The molecular architecture of the nuclear basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

Comprehensive structure and functional adaptations of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

Real-time observation of Cas9 postcatalytic domain motions.

CRISPR-Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease, which has become the most popular genome editing tool. Coordinated domain motions of Cas9 prior to DNA cleavage have been extensively characterized but our understanding of Cas9 conformations postcatalysis is limited. Because Cas9 can remain stably bound to the cleaved DNA for hours, its postcatalytic conformation may influence genome editing mechanisms. Here, we use single-molecule fluorescence resonance energy transfer to characterize the HNH domain motions of Cas9 that are coupled with cleavage activity of the target strand (TS) or nontarget strand (NTS) of DNA substrate. We reveal an NTS-cleavage-competent conformation following the HNH domain conformational activation. The 3' flap generated by NTS cleavage can be rapidly digested by a 3' to 5' single-stranded DNA-specific exonuclease, indicating Cas9 exposes the 3' flap for potential interaction with the DNA repair machinery. We find evidence that the HNH domain is highly flexible post-TS cleavage, explaining a recent observation that the HNH domain was not visible in a postcatalytic cryo-EM structure. Our results illuminate previously unappreciated regulatory roles of DNA cleavage activity on Cas9's conformation and suggest possible biotechnological applications.

Understanding the genomic selection for crop improvement: current progress and future prospects.

Although increased use of modern breeding techniques and technology has resulted in long-term genetic gain, the pace of genetic gain must be sped up to satisfy global agricultural demand. However, marker-assisted selection has proven its potential for improving qualitative traits with large effects regulated by one to few genes. Its contribution to the improvement of the quantitative traits regulated by a number of small-effect genes is modest. In this context, genomic selection (GS) has been regarded as the most promising method for genetically enhancing complicated features that are regulated by several genes, each of which has minor effects. By examining a population's phenotypes and high-density marker scores, genomic selection can forecast the breeding potential of individual lines. The fact that GS uses all marker data in the prediction model prevents skewed marker effect estimations and maximizes the amount of variation caused by small-effect QTL. It has the ability to speed up the breeding cycle and as a consequence of which superior genotypes are selected rapidly. Developing the best GS models while taking into account non-additive effects, genotype-by-environment interaction, and cost-effectiveness will enable the widespread implementation of GS in plants. These steps will also increase heritability estimation and prediction accuracy. This review focuses on the shift from conventional selection methods to GS, underlying statistical tools and methodologies, the state of GS research in agricultural plants, and prospects for its effective use in the creation of climate-resilient crops.

Immunological and safety profile of bacteriophage therapy: A pre-clinical study.

AIMS: Numerous pre-clinical and clinical studies have recently demonstrated the significant role of phage therapy in treating multidrug-resistant bacterial infections. However, only a few researchers have focused on monitoring the phage-mediated adverse reactions during phage therapy. The present study aimed to demonstrated the oral acute and sub-acute toxicity of bacteriophages (Klebsiella pneumoniae XDR strain) in Charles Foster rats with special reference to immunological response and adverse effects. METHODS AND RESULTS: Bacteriophages were orally administered in dosages of 1010  PFU/ml and a 1015  PFU/ml to Charles Foster rats as a single dose (in acute toxicity study) and daily dosage for 28 days (in sub-acute toxicity study). One millilitre suspension of bacteriophages was administered through the oral gavage feeding tube. No adverse effect was observed in any of the experimental as well as in the control animals. Furthermore, an insignificant change in food and water intake and body weight was observed throughout the study period compared with the control group rats. On the 28th day of phage administration, blood was collected to estimate haematological, biochemical and cytokines parameters. The data suggested no difference in the haematological, biochemical and cytokine profiles compared to the control group. No significant change in any of the treatment groups could be observed on the gross and histopathological examinations. The cytokines estimated, interleukin-1 beta (IL-1ß), IL-4, IL-6 and IFN-gamma, were found within the normal range during the experiment. CONCLUSIONS: The results concluded that no adverse effect, including the severe detrimental impact on oral administration of high (1010 PFU/ml) and very high dose (1015  PFU/ml) of the bacteriophages cocktail. SIGNIFICANCE AND IMPACT OF STUDY: The high and long-term oral administration of bacteriophages did not induce noticeable immunological response as well.

Surface water and groundwater interaction in the Kosi River alluvial fan of the Himalayan Foreland.

