Spent coffee grounds as a suitable alternative to standard soil in ecotoxicological tests.

Eisenia andrei is considered in OECD and ISO guidelines to be a suitable replacement for Eisenia fetida in ecotoxicological assays. This suggests that other alternative materials and methods could also be used in standard procedures for toxicity testing. The guidelines also favor using less time-consuming procedures and reducing costs and other limitations to ecotoxicological assessments. In recent years, spent coffee grounds (SCG) have been used to produce vermicompost and biochar and as an additive to organic fertilizers. In addition, the physicochemical characteristics of SCG indicate that the material is a suitable substrate for earthworms, with the organisms performing as well as in natural soil. In the present study, a battery of ecotoxicological tests was established with unwashed and washed SCG and a natural reference soil (LUFA 2.2). The test substrates were spiked with different concentrations of silver nitrate. Survival and reproduction of the earthworm E. andrei were assessed under different conditions, along with substrate basal respiration (SBR) as a proxy for microbial activity. Seedling emergence and the germination index of Lepidium sativum were also determined, following standard guidelines. Exposure to silver nitrate had similar effects on earthworm survival and reproduction, as the estimated effective concentrations (EC10 and EC50) in unwashed SCG and LUFA 2.2 overlapped. A hormetic effect was observed for SBR in LUFA 2.2 spiked with 12.8 mg/kg but not in unwashed SCG. Both SBR and root development were inhibited by similar concentrations of silver nitrate in washed SCG. The findings indicate that unwashed SCG could potentially be used as a substrate in E. andrei toxicity tests and support the eventual inclusion of this material in the standard guidelines.

Mapping secondary substrate-binding sites on the GH11 xylanase from Bacillus subtilis.

Xylanases are of significant interest for biomass conversion technologies. Here, we investigated the allosteric regulation of xylan hydrolysis by the Bacillus subtilis GH11 endoxylanase. Molecular dynamics simulations (MDS) in the presence of xylobiose identified binding to the active site and two potential secondary binding sites (SBS) around surface residues Asn54 and Asn151. Arabinoxylan titration experiments with single cysteine mutants N54C and N151C labeled with the thiol-reactive fluorophore acrylodan or the ESR spin-label MTSSL validated the MDS results. Ligand binding at the SBS around Asn54 confirms previous reports, and analysis of the second SBS around N151C discovered in the present study includes residues Val98/Ala192/Ser155/His156. Understanding the regulation of xylanases contributes to efforts for industrial decarbonization and to establishing a sustainable energy matrix.

Structural insights into the Smirnoff-Wheeler pathway for vitamin C production in the Amazon fruit Camu-Camu.

L-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler (SW) pathway is the primary route for AsA biosynthesis and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focusses on three of them from the model system Myrciaria dubia (camu-camu): GDP-D-mannose 3',5'-epimerase (GME), L-galactose dehydrogenase (L-GalDH), and L-galactono-1,4-lactone dehydrogenase (L-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. MdL-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue from spinach. Finally, the structure of MdL-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with the FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants and the information provided should prove useful for biotechnological applications.

CENTRAL AND PERIPHERAL INVOLVEMENT OF THE RETINA IN THE INITIAL STAGES OF DIABETIC RETINOPATHY.

PURPOSE: To determine the degree of central microvascular closure using optical coherence tomography angiography in eyes of patients with type 2 diabetes with visible lesions only in the central retina or only in the periphery. METHODS: Cross-sectional study. All 127 eyes underwent ultra-widefield fundus photography 200° examinations with OPTOS California (Optos, Dunfermline, United Kingdom) and Cirrus Angioplex optical coherence tomography angiography 3 × 3 mm acquisitions (ZEISS, Dublin, CA). RESULTS: Twenty-five eyes showed visible lesions only in the central retina, 57 only in the peripheral retina, and 45 presented visible lesions in entire retina. The group with visible lesions only in the periphery showed definite closure in the superficial capillary plexus in 49% of the eyes, whereas the group with visible lesions only in the central seven-early treatment diabetic retinopathy study fields area showed a definite closure in 64%. CONCLUSION: Central capillary closure is already present in the initial stages of diabetic retinopathy even when lesions are only visible in the peripheral retina. Capillary closure in the superficial capillary plexus is three times more frequent than in the deep capillary plexus, demonstrating earlier closure of the superficial capillary plexus. Eyes with visible lesions only in the periphery show a milder form of retinopathy.

