Maintenance of Enteral ACE2 Prevents Diabetic Retinopathy in Type 1 Diabetes.

BACKGROUND: We examined components of systemic and intestinal renin-angiotensin system on gut barrier permeability, glucose homeostasis, systemic inflammation, and progression of diabetic retinopathy (DR) in human subjects and mice with type 1 diabetes (T1D). METHODS: T1D individual with (n=18) and without (n=20) DR and controls (n=34) were examined for changes in gut-regulated components of the immune system, gut leakage markers (FABP2 [fatty acid binding protein 2] and peptidoglycan), and Ang II (angiotensin II); Akita mice were orally administered a Lactobacillus paracasei (LP) probiotic expressing humanized ACE2 (angiotensin-converting enzyme 2) protein (LP-ACE2) as either a prevention or an intervention. Akita mice with genetic overexpression of humanAce2 by small intestine epithelial cells (Vil-Cre.hAce2KI-Akita) were similarly examined. After 9 months of T1D, circulatory, enteral, and ocular end points were assessed. RESULTS: T1D subjects exhibit elevations in gut-derived circulating immune cells (ILC1 cells) and higher gut leakage markers, which were positively correlated with plasma Ang II and DR severity. The LP-ACE2 prevention cohort and genetic overexpression of intestinal ACE2 preserved barrier integrity, reduced inflammatory response, improved hyperglycemia, and delayed development of DR. Improvements in glucose homeostasis were due to intestinal MasR activation, resulting in a GSK-3ß (glycogen synthase kinase-3 beta)/c-Myc (cellular myelocytomatosis oncogene)-mediated decrease in intestinal glucose transporter expression. In the LP-ACE2 intervention cohort, gut barrier integrity was improved and DR reversed, but no improvement in hyperglycemia was observed. These data support that the beneficial effects of LP-ACE2 on DR are due to the action of ACE2, not improved glucose homeostasis. CONCLUSIONS: Dysregulated systemic and intestinal renin-angiotensin system was associated with worsening gut barrier permeability, gut-derived immune cell activation, systemic inflammation, and progression of DR in human subjects. In Akita mice, maintaining intestinal ACE2 expression prevented and reversed DR, emphasizing the multifaceted role of the intestinal renin-angiotensin system in diabetes and DR.

Supplementing turmeric rhizome powder in growing Andaman local pigs: a conflated approach for therapy evaluation.

This paper examined the pluripotent effect of supplementation of turmeric rhizome powder (TRP) (Curcuma longa) in growing Andaman local pigs. A total of 48 pigs were randomly allotted into four groups and fed diets containing TRP at 4 concentrations, that is, 0 (control group), 0.05 (treatment 1), 0.1 (treatment 2), and 0.2% (treatment 3) for 30 days. The mean body weight of pigs supplemented with 0.1% and 0.2% TRP was significantly higher than that of the control group (41.66 ± 0.44, 42.59 ± 0.33 vs 40.38 ± 0.30 kg; p ≤ 0.05) which indicated the effect of TRP as growth enhancer. A significant (p ≤ 0.05) decrease in serum concentration of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) was recorded in supplemented groups as compared to the control group. Creatinine kinase (U/l) decreased in all the treatment groups as compared to the control group. Total cholesterol, triglyceride, and low-density lipoprotein cholesterol decreased significantly after supplementation with 0.1% and 0.2% TRP in comparison to the control group. However, there was marked increase in high-density lipoprotein cholesterol (mg/dl) in all TRP-supplemented groups than the control group (27.67 ± 0.60 in T1, 32.76 ± 0.32 in T2, and 34.58 ± 0.37 in T3 vs. 23.73 ± 0.69 in control; p ≤ 0.05). Further, there was increase in antioxidant profile after TRP supplementation. Anti-inflammatory potentiality of TRP could also be appreciated since TRP supplementation downregulated (p ≤ 0.05) expression of IL-6, IL-1ß, and IFN-γ. Therefore, we perceive that this conflated approach is an example of its own kind to focus on modification of health status of pigs for more productivity and augmentation of immune response.

ADAM17 mediates ectodomain shedding of the soluble VLDL receptor fragment in the retinal epithelium.