We report the isotopic composition of the surface water and groundwater of the Kosi River fan on the Himalayan Foreland, India. We have collected 65 water samples from surface water (Kosi River (n = 2), streams (n = 9), waterlogging (n = 29), and canal (n = 4)), and groundwater (n = 21) for δ18O and δ2H analysis during December 2019. We obtained groundwater level data measured at the observation wells from the Central Groundwater Board, India, for 1996 and 2017. The groundwater level varies from 1.0 to 8.1 m below ground level (bgl) and from 0.5 to 9.0 m bgl during 1996 and 2017, respectively. We have used water table fluctuation approach to estimate the recharge rate. The recharge rate in the Kosi Fan varies from 0.7 to 21.4 mm/year from 1996 to 2017. Further, we have used δ18O and δ2H values of water samples to identify the source and the interaction between surface water and groundwater. The δ18O value of groundwater shows a wide variation (from -9.3‰ to -5.6‰) compared to the surface water, i.e., streams (-7.8‰ to -6.4‰) and canals (-6.9‰ to -6.0‰), suggesting mixing in groundwater during recharge processes. Furthermore, we have used a two-component mixing model to assess the fraction contribution from streams and precipitation to groundwater. The estimated fraction contribution from stream water to groundwater ranges from 45 to 83%. We also suggest higher recharge is limited up to the depth of 6 m bgl. We suggest precipitation and surface water actively recharge groundwater. We conclude that marked spatial variation in the isotopic composition of groundwater is mainly due to the local recharge sources and interaction between surface water and groundwater.

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding.

Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5' end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5' end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a).

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5' guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

Ethambutol Optic Neuropathy: Vigilance and Screening, the Keys to Prevent Blindness with the Revised Anti-tuberculous Therapy Regimen.

There has been change in the guidelines for the management of tuberculosis in India. The new guidelines advocate the daily use of Ethambutol for both intensive and continuation phase of the treatment. This may be a matter of concern as increased cumulative dose may lead to increase in incidence of toxic optic neuropathy due to ethambutol. Indian Neuro-Ophthalmology Society has taken cognizance of the issue and has come-up with guidelines for prevention and early detection of the toxic optic neuropathy.

The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays.

Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9-RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.

Steroids versus No Steroids in Nonarteritic Anterior Ischemic Optic Neuropathy: A Randomized Controlled Trial.

PURPOSE: To examine the role of oral steroid therapy in the treatment of nondiabetic cases of acute nonarteritic anterior ischemic optic neuropathy (NAAION). DESIGN: Randomized double-blind clinical trial. PARTICIPANTS: Thirty-eight patients with acute nondiabetic NAAION divided into 2 arms of 19 patients each. One arm constituted the cases and the other constituted the controls. METHODS: Cases received oral steroid therapy and were designated the steroid group, whereas controls received placebo and were designated the nonsteroid group. Best-corrected visual acuity (BCVA), visual evoked response (VER), and OCT were performed at baseline, 1 month, 3 months, and 6 months after recruitment into the trial. MAIN OUTCOME MEASURES: Best-corrected visual acuity, VER, and retinal nerve fiber layer changes on OCT. RESULTS: Both groups showed significant improvement in BCVA, VER latency, and resolution of disc edema on OCT parameters over 6 months. Final outcome showed no statistically significant difference with regard to visual acuity, although VER was better in the steroid group (P = 0.011). Best-corrected visual acuity, VER amplitude, and VER latency (P = 0.02, P = 0.02, and P = 0.04, respectively) showed a greater percentage improvement in the steroid group, which also saw a faster resolution of disc edema on OCT (1-month follow-up). CONCLUSIONS: Oral steroids in acute NAAION did not improve the visual acuity significantly at 6 months. However, they improved resolution of disc edema significantly and enabled a greater improvement in VER parameters. This subtle benefit of oral steroids in NAAION is clinically unimportant and does not provide support for its use.

Neuro-Ophthalmology at a Tertiary Eye Care Centre in India.

BACKGROUND: Neuro-ophthalmology as a specialty is underdeveloped in India. The aim of our study was to determine the spectrum and profile of patients presenting to a tertiary eye care center with neuro-ophthalmic disorders. METHODS: A retrospective hospital-based study was conducted, and records of all patients seen at the neuro-ophthalmology clinic of Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India, over a 1-year period were retrieved and evaluated. RESULTS: Of a total of 30,111 patients referred to various specialty clinics in a span of 1 year, 1597 (5%) were referred for neuro-ophthalmology evaluation. The mean patient age was 30.8 ± 19.5 years, with a male dominance (M:F = 2.02:1). Among these patients, optic nerve disorders were noted in 63.8% (n = 1,020), cranial nerve palsy in 7% (n = 114), cortical visual impairment in 6.5% (n = 105), and others (eye/optic nerve hypophasia, blepharospasm, and optic disc drusen) in 6% (n = 95). Among the patients with optic nerve disorders, optic neuropathy without disc edema/(traumatic optic neuropathy, hereditary, tumor-related, retrobulbar neuritis, toxic, and idiopathic) was noted in 42.8% (n = 685) and optic neuropathy with disc edema (ischemic optic neuropathy, papilledema, post-papilledema optic atrophy, papillitis, neuroretinitis, and inflammatory optic neuropathy) in 20.9% (n = 335). Sixteen percent of patients (n = 263) were incorrect referrals. CONCLUSION: The neuro-ophthalmic clinic constitutes a significant referral unit in a tertiary eye care center in India. Traumatic and ischemic optic neuropathies are the most common diagnoses. Neuro-ophthalmology requires further development as a subspecialty in India to better serve the nation's population.