Retinal Capillary Nonperfusion in Preclinical Diabetic Retinopathy.

INTRODUCTION: The aim of the study was to identify retinal microvascular changes using optical coherence tomography angiography (OCTA) in type 2 diabetes (T2D) patients with preclinical retinopathy identified by ultra-widefield fundus photography (UWF-FP). METHODS: This is a cross-sectional observational study. All patients underwent UWF-FP 200° examinations with OPTOS California (Optos, Dunfermline, UK) and Cirrus AngioPlex® spectral-domain (SD)-OCTA 3 × 3 mm acquisitions (ZEISS, Dublin, CA, USA). The absence of visible lesions was identified using UWF-FP. RESULTS: One hundred and ninety three eyes of individuals with T2D with no visible lesions in the fundus and identified in a screening setting were included in the study. Skeletonized vessel density (SVD), perfusion density (PD), and areas of capillary nonperfusion (CNP) values on SD-OCTA were significantly decreased when compared with healthy population (p < 0.001). SVD and CNP values of the superficial capillary plexus (SCP) were more frequently decreased (35% and 45%, respectively) than SVD values of the deep capillary plexus (DCP) (9% and 15%, respectively), demonstrating that diabetic microvascular changes occur earlier in the SCP than in the DCP. The ischemic phenotype, identified by a definite decrease in SVD or CNP in the SCP may, therefore, be identified in the preclinical stage of diabetic retinal disease. CONCLUSIONS: Retinal capillary nonperfusion detected by OCTA metrics of SVD and CNP can be identified in the central retina in eyes with T2D before development of visible lesions in the retina. Our findings confirm the relevance of OCTA to identify macular microvascular changes in the initial stages of diabetic retinopathy, allowing the identification of its ischemic phenotype very early in the disease process.

Retinal neurodegeneration in eyes with NPDR risk phenotypes: A two-year longitudinal study.

INTRODUCTION: Diabetic retinopathy (DR) is both a microangiopathy and a neurodegenerative disease. However, the connections between both changes are not well known. PURPOSE: To characterise the longitudinal retinal ganglion cell layer + inner plexiform layer (GCL + IPL) changes and their association with microvascular changes in type-2 diabetes (T2D) patients with nonproliferative diabetic retinopathy (NPDR). METHODS: This two-year prospective study (CORDIS, NCT03696810) included 122 T2D individuals with NPDR identified as risk phenotypes B and C, which present a more rapid progression. Phenotype C was identified by decreased VD ≥ 2SD in healthy controls, and phenotype B, identified by subclinical macular oedema with only minimal vascular closure. The GCL + IPL thickness, vessel density, perfusion density and area of intercapillary spaces (AIS) were assessed by optical coherence tomography (OCT) and OCT angiography (OCTA). Linear mixed effects models were employed to evaluate the retinal GCL + IPL progression and its associations. RESULTS: Regarding GCL + IPL thickness, T2D individuals presented on average 80.1 ± 7.49 µm, statistically significantly lower than the healthy control group, 82.5 ± 5.71 (p = 0.022), with only phenotype C differing significantly from controls (p = 0.006). GCL + IPL thickness steadily decreased during the two-year period in both risk phenotypes, with an annual decline rate of -0.372 µm/year (p < 0.001). Indeed, phenotype C showed a higher rate of progression (-0.459 µm/year, p < 0.001) when compared to phenotype B (-0.296 µm/year, p = 0.036). Eyes with ETDRS grade 20 showed GCL + IPL thickness values comparable to those of healthy control group (83.3 ± 5.80 and 82.7 ± 5.50 µm, respectively, p = 0.880), whereas there was a progressive decrease in GCL + IPL thickness in ETDRS grades 35 and 43-47 associated with the increase in severity of the retinopathy (-0.276 µm/year, p = 0.004; -0.585 µm/year, p = 0.013, respectively). Furthermore, the study showed statistically significant associations between the progressive thinning of GCL + IPL and the progressive increase in retinal capillary non-perfusion, with particular relevance for AIS (p < 0.001). CONCLUSIONS: Our findings showed that, in eyes with NPDR and at risk for progression, retinal neurodegeneration occurs at different rates in different risk phenotypes, and it is associated with retinal microvascular non-perfusion.

