and cataract who had uneventful phacoemulsification with endocapsular implantation of an IOL and completed a 1-year follow-up. RESULTS: Preoperatively, loss of the iris collarette and fine keratic precipitates were seen in all 20 eyes, iris heterochromia in 16, iris atrophy in 12, and iris nodules in 2. Fourteen eyes had mild or negligible preoperative anterior chamber inflammation. No eye had increased intraocular pressure. An acrylic IOL was implanted in 10 eyes, a silicone IOL in 4, and a PMMA IOL in 6. There were no significant differences in outcomes among the 3 IOL groups. Three patients had intraoperative hyphema that resolved spontaneously. Postoperatively, 16 eyes had mild anterior chamber reaction. Four patients had significant anterior chamber inflammation necessitating the use of frequent topical steroids. No case had secondary glaucoma or posterior synechias. The best corrected visual acuity was 6/5 in 6 eyes, 6/6 in 11, and 6/9 in 3. CONCLUSIONS: Uneventful phacoemulsification with endocapsular IOL implantation visually rehabilitated patients with FHU. All 3 IOLs yielded similar results except for increased early postoperative inflammation and late dense anterior capsule opacification in the silicone group. Further studies comparing the outcome of different IOL materials are required to determine their effectiveness in patients with FHU.

Aspergillus niger endophthalmitis after cataract surgery.

We report a 65-year-old diabetic woman who developed Aspergillus niger endophthalmitis after cataract surgery. She presented 9 weeks after extracapsular cataract extraction with a black growth covering the cornea and moderate echoes in the vitreous on ultrasonography. After microbiological confirmation of fungal endophthalmitis, the patient received intravitreal amphoterecin B 5 micro g, topical natamycin 5% hourly, atropine 1% 3 times, and oral antifungal therapy. The patient was told the visual prognosis and was advised to have penetrating keratoplasty and vitrectomy, which she refused.

Comparison of the thermal effect on clear corneal incisions during phacoemulsification with different generation machines.

BACKGROUND AND OBJECTIVE: Older generation phacoemulsification units are widely employed where economics do not permit frequent upgrades. Thermal effects on the cornea while performing phacoemulsification with different generation machines were evaluated. PATIENTS AND METHODS: The thermal effect on the clear corneal incision during uncomplicated phacoemulsification performed by the Series 10,000 (Alcon Laboratories, Fort Worth, TX) and the Diplomax (Allergan, Irvine, CA) machines was clinically evaluated and correlated with the effective phacoemulsification time required. The clinical outcome was analyzed with respect to wound closure and surgically induced corneal astigmatism. RESULTS: The average effective phacoemulsification time required to emulsify a grade IV cataract was 88.33 +/- 15.81 seconds with the Series 10,000 machine and 61.67 +/- 21.79 seconds with the Diplomax machine. A grade 2 or more thermal effect was seen in 19.33% of patients operated on with the Series 10,000 machine and 3.22% of patients operated on with the Diplomax machine. The resultant tissue retraction affected wound closure and mean surgically induced corneal astigmatism (1.71 +/- 1.05 D with the Diplomax machine and 2.21 +/- 1.32 D with the Series 10,000 machine). CONCLUSIONS: Newer generation machines require less phacoemulsification power to emulsify hard cataracts, and the clinical outcomes are more favorable. Incision burns must be kept in mind when working with older generation phacoemulsification machines.

Phacoemulsification in vitrectomized eyes: results using a 'phaco chop' technique.

PURPOSE: To evaluate the intraoperative and early postoperative complications of phacoemulsification using a 'phaco chop' technique in previously vitrectomized eyes. METHODS: A prospective interventional case series. Seventy-five previously vitrectomized eyes of 73 consecutive patients underwent phacoemulsification using a 'phaco chop' technique via clear corneal incision. Patients were observed for any intraoperative or early postoperative complications. RESULTS: Fifty-four eyes (72%) had predominant nuclear sclerosis or posterior subcapsular cataract. The intraoperative findings included intraoperative miosis [seen in 21 eyes (28%)] and posterior capsule plaques [in 15 eyes (20%)]. No other significant intraoperative complication (posterior capsule rupture, zonular lysis or dropped nuclei) was observed. In the early postoperative period, one patient had massive serous choroidal detachment, which resolved with conservative treatment. Mean preoperative visual acuity (LogMar scale) was 0.74, which improved to 0.36 postoperatively (p < 0.001). CONCLUSION: Phacoemulsification using a 'phaco chop' technique is a safe procedure in vitrectomized eyes.

Anomalous bilateral lateral rectus muscles and anterior polar cataract with dysmorphic features.

Congenital anomalies, such as absence, hypoplasia, bifurcation, or reduplication of various extraocular muscles, including lateral rectus muscles have been reported previously. We report a 5-year-old girl who had posteriorly malinserted lateral rectus at 15 mm from the limbus in the right eye with multiple associated ocular and systemic congenital malformations.

Results of intraocular lens implantation with capsular tension ring in subluxated crystalline or cataractous lenses in children.

PURPOSE: To evaluate the outcome of intraocular lens (IOL) implantation using capsular tension ring (CTR) in subluxated crystalline or cataractous lenses in children. SETTING: Tertiary care setting. MATERIALS AND METHODS: We prospectively studied 18 eyes of 15 children with subluxation of crystalline or cataractous lenses between 90 degrees up to 210 degrees after phacoemulsification, CTR and IOL implantation. Each child was examined for IOL centration, zonular dehiscence and posterior capsular opacification (PCO). RESULTS: Age of the patient ranged between five to 15 years. Out of 18 eyes, seven had traumatic and 11 had spontaneous subluxation of crystalline or cataractous lens. Phacoemulsification was successfully performed with CTR implantation in the capsular bag. Intraoperative zonular dialysis occurred in two eyes. Anterior vitrectomy was performed in six eyes to manage vitreous prolapse. IOL implanted was polymethyl methacrylate (PMMA) in eight eyes, hydrophobic acrylic in seven and hydrophilic acrylic in three. Follow-up ranged from 24 months to 72 months. Sixteen eyes had a best corrected visual acuity of 20/40 or better. Nine eyes developed significant PCO and were managed with Neodymium Yttrium Aluminum Garnet (Nd:YAG) laser posterior capsulotomy. One eye with acrylic IOL in the capsular bag had IOL dislocation after two years which was managed with vitrectomy and secondary trans-scleral fixation of IOL. CONCLUSIONS: Phacoaspiration with CTR implantation makes capsular bag IOL fixation possible in most of the eyes with subluxated crystalline or cataractous lenses. PCO still remains a challenge in children with successful phacoaspiration with CTR implantation.