An improved surface passivation method for single-molecule studies.

We report a surface passivation method based on dichlorodimethylsilane (DDS)-Tween-20 for in vitro single-molecule studies, which, under the conditions tested here, more efficiently prevented nonspecific binding of biomolecules than the standard poly(ethylene glycol) surface. The DDS-Tween-20 surface was simple and inexpensive to prepare and did not perturb the behavior and activities of tethered biomolecules. It can also be used for single-molecule imaging in the presence of high concentrations of labeled species in solution.

Stereoacuity changes after laser in situ keratomileusis.

PURPOSE: To study changes in near and distance stereoacuity after laser in situ keratomileusis (LASIK). METHODS: A prospective interventional study was conducted at an apex tertiary care ophthalmology center in India. Near and distance stereoacuity was tested in 40 patients (80 eyes) who underwent LASIK for myopic correction and got unaided vision of 0.67 or better in each eye. Stereoacuity was tested with best spectacle correction before LASIK, and post-LASIK stereoacuity was tested with unaided eye near and distance Randot tests. RESULTS: Forty patients (80 eyes) had a mean (±SD) pre-LASIK refractive error of -4.70 (±1.72) DS OD and -4.59 (±1.58) DS OS and a mean (±SD) anisometropia of 0.55 (±0.51) DS. The median pre-LASIK near stereoacuity was 70 arcsec and distance stereoacuity was 200 arcsec, both of which improved after LASIK to 30 and 60 arcsec, respectively (p < 0.001, both). Amount of refractive error was not associated with stereoacuity but anisometropia of greater than or equal to 1 diopter had significantly worse distance stereoacuity in both the pre-LASIK and post-LASIK period. The post-LASIK near stereoacuity and distance stereoacuity were strongly associated (r = 0.706, p < 0.001) unlike the change in stereoacuity. CONCLUSIONS: Near and distance stereoacuity shows significant improvement after LASIK. Stereoacuity is associated with the degree of anisometropia but not the amount of refractive error corrected.

Correlation Between Structural and Functional Retinal Changes in Parkinson Disease.

BACKGROUND: To evaluate structural changes in the retina and correlate those with visual function measurements in patients with Parkinson disease (PD). METHODS: A cross-sectional comparative study of 20 patients with PD and 20 age-matched healthy controls was conducted. Visual acuity, color vision, contrast sensitivity, visual fields, pattern visual-evoked response (VER), and multifocal electroretinogram were recorded to determine functional change, whereas structural changes were evaluated with retinal nerve fiber layer (RNFL) thickness, macular thickness, macular volume, and ganglion cell-inner plexiform layer complex (GCL-IPL) thickness using spectral domain ocular coherence tomography (SD-OCT). RESULTS: PD patients ranged from Stage 1-3, with median Stage 2 (Hoehn and Yahr Classification) with mean Unified Parkinson Disease Rating Scale III score of 19 ± 10.42, and average disease duration of 5.8 ± 2.78 years. Visual acuity, color vision, and visual fields were unaffected but contrast sensitivity was significantly worse than controls (P < 0.001). Multifocal electroretinogram values in the central 2° field revealed decreased foveal electrical activity, with increased pattern VER amplitude and latency. Significant RNFL thinning was observed in the average RNFL (P = 0.033), superior (P = 0.018), and temporal (P = 0.036) quadrants. Significant ganglion cell layer loss was captured on SD-OCT with average, minimum GCL-IPL, and all 6 sectors showing thinning (P ≤ 0.003). The functional changes correlated significantly with structural changes, disease duration, and severity. There was no correlation between structural changes in the retina and disease duration or severity. CONCLUSIONS: Subclinical visual dysfunction was observed in patients with PD with good structural-functional correlation. GCL-IPL thinning may be a more reliable parameter than RNFL thickness for structural alterations of the retina in patients with PD.

Cognitive impairment and neurogenotoxic effects in rats exposed to low-intensity microwave radiation.