Different Risk Profiles for Progression of Nonproliferative Diabetic Retinopathy: A 2-Year Study.

INTRODUCTION: Characterization of 2-year progression of different risk phenotypes in eyes with mild and moderate nonproliferative diabetic retinopathy (NPDR) in type 2 diabetes (T2D). METHODS: A 2-year prospective longitudinal cohort study (CORDIS, NCT03696810) was conducted. Ophthalmological examinations were performed including best corrected visual acuity, color fundus photography and optical coherence tomography (OCT and OCTA). OCT metrics, central retinal thickness and ganglion cell layer + inner plexiform layer (GCL + IPL) thickness were analyzed. OCTA metrics, vessel density (VD), perfusion density (PD) and area of intercapillary spaces (AIS) were obtained from superficial and deep capillary plexus (SCP, DCP). Only phenotype C identified by decreased VD ≥ 2 SD of healthy controls and phenotype B identified by subclinical macular edema with decreased VD < 2 SD of healthy controls were included. RESULTS: One hundred twenty-two eyes from T2D individuals were included in study; 65 eyes (53%) were classified as phenotype B and 57 eyes (47%) as phenotype C. For phenotype B, progression was associated with thinning of the GCL + IPL (ETDRS 35, 1 year p = 0.013, 2 year p < 0.001; ETDRS 43-47, 2 year p = 0.003) and vessel closure involving mainly the DCP for both ETDRS grades (ETDRS 35, 1 year p = 0.025, 2 year p = 0.034; ETDRS 43-47, 1 year p = 0.011). For phenotype C there was also progressive thinning of the GCL + IPL (ETDRS 35, in both years p ≤ 0.001; ETDRS 43-47, 1 year p = 0.002, 2 year p = 0.001), with vessel closure involving mainly SCP (ETDRS 35, 1 year p = 0.012, 2 year p = 0.023 in full-retina), which appeared to stabilize at maximal values in ETDRS grade 43-47 at the end of 2 years. ETDRS severity changes at the end of the 2-year period showed that worsening was associated with phenotype C with changes involving predominantly the SCP (VD, p = 0.005; PD, p = 0.008; AIS, p = 0.005). CONCLUSIONS: Association between ETDRS classification of NPDR severity and identification of different risk phenotypes offers new perspective to predict disease progression in T2D individuals with NPDR.

Use of artificial intelligence in ophthalmology: a narrative review

ABSTRACT BACKGROUND: Artificial intelligence (AI) deals with development of algorithms that seek to perceive one's environment and perform actions that maximize one's chance of successfully reaching one's predetermined goals. OBJECTIVE: To provide an overview of the basic principles of AI and its main studies in the fields of glaucoma, retinopathy of prematurity, age-related macular degeneration and diabetic retinopathy. From this perspective, the limitations and potential challenges that have accompanied the implementation and development of this new technology within ophthalmology are presented. DESIGN AND SETTING: Narrative review developed by a research group at the Universidade Federal de São Paulo (UNIFESP), São Paulo (SP), Brazil. METHODS: We searched the literature on the main applications of AI within ophthalmology, using the keywords "artificial intelligence", "diabetic retinopathy", "macular degeneration age-related", "glaucoma" and "retinopathy of prematurity," covering the period from January 1, 2007, to May 3, 2021. We used the MEDLINE database (via PubMed) and the LILACS database (via Virtual Health Library) to identify relevant articles. RESULTS: We retrieved 457 references, of which 47 were considered eligible for intensive review and critical analysis. CONCLUSION: Use of technology, as embodied in AI algorithms, is a way of providing an increasingly accurate service and enhancing scientific research. This forms a source of complement and innovation in relation to the daily skills of ophthalmologists. Thus, AI adds technology to human expertise.

Characterization of two-year progression of different phenotypes of nonproliferative diabetic retinopathy.