Very low-density lipoprotein receptor (VLDLR) is a multifunctional transmembrane protein. Beyond the function of the full-length VLDLR in lipid transport, the soluble ectodomain of VLDLR (sVLDLR) confers anti-inflammatory and antiangiogenic roles in ocular tissues through inhibition of canonical Wnt signaling. However, it remains unknown how sVLDLR is shed into the extracellular space. In this study, we present the first evidence that a disintegrin and metalloprotease 17 (ADAM17) is responsible for sVLDLR shedding in human retinal pigment epithelium cells using pharmacological and genetic approaches. Among selected proteinase inhibitors, an ADAM17 inhibitor demonstrated the most potent inhibitory effect on sVLDLR shedding. siRNA-mediated knockdown or CRISPR/Cas9-mediated KO of ADAM17 diminished, whereas plasmid-mediated overexpression of ADAM17 promoted sVLDLR shedding. The amount of shed sVLDLR correlated with an inhibitory effect on the Wnt signaling pathway. Consistent with these in vitro findings, intravitreal injection of an ADAM17 inhibitor reduced sVLDLR levels in the extracellular matrix in the mouse retina. In addition, our results demonstrated that ADAM17 cleaved VLDLR only in cells coexpressing these proteins, suggesting that shedding occurs in a cis manner. Moreover, our study demonstrated that aberrant activation of Wnt signaling was associated with decreased sVLDLR levels, along with downregulation of ADAM17 in ocular tissues of an age-related macular degeneration model. Taken together, our observations reveal the mechanism underlying VLDLR cleavage and identify a potential therapeutic target for the treatment of disorders associated with dysregulation of Wnt signaling.

Fasting and fasting-mimicking treatment activate SIRT1/LXRα and alleviate diabetes-induced systemic and microvascular dysfunction.

AIMS/HYPOTHESIS: Homo sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting-mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage. METHODS: The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as liver X receptor alpha [LXRα]) acetylation was measured via western blot analysis. RESULTS: IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression. CONCLUSIONS/INTERPRETATION: Taken together, activation of SIRT1 signalling by IF or through pharmacological activation represents an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimens and prevention of microvascular and bone marrow dysfunction in diabetes.

Novel molecular mechanisms for Prph2-associated pattern dystrophy.

Mutations in peripherin 2 (PRPH2) have been associated with retinitis pigmentosa (RP) and macular/pattern dystrophies, but the origin of this phenotypic variability is unclear. The majority of Prph2 mutations are located in the large intradiscal loop (D2), a region that contains seven cysteines involved in intra- and intermolecular disulfide bonding and protein folding. A mutation at cysteine 213, which is engaged in an intramolecular disulfide bond, leads to butterfly-shaped pattern dystrophy in humans, in sharp contrast to mutations in the adjacent cysteine at position 214 which result in RP. To help understand this unexpected phenotypic variability, we generated a knockin mouse line carrying the C213Y disease mutation. The mutant Prph2 protein lost the ability to oligomerize with rod outer segment membrane protein 1 (Rom1), but retained the ability to form homotetramers. C213Y heterozygotes had significantly decreased overall Prph2 levels as well as decreased rod and cone function. Critically, supplementation with extra wild-type Prph2 protein elicited improvements in Prph2 protein levels and rod outer segment structure, but not functional rescue in rods or cones. These findings suggest that not all interruptions of D2 loop intramolecular disulfide bonding lead to haploinsufficiency-related RP, but rather that more subtle changes can lead to mutant proteins stable enough to exert gain-of-function defects in rods and cones. This outcome highlights the difficulty in targeting Prph2-associated gain-of-function disease and suggests that elimination of the mutant protein will be a pre-requisite for any curative therapeutic strategy.

Estimation of (Co)Variance components and genetic parameters of fibre traits in Rambouillet sheep using multi-trait analysis.

The present study aimed to estimate the genetic parameters of different fibre traits, viz., greasy fleece weight, staple length, and fibre diameter in Rambouillet sheep population using a multi-trait animal model. Data, spanning over 10 years (1998-2007) and pertaining to fibre traits at first clip, were collected for a total of 4186 Rambouillet sheep maintained at an organized farm. (Co)Variance structure and genetic parameters were estimated using a multi-trait animal model. The genetic analysis of data was performed based on restricted maximum likelihood (REML) procedure using WOMBAT software. The model incorporated sex of lamb (i = 1, 2), year of birth (j = 1-10), season of birth (k = 1-2), and litter size (l = 1-2) as fixed effects while direct additive genetic and maternal genetic effects were included as random effects. The direct additive genetic heritability estimates were 0.120±0.034, 0.136±0.037, and 0.356±0.070 for greasy fleece weight, staple length, and fibre diameter, respectively. The maternal genetic heritability of all fibre traits under study was very low. Additive genetic correlation was positive and low between greasy fleece weight and staple length; and between staple length and fibre diameter. In conclusion, fibre diameter was moderately heritable which implies that selection may lead to moderate improvement in this trait. The results from the present study will help in formulating optimal breeding plans for improvement of fibre traits in Rambouillet sheep.

