A map of the rubisco biochemical landscape.

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

Contributory factors for developing foveal neovascularization in proliferative diabetic retinopathy.

PURPOSE: Foveal neovascularisation (NV) in proliferative diabetic retinopathy (PDR) is uncommon. The study aim is to analyse a series of cases of foveal NV in PDR and ascertain the factors leading to its development. METHODS: In this retrospective case-control study, optical coherence tomography (OCT) and OCT-angiography (OCTA) images of PDR cases with/without foveal NV diagnosed on fluorescein angiography were analysed. RESULTS: From 124 consecutive PDR eyes, foveal NV was identified in 12 (10%) eyes. Eyes with foveal NV showed thin choroid (p = 0.001), increased FAZ area and reduced vessel density at the macula compared to control group on OCT and OCTA. After regression analysis, an increased FAZ in the superficial capillary plexus slab (p = 0.002) was associated with foveal NV development. CONCLUSION: Our case series suggest that foveal NV is an uncommon finding, occurring due to reduced choroidal and inner retinal perfusion at the macula. Further studies are required to assess the treatment outcomes in such eyes.

The preproliferative stage in type 2 macular telangiectasia (MacTel type 2).

PURPOSE: To study clinical and imaging features of various stages of macular telangiectasia (MacTel type 2). METHODS: In this retrospective study, cases of MacTel type 2 with fluorescein angiography (FA), optical coherence tomography (OCT) and OCT-angiography (OCTA) imaging were included. Based on angiographic perifoveal fluorescence, two groups were formed: group 1: diffuse hyperfluoroscence and group 2: diffuse + focal hyperfluoroscence. Later, based on OCT features, group 2 was subdivided into group 2A: without SRNVM and group 2B: with SRNVM. Clinical, FA, OCT and OCTA features were analysed. Eyes showing conversion to the proliferative stage at final visit were noted. RESULTS: Ninety-four eyes of 48 patients were included. Group 1 (n = 28) showed diffuse perifoveal hyperfluoroscence, hyperreflective middle retinal layers, absent SRNVM (p = 0.006) on OCT and dilated perifoveal capillaries in deep capillary plexus (DCP) on OCTA. Group 2A (n = 40) showed diffuse + focal perifoveal hyperfluoroscence, hyperreflective middle retinal layers (p = 0.001), hyporeflective outer retina cavities (p = 0.021), absent SRNVM with dilated and bunching perifoveal capillaries (p = 0.004) in DCP. Group 2B (n = 26) showed late diffuse + focal perifoveal hyperfluoroscence, foveal contour irregularity (p = 0.002), retinal pigment clumps (p = 0.015) and SRNVM on OCT with bunching of capillaries in DCP and vessels in outer retina (p = 0.002). Five eyes showed conversion to group 2B at final visit. CONCLUSION: There exists a distinct disease stage called "preproliferative" MacTel type 2 showing clinical features of non-proliferative disease, diffuse + focal perifoveal hyperfluoroscence on FA, absent SRNVM on OCT and bunching perifoveal capillaries in DCP on OCTA. Its identification is important for suspecting proliferative disease, planning management and follow-up visit accordingly.

Indocyanine green angiography imaging findings in artery occlusions.

PURPOSE: To describe the multimodal imaging features including indocyanine green angiography (ICGA) in cases diagnosed clinically as central retinal artery occlusion (CRAO) at its different disease stages. METHODS: In this retrospective observational study, patients diagnosed clinically as CRAO or hemi-CRAO were included. All patients underwent multimodal imaging with optical coherence tomography (OCT), fundus fluorescein angiography (FFA) and indocyanine green angiography (ICGA) were studied. Analysis of ICGA images in different stages of artery occlusions and its correlation with accompanying FFA and OCT images was done. RESULTS: Eight such studies in five patients were available for analysis. The most important observation noted on ICGA was the presence of hypercyanescent spots seen during the acute stages of the disease in four of the five cases. The spots were accompanied by retinal vessel staining on FFA in the corresponding region. As the disease showed signs of resolution, the hypercyanescent spots on ICGA and retinal vessel staining on FFA disappeared. The hypercyanescent spots seen on the ICGA were noted due to the red blood cell aggregation or 'rouleaux' formation. In addition, choroidal perfusion abnormalities were noted on ICGA in all five cases in the acute stage. CONCLUSION: Choroidal perfusion changes can be identified in acute phase of retinal artery occlusion. Rouleaux formation in the retinal circulation occurs due to the slowing of the blood flow following artery occlusion. These are seen as hypercyanescent spots in the late phase on ICGA.

Large scale changes in the transcriptome of Eisenia fetida during regeneration.

Earthworms show a wide spectrum of regenerative potential with certain species like Eisenia fetida capable of regenerating more than two-thirds of their body while other closely related species, such as Paranais litoralis seem to have lost this ability. Earthworms belong to the phylum Annelida, in which the genomes of the marine oligochaete Capitella telata and the freshwater leech Helobdella robusta have been sequenced and studied. Herein, we report the transcriptomic changes in Eisenia fetida (Indian isolate) during regeneration. Following injury, E. fetida regenerates the posterior segments in a time spanning several weeks. We analyzed gene expression changes both in the newly regenerating cells and in the adjacent tissue, at early (15days post amputation), intermediate (20days post amputation) and late (30 days post amputation) by RNAseq based de novo assembly and comparison of transcriptomes. We also generated a draft genome sequence of this terrestrial red worm using short reads and mate-pair reads. An in-depth analysis of the miRNome of the worm showed that many miRNA gene families have undergone extensive duplications. Sox4, a master regulator of TGF-beta mediated epithelial-mesenchymal transition was induced in the newly regenerated tissue. Genes for several proteins such as sialidases and neurotrophins were identified amongst the differentially expressed transcripts. The regeneration of the ventral nerve cord was also accompanied by the induction of nerve growth factor and neurofilament genes. We identified 315 novel differentially expressed transcripts in the transcriptome, that have no homolog in any other species. Surprisingly, 82% of these novel differentially expressed transcripts showed poor potential for coding proteins, suggesting that novel ncRNAs may play a critical role in regeneration of earthworm.

A Novel Long Non-coding RNA, durga Modulates Dendrite Density and Expression of kalirin in Zebrafish.

Kalirin, a key player in axonal development, nerve growth and synaptic re-modeling, is implicated in many pathological conditions like schizophrenia and autism-spectrum disorders. Alternative promoters and splicing lead to functionally distinct isoforms, but the post-transcriptional regulation of Kalirin has not been studied. Here, we report a novel non-coding RNA, which we name durga, arising from the first exon of kalirin a (kalrna) in the antisense orientation in zebrafish. The kalrna and durga transcripts are barely detectable during early development, but steadily increase by 24 hours post-fertilization (hpf) as the brain develops. Over-expression of durga in the zebrafish embryo led to an increase in kalrna expression. The morphology of the neurons cultured from durga injected embryos had significantly fewer and shorter dendrites. Although durga has no apparent sequence homolog in mammals, based on gene synteny, we found a non-coding RNA arising from the 5' end of the human Kalrn gene and expressed in the human neuronal cell line, SH-SY5Y. We propose that the zebrafish lncRNA durga maintains dendritic length and density through regulation of kalrna expression and this may have further implications in mammalian systems.