INTRODUCTION: To characterize the two-year progression of risk phenotypes of nonproliferative diabetic retinopathy (NPDR) in type 2 diabetes (T2D) Phenotype C, or ischemic phenotype, identified by decreased skeletonized retinal vessel density (VD), ≥ 2 SD over normal values, and Phenotype B, or edema phenotype, identified by increased retinal thickness, i.e. subclinical macular edema, and no significant decrease in VD. METHODS: A prospective longitudinal cohort study (CORDIS, NCT03696810) was conducted with 4 visits (baseline, 6-months, one-year and two-year). Ophthalmological examinations included best corrected visual acuity, color fundus photography (CFP) and optical coherence tomography (OCT) and OCT Angiography. Early Treatment Diabetic Retinopathy Study grading was performed at the baseline and last visits based on 7-fields CFP. RESULTS: One hundred and twenty-two eyes from T2D individuals with NPDR fitted in the categories of phenotype B and C and completed the two-years follow-up. Sixty-five (53%) of the eyes were classified as phenotype B and 57 (47%) eyes as phenotype C. Neurodegeneration represented by thinning of the ganglion cell layer and inner plexiform layer was present in both phenotypes and showed significant progression over the two-year period (p<0.001). In phenotype C, significant progression in the two-year period was identified in decreased skeletonized VD (p=0.01), whereas in phenotype B microvascular changes involved preferentially decreases in perfusion density (PD, p=0.012). Phenotype B with changes in VD and PD (flow) and preferential involvement of the deep capillary plexus (p<0.001) is associated with development of center-involved macular edema. DISCUSSION: In the two-year period of follow-up both phenotypes B and C showed progression in retinal neurodegeneration, with changes at the microvascular level characterized by decreases in PD in phenotype B and decreases in VD in phenotype C.

Use of artificial intelligence in ophthalmology: a narrative review.

BACKGROUND: Artificial intelligence (AI) deals with development of algorithms that seek to perceive one's environment and perform actions that maximize one's chance of successfully reaching one's predetermined goals. OBJECTIVE: To provide an overview of the basic principles of AI and its main studies in the fields of glaucoma, retinopathy of prematurity, age-related macular degeneration and diabetic retinopathy. From this perspective, the limitations and potential challenges that have accompanied the implementation and development of this new technology within ophthalmology are presented. DESIGN AND SETTING: Narrative review developed by a research group at the Universidade Federal de São Paulo (UNIFESP), São Paulo (SP), Brazil. METHODS: We searched the literature on the main applications of AI within ophthalmology, using the keywords "artificial intelligence", "diabetic retinopathy", "macular degeneration age-related", "glaucoma" and "retinopathy of prematurity," covering the period from January 1, 2007, to May 3, 2021. We used the MEDLINE database (via PubMed) and the LILACS database (via Virtual Health Library) to identify relevant articles. RESULTS: We retrieved 457 references, of which 47 were considered eligible for intensive review and critical analysis. CONCLUSION: Use of technology, as embodied in AI algorithms, is a way of providing an increasingly accurate service and enhancing scientific research. This forms a source of complement and innovation in relation to the daily skills of ophthalmologists. Thus, AI adds technology to human expertise.

Comparative Metabolomics Study of the Impact of Articaine and Lidocaine on the Metabolism of SH-SY5Y Neuronal Cells.

Articaine (ATC) and lidocaine (LDC) are the local anesthetics (LAs) currently most employed in dentistry. Cases of paresthesia, reported more frequently for ATC, have raised concerns about their potential neurotoxicity, calling for further investigation of their biological effects in neuronal cells. In this work, the impact of ATC and LDC on the metabolism of SH-SY5Y cells was investigated through 1H NMR metabolomics. For each LA, in vitro cultured cells were exposed to concentrations causing 10 and 50% reductions in cell viability, and their metabolic intracellular and extracellular profiles were characterized. Most effects were common to ATC and LDC, although with varying magnitudes. The metabolic variations elicited by the two LAs suggested (i) downregulation of glycolysis and of glucose-dependent pathways (e.g., one-carbon metabolism and hexosamine biosynthetic pathway), (ii) disturbance of branched chain amino acids (BCAA) catabolism, (iii) downregulation of TCA cycle anaplerotic fueling and activation of alternative energy producing pathways, (iv) interference with choline metabolism and (v) lipid droplet build-up. Interestingly, LDC had a greater impact on membrane phospholipid turnover, as suggested by higher phosphatidylcholine to phosphocholine conversion. Moreover, LDC elicited an increase in triglycerides, whereas cholesteryl esters accumulated in ATC-exposed cells, suggesting a different composition and handling of lipid droplets.