Retinal Vascular Abnormalities and Microglia Activation in Mice with Deficiency in Cytochrome P450 46A1-Mediated Cholesterol Removal.

CYP46A1 is the cytochrome P450 enzyme that converts cholesterol to 24-hydroxycholesterol, a cholesterol elimination product and a potent liver X receptor (LXR) ligand. We conducted retinal characterizations of Cyp46a1-/- mice that had normal fasting blood glucose levels but up to a 1.8-fold increase in retinal cholesterol. The retina of Cyp46a1-/- mice exhibited venous beading and tortuosity, microglia/macrophage activation, and increased vascular permeability, features commonly associated with diabetic retinopathy. The expression of Lxrα and Lxrß was increased in both the whole Cyp46a1-/- retina and retinal macroglia/macrophages. The LXR-target genes were affected as well, primarily in activated microglial cells and macrophages. In the latter, the LXR-transactivated genes (Abca1, Abcg1, Apod, Apoe, Mylip, and Arg2) were up-regulated; similarly, there was an up-regulation of the LXR-transrepressed genes (Ccl2, Ptgs2, Cxcl1, Il1b, Il6, Nos2, and Tnfa). For comparison, gene expression was investigated in bone marrow-derived macrophages from Cyp46a1-/- mice as well as retinal and bone marrow-derived macrophages from Cyp27a1-/- and Cyp27a1-/-Cyp46a1-/- mice. CYP46A1 expression was detected in retinal endothelial cells, and this expression was increased in the proinflammatory environment. Retinal Cyp46a1-/- phosphoproteome revealed altered phosphorylation of 30 different proteins, including tight junction protein zonula occludens 1 and aquaporin 4. Collectively, the data obtained establish metabolic and regulatory significance of CYP46A1 for the retina and suggest pharmacologic activation of CYP46A1 as a potential therapeutic approach to dyslipidemia-induced retinal damage.

Reassessment of temperature-humidity index for measuring heat stress in crossbred dairy cattle of a sub-tropical region.

The temperature-humidity index (THI) has been extensively applied for assessing heat stress in moderate to hot conditions in dairy cattle. However, there exist wide variation between researchers in defining an appropriate range of THI values for denoting different levels of stress. The present study was aimed to reassess previously described heat stress indicators of dairy cattle of sub-tropical region of India. From comparative evaluation of meteorological data over previous four years (2014-2017) the period of year when high THI prevailed in the region was determined. Accordingly, the time period of sample collection and observation on animals was decided, so that a THI range of 68-86 could be covered. After analyzing physiological, biochemical parameters and expression profile of heat shock response (HSR) genes of animals in response to different THI, it was evident from the study that animal undergoes few or little changes at THI 72, but major physiological changes occurred after THI reached 74. At THI range 74-79, no drastic change in these parameters occurred suggesting animals undergo transient acclimatization in this range to maintain homeostasis. Once THI reached and crossed 80, this homeostasis was perturbed and animals experienced major physiological changes again. Overall, the study suggests that THI values indicating level of heat stress are dependent on the geographic location, as well as type of animal and therefore, existing THI should be recalibrated for different climatic region for accurate assessment of the heat stress.

The K153Del PRPH2 mutation differentially impacts photoreceptor structure and function.

Peripherin 2 (Prph2) is a photoreceptor tetraspanin, and deletion of codon 153 (K153Δ) leads to retinitis pigmentosa, pattern dystrophy, and fundus flavimaculatus in the same family. To study this variability, we generated a K153Δ-Prph2 knockin mouse. K153Δ-Prph2 cannot form the complexes required for outer segment formation, and in cones cannot interact with its binding partner rod outer segment membrane protein 1. K153Δ causes dominant defects in rod and cone function; however, rod but not cone ultrastructure is improved by the presence of K153Δ-Prph2. Likewise, supplementation of K153Δ heterozygotes with WT-Prph2 results in structural but not functional improvements. These results support the idea that mutations may differentially affect Prph2's role as a structural component, and its role as a functional protein key for organizing membrane domains for cellular signalling. These roles may be different in rods and cones, thus contributing to the phenotypic heterogeneity that characterizes diseases associated with Prph2 mutations.