The health hazard of microwave radiation (MWR) has become a recent subject of interest as a result of the enormous increase in mobile phone usage. The present study aimed to investigate the effects of chronic low-intensity microwave exposure on cognitive function, heat shock protein 70 (HSP70), and DNA damage in rat brain. Experiments were performed on male Fischer rats exposed to MWR for 180 days at 3 different frequencies, namely, 900, 1800 MHz, and 2450 MHz. Animals were divided into 4 groups: group I: sham exposed; group II: exposed to MWR at 900 MHz, specific absorption rate (SAR) 5.953 × 10(-4) W/kg; group III: exposed to 1800 MHz, SAR 5.835 × 10(-4) W/kg; and group IV: exposed to 2450 MHz, SAR 6.672 × 10(-4) W/kg. All the rats were tested for cognitive function at the end of the exposure period and were subsequently sacrificed to collect brain. Level of HSP70 was estimated by enzyme-linked immunotarget assay and DNA damage was assessed using alkaline comet assay in all the groups. The results showed declined cognitive function, elevated HSP70 level, and DNA damage in the brain of microwave-exposed animals. The results indicated that, chronic low-intensity microwave exposure in the frequency range of 900 to 2450 MHz may cause hazardous effects on the brain.

Long-term success of mitomycin-augmented trabeculectomy for glaucoma after vitreoretinal surgery with silicone oil insertion: a prospective case series.

PURPOSE: To evaluate the long-term success of trabeculectomy with mitomycin C for glaucoma after vitreoretinal surgery with silicone oil insertion. METHODS: Prospective evaluation of patients who underwent trabeculectomy with mitomycin C (superior or inferior site) for glaucoma after vitreoretinal surgery. Parameters examined included intraocular pressure (IOP), visual acuity, and glaucomatous neuropathy status, preoperatively and at multiple follow-up visits postoperatively till 12 months. Success, both absolute (IOP <21 mmHg) and qualified (IOP <21 mmHg with the use of medications and/or needling), was determined at each follow-up visit. RESULTS: Nineteen patients with mean age of 29 ± 16 years had a mean untreated baseline IOP of 42.5 ± 10.66 mmHg, which reduced to 23.82 ± 7.58 at 1 year (P = 0.008). Preoperative decimal visual acuity was 0.12 ± 0.21, which worsened to 0.07 ± 0.08 at the final follow-up (P = 0.81). Total success rate was 36.9% at the end of 1 year, whereas absolute success rate was only 15.8%. Duration between vitreoretinal surgery and silicone oil removal, preoperative IOP, or site of surgery did not determine success rate. CONCLUSION: Twelve-month success rate for trabeculectomy with mitomycin C in glaucoma after vitreoretinal surgery with silicone oil insertion is lower than reported for most refractory glaucomas.

Two-step technique for posterior optic buttonholing of intraocular lens.

PURPOSE: This study aims to describe a two-step surgical technique for placement of a posterior chamber intraocular lens (IOL) in cases with crystalline lens subluxation resulting from non-progressive zonular dialysis. METHODS: The first stage entails a phacoemulsification with creation of a 4-mm posterior capsular opening using an automated vitrector. The second stage performed 6 weeks later includes an anterior vitrectomy and injection of a foldable three-piece IOL in the sulcus. The haptics of IOL are positioned in the sulcus while the optic is pushed behind the posterior capsular opening therefore "buttonholing" the IOL. RESULTS: Seven eyes of seven patients with posttraumatic zonular dialysis were operated using this technique. Follow-up of all cases revealed a well-centered IOL with good postoperative visual acuity (20/20 to 20/80). CONCLUSIONS: Our two-stage surgical technique precludes the insertion of capsular tension ring in cases with non-progressive zonular dialysis. The technique is recommended in the presence of less than or equal to 6 clock hours of zonular dialysis with preexisting posterior capsular tear or herniation of vitreous in the anterior chamber.

Schizophillum commune causing sinusitis with nasal polyposis in the sub-Himalayan region: first case report and review.

Schizophillum commune is an environmental fungus rarely causing human infections of diverse nature. Sinusitis occurs in immunocompromised persons and seldom in healthy subjects. Though easily isolated, the lack of awareness of its virulence is a bottleneck in the diagnosis of this infection. We report the first case of S. commune sinusitis with nasal polyps in an immunocompetent male from the sub-Himalayan region. The computerized tomography scan findings established the clinical diagnosis, and causative agent was confirmed as S. commune. A white, woolly mold with septate, hyaline hyphae and characteristic spicules but unclamped connections suggested a monokaryotic isolate. Patient was treated successfully with fiberoptic endoscopic sinus surgery, and no antifungal therapy was instituted. There was no recurrence at review after 1 year.