Cytotoxicity Assessment of Nanoplastics and Plasticizers Exposure in In Vitro Lung Cell Culture Systems-A Systematic Review.

Emerging contaminants such as nanoplastics (NPs), as well as manufacturing by-products such as plasticizers, have gained global attention and concern due to their limited biodegradability and their potential impact on human health, in particular the effects on respiratory tissue. In parallel, in vitro cell culture techniques are key to the assessment and characterization of toxic effects and cellular mechanisms in different types of tissues and should provide relevant information to understand the hazardous potential of these emergent contaminants. This systematic review presents the main results on the current knowledge of the effects of NPs and plasticizers on lung cells, as assessed with the use of in vitro cell culture techniques. From the selected studies (n = 10), following the PRISMA approach, it was observed that cell viability was the most frequently assessed endpoint and that most studies focused on epithelial cells and exposures to polystyrene (PS). It was observed that exposure to NPs or plasticizers induces cytotoxicity in a dose-dependent manner, regardless of the size of the NPs. Furthermore, there is evidence that the characteristics of NPs can affect the toxic response by promoting the association with other organic compounds. As such, further in vitro studies focusing on the combination of NPs with plasticizers will be essential for the understanding of mechanisms of NPs toxicity.

Characterization of the RNA-dependent RNA polymerase from Chikungunya virus and discovery of a novel ligand as a potential drug candidate.

Chikungunya virus (CHIKV) is the causative agent of Chikungunya fever, an acute febrile and arthritogenic illness with no effective treatments available. The development of effective therapeutic strategies could be significantly accelerated with detailed knowledge of the molecular components behind CHIKV replication. However, drug discovery is hindered by our incomplete understanding of their main components. The RNA-dependent RNA-polymerase (nsP4-CHIKV) is considered the key enzyme of the CHIKV replication complex and a suitable target for antiviral therapy. Herein, the nsP4-CHIKV was extensively characterized through experimental and computational biophysical methods. In the search for new molecules against CHIKV, a compound designated LabMol-309 was identified as a strong ligand of the nsp4-CHIKV and mapped to bind to its active site. The antiviral activity of LabMol-309 was evaluated in cellular-based assays using a CHIKV replicon system and a reporter virus. In conclusion, this study highlights the biophysical features of nsP4-CHIKV and identifies a new compound as a promising antiviral agent against CHIKV infection.

Eye diseases during pregnancy: a study with the medical data warehouse in the eye clinic of the Ludwig-Maximilians-Universität München in Munich in Germany.

OBJECTIVE: To analyze the most common ophthalmologic disorders in pregnant women seen in a hospital in Munich in Germany using a big data analysis system, as well as to compare the results obtained with those from other epidemiological studies that used different data acquisition methods. METHODS: We retrospectively analyzed electronic health records of pregnant women who were seen at the ophthalmology department from 2003 to 2019 at the Ludwig-Maximilians-Universität München hospital. The main complaints that led to ophthalmic consultations during this period were evaluated, and also the variation in intraocular pressure of patients throughout gestational trimesters by analyzing data from the data warehouse system. RESULTS: A total of 27,326 electronic health records were analyzed. Of participants, 149 (0.54%) required eye care during pregnancy. Their mean intraocular pressure was 17mmHg in the first trimester, 12mmHg in the second trimester, and 14mmHg in the third trimester. The most prevalent findings were dry eye (29.3%) and conjunctivitis (16%), and ametropia (16%). The most common posterior segment problem was diabetic retinopathy (4.6%). The lower mean intraocular pressure in the second and third trimester found in our study is in accordance with other studies that used other method for data acquisition. CONCLUSION: The most common ophthalmic conditions found in this study population were dry eye, conjunctivitis, and ametropia. The use of data warehouse proved to be useful for acquiring and analyzing data from many patients. This study results are comparable with other studies in published literature that adopted different methodology.