A Dominant Mutation in Rpe65, D477G, Delays Dark Adaptation and Disturbs the Visual Cycle in the Mutant Knock-In Mice.

RPE65 is an indispensable component of the retinoid visual cycle in vertebrates, through which the visual chromophore 11-cis-retinal (11-cis-RAL) is generated to maintain normal vision. Various blinding conditions in humans, such as Leber congenital amaurosis and retinitis pigmentosa (RP), are attributed to either homozygous or compound heterozygous mutations in RPE65. Herein, we investigated D477G missense mutation, an unprecedented dominant-acting mutation of RPE65 identified in patients with autosomal dominant RP. We generated a D477G knock-in (KI) mouse and characterized its phenotypes. Although RPE65 protein levels were decreased in heterozygous KI mice, their scotopic, maximal, and photopic electroretinography responses were comparable to those of wild-type (WT) mice in stationary condition. As shown by high-performance liquid chromatography analysis, levels of 11-cis-RAL in fully dark-adapted heterozygous KI mice were similar to that in WT mice. However, kinetics of 11-cis-RAL regeneration after light exposure were significantly slower in heterozygous KI mice compared with WT and RPE65 heterozygous knockout mice. Furthermore, heterozygous KI mice exhibited lower A-wave recovery compared with WT mice after photobleaching, suggesting a delayed dark adaptation. Taken together, these observations suggest that D477G acts as a dominant-negative mutant of RPE65 that delays chromophore regeneration. The KI mice provide a useful model for further understanding of the pathogenesis of RP associated with this RPE65 mutant and for the development of therapeutic strategies.

Insights into the mechanisms of macular degeneration associated with the R172W mutation in RDS.

Mutations in the photoreceptor tetraspanin gene peripherin-2/retinal degeneration slow (PRPH2/RDS) cause both rod- and cone-dominant diseases. While rod-dominant diseases, such as autosomal dominant retinitis pigmentosa, are thought to arise due to haploinsufficiency caused by loss-of-function mutations, the mechanisms underlying PRPH2-associated cone-dominant diseases are unclear. Here we took advantage of a transgenic mouse line expressing an RDS mutant (R172W) known to cause macular degeneration (MD) in humans. To facilitate the study of cones in the heavily rod-dominant mouse retina, R172W mice were bred onto an Nrl(-/-) background (in which developing rods adopt a cone-like fate). In this model the R172W protein and the key RDS-binding partner, rod outer segment (OS) membrane protein 1 (ROM-1), were properly expressed and trafficked to cone OSs. However, the expression of R172W led to dominant defects in cone structure and function with equal effects on S- and M-cones. Furthermore, the expression of R172W in cones induced subtle alterations in RDS/ROM-1 complex assembly, specifically resulting in the formation of abnormal, large molecular weight ROM-1 complexes. Fundus imaging demonstrated that R172W mice developed severe clinical signs of disease nearly identical to those seen in human MD patients, including retinal degeneration, retinal pigment epithlium (RPE) defects and loss of the choriocapillaris. Collectively, these data identify a primary disease-causing molecular defect in cone cells and suggest that RDS-associated disease in patients may be a result of this defect coupled with secondary sequellae involving RPE and choriocapillaris cell loss.

A Dhdds K42E knock-in RP59 mouse model shows inner retina pathology and defective synaptic transmission.

Retinitis pigmentosa (RP) defines a group of hereditary progressive rod-cone degenerations that exhibit a common phenotype caused by variants in over 70 genes. While most variants in the dehydrodolichyl diphosphate synthase (DHDDS) gene result in syndromic abnormalities, some variants cause non-syndromic RP (RP59). DHDDS encodes one subunit of the enzyme cis-prenyltransferase (CPT), which is required for the synthesis of dolichol (Dol), that is a necessary protein glycosylation cofactor. We previously reported the creation and initial characterization of a knock-in (KI) mouse model harboring the most prevalent RP59-associated DHDDS variant (K42E) to understand how defects in DHDDS lead to retina-specific pathology. This model exhibited no profound retinal degeneration, nor protein N-glycosylation defects. Here, we report that the Dol isoprenylogue species in retina, liver, and brain of the K42E mouse model are statistically shorter than in the corresponding tissues of age-matched controls, as reported in blood and urine of RP59 patients. Retinal transcriptome analysis demonstrated elevation of many genes encoding proteins involved in synaptogenesis and synaptic function. Quantitative retinal cell layer thickness measurements demonstrated a significant reduction in the inner nuclear layer (INL) and total retinal thickness (TRT) beginning at postnatal (PN) ∼2 months, progressively increasing to PN 18-mo. Histological analysis revealed cell loss in the INL, outer plexiform layer (OPL) disruption, and ectopic localization of outer nuclear layer (ONL) nuclei into the OPL of K42E mutant retinas, relative to controls. Electroretinograms (ERGs) of mutant mice exhibited reduced b-wave amplitudes beginning at PN 1-mo, progressively declining through PN 18-mo, without appreciable a-wave attenuation, relative to controls. Our results suggest that the underlying cause of DHDDS K42E variant driven RP59 retinal pathology is defective synaptic transmission from outer to inner retina.

Sustained ACE2 Expression by Probiotic Improves Integrity of Intestinal Lymphatics and Retinopathy in Type 1 Diabetic Model.

Intestinal lymphatic, known as lacteal, plays a critical role in maintaining intestinal homeostasis by regulating several key functions, including the absorption of dietary lipids, immune cell trafficking, and interstitial fluid balance in the gut. The absorption of dietary lipids relies on lacteal integrity, mediated by button-like and zipper-like junctions. Although the intestinal lymphatic system is well studied in many diseases, including obesity, the contribution of lacteals to the gut-retinal axis in type 1 diabetes (T1D) has not been examined. Previously, we showed that diabetes induces a reduction in intestinal angiotensin-converting enzyme 2 (ACE2), leading to gut barrier disruption. However, when ACE2 levels are maintained, a preservation of gut barrier integrity occurs, resulting in less systemic inflammation and a reduction in endothelial cell permeability, ultimately retarding the development of diabetic complications, such as diabetic retinopathy. Here, we examined the impact of T1D on intestinal lymphatics and circulating lipids and tested the impact of intervention with ACE-2-expressing probiotics on key aspects of gut and retinal function. Akita mice with 6 months of diabetes were orally gavaged LP-ACE2 (3x/week for 3 months), an engineered probiotic (Lactobacillus paracasei; LP) expressing human ACE2. After three months, immunohistochemistry (IHC) was used to evaluate intestinal lymphatics, gut epithelial, and endothelial barrier integrity. Retinal function was assessed using visual acuity, electroretinograms, and enumeration of acellular capillaries. LP-ACE2 significantly restored intestinal lacteal integrity as assessed by the increased expression of lymphatic vessel hyaluronan receptor 1 (LYVE-1) expression in LP-ACE2-treated Akita mice. This was accompanied by improved gut epithelial (Zonula occludens-1 (ZO-1), p120-catenin) and endothelial (plasmalemma vesicular protein -1 (PLVAP1)) barrier integrity. In Akita mice, the LP-ACE2 treatment reduced plasma levels of LDL cholesterol and increased the expression of ATP-binding cassette subfamily G member 1 (ABCG1) in retinal pigment epithelial cells (RPE), the population of cells responsible for lipid transport from the systemic circulation into the retina. LP-ACE2 also corrected blood-retinal barrier (BRB) dysfunction in the neural retina, as observed by increased ZO-1 and decreased VCAM-1 expression compared to untreated mice. LP-ACE2-treated Akita mice exhibit significantly decreased numbers of acellular capillaries in the retina. Our study supports the beneficial role of LP-ACE2 in the restoration of intestinal lacteal integrity, which plays a key role in gut barrier integrity and systemic lipid metabolism and decreased diabetic retinopathy severity.

Differences in RDS trafficking, assembly and function in cones versus rods: insights from studies of C150S-RDS.