Tetramethyl-phenanthroline copper complexes in the development of drugs to treat cancer: synthesis, characterization and cytotoxicity studies of a series of copper(II)-L-dipeptide-3,4,7,8-tetramethyl-phenanthroline complexes.

New compounds to fight cancer are needed due to cancer high incidence and lack of curative treatments for several classes of this disease. Metal-based coordination compounds offer a variety of molecules that can turn into drugs. Among them, coordination copper complexes are emerging as an attractive class of compounds for cancer treatment. A series of [Cu(L-dipeptide)(tmp)] (tmp = 3,4,7,8-tetramethyl-1,10-phenanthroline) complexes were synthesized and characterized in the solid state, including the determination of the crystalline structure of [Cu(Gly-Gly)(tmp)]·3.5 H2O and [Cu2Cl4(tmp)2]. The complexes were studied in solution, where the major species are also ternary ones. The lipophilicity of the complexes was determined and the binding to the DNA was evaluated, suggesting that it occurs in the DNA's major groove. The cytotoxicity of the complexes was evaluated on different cancer cell lines: human metastatic breast adenocarcinoma MDA-MB-231 (triple negative, ATCC: HTB-26), MCF-7 (ATCC: HTB-22), SK-BR-3 (ATCC: HTB-30), human lung epithelial carcinoma A549 (ATCC: CCL-185), cisplatin resistant-human ovarian carcinoma A2780cis (SIGMA) and nontumoral cell lines: MRC-5 (lung; ATCC: CCL-171) and MCF-10A (breast, ATCC: CRL-10317). [Cu(L-dipeptide)(tmp)] complexes are highly cytotoxic as compared to [Cu(L-dipeptide)(phenanthroline)] and cisplatin. Therefore, [Cu(L-dipeptide)(tmp)] complexes are promising candidates to have their in vivo activity further studied toward new treatments for triple negative breast cancer and other aggressive tumors for which there is no curative pharmacological treatment to the date.

Co-Exposure of Nanopolystyrene and Other Environmental Contaminants-Their Toxic Effects on the Survival and Reproduction of Enchytraeus crypticus.

Plastics in all shapes and sizes have become widespread across ecosystems due to intense anthropogenic use. As such, they can interact with other contaminants that accumulate in the terrestrial environment, such as pharmaceuticals, metals or nanomaterials (NMs). These interactions can potentiate combined toxic effects in the exposed soil organisms, with hazardous long-term consequences to the full ecosystem. In the present study, a terrestrial model species, Enchytraeus crypticus (oligochaeta), was exposed through contaminated soil with nanopolystyrene (representative of nanoplastics (NPls)), alone and in combination with diphenhydramine (DPH, representative of pharmaceuticals), silver nitrate (AgNO3, representative of metals) and vanadium nanoparticles (VNPs, representative of NMs). AgNO3 and VNPs decreased E. crypticus reproduction at 50 mg/kg, regardless of the presence of NPls. Moreover, at the same concentration, both single and combined VNP exposures decreased the E. crypticus survival. On the other hand, DPH and NPls individually caused no effect on organisms' survival and reproduction. However, the combination of DPH (10 and 50 mg/kg) with 300 mg NPls/kg induced a decrease in reproduction, showing a relevant interaction between the two contaminants (synergism). Our findings indicate that the NPls can play a role as vectors for other contaminants and can potentiate the effects of pharmaceuticals, such as DPH, even at low and sub-lethal concentrations, highlighting the negative impact of mixtures of contaminants (including NPls) on soil systems.

Association between Neurodegeneration and Macular Perfusion in the Progression of Diabetic Retinopathy: A 3-Year Longitudinal Study.