Cysteine 150 of retinal degeneration slow protein (RDS) mediates the intermolecular disulfide bonding necessary for large RDS complex assembly and morphogenesis of the rim region of photoreceptor outer segments. Previously, we showed that cones have a different requirement for RDS than rods, but the nature of that difference was unclear. Here, we express oligomerization-incompetent RDS (C150S-RDS) in the cone-dominant nrl(-/-) mouse. Expression of C150S-RDS leads to dominant functional abnormalities, ultrastructural changes, biochemical anomalies and protein mislocalization in cones. These data suggest that RDS complexes in cones are more susceptible to disruption than those in rods, possibly due to structural or microenvironmental differences in the two cell types. Furthermore, our results suggest that RDS intermolecular disulfide bonding may be part of RDS inner-segment assembly in cones but not in rods. These data highlight significant differences in assembly, trafficking and function of RDS in rods versus cones.

Applications of Omics Technology for Livestock Selection and Improvement.

Conventional animal selection and breeding methods were based on the phenotypic performance of the animals. These methods have limitations, particularly for sex-limited traits and traits expressed later in the life cycle (e.g., carcass traits). Consequently, the genetic gain has been slow with high generation intervals. With the advent of high-throughput omics techniques and the availability of multi-omics technologies and sophisticated analytic packages, several promising tools and methods have been developed to estimate the actual genetic potential of the animals. It has now become possible to collect and access large and complex datasets comprising different genomics, transcriptomics, proteomics, metabolomics, and phonemics data as well as animal-level data (such as longevity, behavior, adaptation, etc.,), which provides new opportunities to better understand the mechanisms regulating animals' actual performance. The cost of omics technology and expertise of several fields like biology, bioinformatics, statistics, and computational biology make these technology impediments to its use in some cases. The population size and accurate phenotypic data recordings are other significant constraints for appropriate selection and breeding strategies. Nevertheless, omics technologies can estimate more accurate breeding values (BVs) and increase the genetic gain by assisting the section of genetically superior, disease-free animals at an early stage of life for enhancing animal productivity and profitability. This manuscript provides an overview of various omics technologies and their limitations for animal genetic selection and breeding decisions.

Microbial Signatures in The Rodent Eyes With Retinal Dysfunction and Diabetic Retinopathy.

Purpose: The gut microbiome has been linked to disease pathogenesis through their interaction in metabolic, endocrine, and immune functions. The goal of this study was to determine whether the gut and plasma microbiota could transfer microbes to the retina in type 1 diabetic mice with retinopathy. Methods: We analyzed the fecal, plasma, whole globe, and retina microbiome in Akita mice and compared with age-matched wild-type (WT) mice using 16S rRNA sequencing and metatranscriptomic analysis. To eliminate the contribution of the ocular surface and plasma microbiome, mice were perfused with sterile saline solution, the whole globes were extracted, and the neural retina was removed under sterile conditions for retinal microbiome. Results: Our microbiome analysis revealed that Akita mice demonstrated a distinct pattern of microbes within each source: feces, plasma, whole globes, and retina. WT mice and Akita mice experienced transient bacteremia in the plasma and retina. Bacteria were identified in the retina of the Akita mice, specifically Corynebacterium, Pseudomonas, Lactobacillus, Staphylococcus, Enterococcus, and Bacillus. Significantly increased levels of peptidoglycan (0.036 ± 0.001 vs. 0.023 ± 0.002; P < 0.002) and TLR2 (3.47 ± 0.15 vs. 1.99 ± 0.07; P < 0.0001) were observed in the retina of Akita mice compared to WT. Increased IBA+ cells in the retina, reduced a- and b-waves on electroretinography, and increased acellular capillary formation demonstrated the presence of retinopathy in the Akita cohort compared to WT mice. Conclusions: Together, our findings suggest that transient bacteremia exists in the plasma and retina of both cohorts. The bacteria found in Akita mice are distinct from WT mice and may contribute to development of retinal inflammation and barrier dysfunction in retinopathy.

Specific mesoderm subset derived from human pluripotent stem cells ameliorates microvascular pathology in type 2 diabetic mice.

Human induced pluripotent stem cells (hiPSCs) were differentiated into a specific mesoderm subset characterized by KDR+CD56+APLNR+ (KNA+) expression. KNA+ cells had high clonal proliferative potential and specification into endothelial colony-forming cell (ECFCs) phenotype. KNA+ cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient mice and when injected into the vitreous of type 2 diabetic mice (db/db mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA+ cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA+ cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of db/db mice injected with either control or diabetic donor-derived KNA+ cells showed correction of aberrant signaling in db/db retinas toward normal healthy retina. These data provide "proof of principle" that KNA+ cells restore perfusion and correct vascular dysfunction in db/db mice.