OBJECTIVE AND PURPOSE: The aim of this study was to explore the relation between retinal neurodegenerative changes and vessel closure (VC) in individuals with nonproliferative diabetic retinopathy (NPDR) in a follow-up period of 3 years. DESIGN: This is a 3-year prospective longitudinal study with four annual visits. PARTICIPANTS: This study involved 74 individuals with type 2 diabetes, NPDR, and Early Treatment Diabetic Retinopathy Study grades from 10 to 47, one eye/person. An age-matched healthy control population of 84 eyes was used as control group. METHODS: Participants were annually examined by color fundus photography, spectral domain-optical coherence tomography (SD-OCT) and OCT-angiography (OCTA). VC was assessed by OCTA vessel density maps. SD-OCT segmentations were performed to access central retinal thickness (CRT) and retinal neurodegeneration considered as thinning of the ganglion cell plus inner plexiform layer (GCL + IPL). RESULTS: Type 2 diabetic individuals presented significantly higher CRT (p = 0.001), GCL + IPL thinning (p = 0.042), and decreased vessel density at the superficial capillary plexus (p < 0.001) and full retina (FR) (p = 0.001). When looking at changes occurring over the 3-year period of follow-up (Table 2), there were statistically significant decreases in GCL + IPL thickness (-0.438 µm/year; p = 0.038), foveal avascular zone circularity (-0.009; p = 0.047), and vessel density in superficial capillary plexus (-0.172 mm-1/year; p < 0.001), deep capillary plexus (DCP) (-0.350 mm-1/year; p < 0.001), and FR (-0.182 mm-1/year; p < 0.001). A statistically significant association was identified between GCL + IPL thinning and decrease in DCP vessel density (ß = 0.196 [95% confidence interval: 0.037, 0.355], z = 2.410, p = 0.016), after controlling for age, gender, diabetes duration, hemoglobin A1c level, and CRT. CONCLUSIONS: Retinal neurodegenerative changes show a steady progression during a 3-year period of follow-up in eyes with NPDR and appear to be directly associated with progression in decreased vessel density including vascular closure through preferential involvement of the DCP. Our findings provide evidence that retinal neuropathy is linked with microvascular changes occurring in diabetic patients.

A gold revision of the Golgi Dynamics (GOLD) domain structure and associated cell functionalities.

The classical secretory pathway is the key membrane-based delivery system in eukaryotic cells. Several families of proteins involved in the secretory pathway, with functionalities going from cargo sorting receptors to the maintenance and dynamics of secretory organelles, share soluble globular domains predicted to mediate protein-protein interactions. One of them is the 'Golgi Dynamics' (GOLD) domain, named after its strong association with the Golgi apparatus. There are many GOLD-containing protein families, such as the transmembrane emp24 domain-containing proteins (TMED/p24 family), animal SEC14-like proteins, human Golgi resident protein ACBD3, a splice variant of TICAM2 called TRAM with GOLD domain, and FYCO1. Here, we critically review the state-of-the-art knowledge of the structures and functions of the main representatives of GOLD-containing proteins in vertebrates. We provide the first unified description of the GOLD domain structure across different families since the first high-resolution structure was determined. With a brand-new update on the definition of the GOLD domain, we also discuss how its tertiary structure fits the ß-sandwich-like fold map and give exciting new directions for forthcoming studies.

Resurrecting Golgi proteins to grasp Golgi ribbon formation and self-association under stress.

The Golgi complex is an essential organelle of the eukaryotic exocytic pathway. A subfamily of Golgi matrix proteins, called GRASPs, is central in stress-induced unconventional secretion, Golgi dynamics during mitosis/apoptosis, and Golgi ribbon formation. The Golgi ribbon is vertebrate-specific and correlates with the appearance of two GRASP paralogues and two Golgins (GM130/Golgin45), which form specific GRASP-Golgin pairs. The molecular details of their appearance only in Metazoans are unknown. Moreover, despite new functionalities supported by GRASP paralogy, little is known about their structural and evolutionary differences. Here, we used ancestor sequence reconstruction and biophysical/biochemical approaches to assess the evolution of GRASPs structure/dynamics, fibrillation, and how they started anchoring their Golgin partners. Our data showed that a GRASP ancestor anchored Golgins before gorasp gene duplication in Metazoans. After gene duplication, variations within the GRASP binding pocket determined which paralogue would recruit which Golgin. These interactions are responsible for their specific Golgi location and Golgi ribbon appearance. We also suggest that GRASPs have a long-standing capacity to form supramolecular structures, affecting their participation in stress-induced processes.