Differential requirements for retinal degeneration slow intermolecular disulfide-linked oligomerization in rods versus cones.

It is commonly assumed that the ultrastructural organization of the rim region of outer segment (OS) discs in rods and lamellae in cones requires functional retinal degeneration slow/rod outer segment membrane protein 1 (Rds/Rom-1) complexes. Cysteine-150 (C150) in Rds has been implicated in intermolecular disulfide bonding essential for functional Rds complexes. Transgenic mice containing the Rds C150S mutation (C150S-Rds) failed to form higher-order Rds oligomers, although interactions between C150S-Rds and Rom-1 occurred in rods, but not in cones. C150S-Rds mice exhibited marked early-onset reductions in cone function and abnormal OS structure. In contrast, C150S-Rds expression in rods partly rescued the rds(+/-) phenotype. Although C150S-Rds was detected in the OSs in rods and cones, a substantial percentage of C150S-Rds and cone opsins were mislocalized to different cellular compartments in cones. The results of this study provide novel insights into the importance of C150 in Rds oligomerization and the differences in Rds requirements in rods versus cones. The apparent OS structural differences between rods and cones may cause cones to be more susceptible to the elimination of higher-order Rds/Rom-1 oligomers (e.g. as mediated by mutation of the Rds C150 residue).

The function of oligomerization-incompetent RDS in rods.

The photoreceptor-specific tetraspanin glycoprotein RDS (retinal degeneration slow) is associated with many forms of inherited retinal disease. RDS shares features in common with other tetraspanin proteins, including the existence of a large intradiscal D2 loop containing several cysteines. While these cysteines are used only for intramolecular disulfide bonds in most tetraspanins, RDS expresses a seventh, unpaired cysteine (C150) used for intermolecular disulfide bonding in the formation of large RDS oligomers. To study oligomerization-dependent vs. oligomerization-independent RDS functions in rods, we generated a transgenic mouse line harboring a point mutation that replaces this Cys with Ser (C150S), leading to the expression of an RDS protein that cannot form intermolecular disulfide bonds. The mouse opsin promoter (MOP) was used to direct C150S RDS expression specifically in rods in these transgenic mice (MOP-T). Here we report improvement in scotopic ERGs in MOP-T/rds ( +/- ) mice (compared to non-transgenic rds ( +/- ) controls) and the appearance of malformed outer segments (OSs) in MOP-T mice that do not express native RDS (MOP-T/rds ( -/- )). These results suggest that while normal OS structure and function require RDS oligomerization, some RDS function is retained in the absence of C150. Since one of the functions of other tetraspanin proteins is to promote assembly of a membrane microdomain known as the "tetraspanin web", future studies may investigate whether assembly of this web is one of RDS's oligomerization-independent functions.

Characterizing the Retinal Phenotype in the High-Fat Diet and Western Diet Mouse Models of Prediabetes.

We sought to delineate the retinal features associated with the high-fat diet (HFD) mouse, a widely used model of obesity. C57BL/6 mice were fed either a high-fat (60% fat; HFD) or low-fat (10% fat; LFD) diet for up to 12 months. The effect of HFD on body weight and insulin resistance were measured. The retina was assessed by electroretinogram (ERG), fundus photography, permeability studies, and trypsin digests for enumeration of acellular capillaries. The HFD cohort experienced hypercholesterolemia when compared to the LFD cohort, but not hyperglycemia. HFD mice developed a higher body weight (60.33 g vs. 30.17g, p < 0.0001) as well as a reduced insulin sensitivity index (9.418 vs. 62.01, p = 0.0002) compared to LFD controls. At 6 months, retinal functional testing demonstrated a reduction in a-wave and b-wave amplitudes. At 12 months, mice on HFD showed evidence of increased retinal nerve infarcts and vascular leakage, reduced vascular density, but no increase in number of acellular capillaries compared to LFD mice. In conclusion, the HFD mouse is a useful model for examining the effect of prediabetes and hypercholesterolemia on the retina. The HFD-induced changes appear to occur slower than those observed in type 2 diabetes (T2D) models but are consistent with other retinopathy models, showing neural damage prior to vascular changes.