Analytical Post-Embedding Immunogold-Electron Microscopy with Direct Gold-Labelled Monoclonal Primary Antibodies against RIBEYE A- and B-Domain Suggests a Refined Model of Synaptic Ribbon Assembly.

Synaptic ribbons are the eponymous specializations of continuously active ribbon synapses. They are primarily composed of the RIBEYE protein that consists of a unique amino-terminal A-domain and carboxy-terminal B-domain that is largely identical to the ubiquitously expressed transcriptional regulator protein CtBP2. Both RIBEYE A-domain and RIBEYE B-domain are essential for the assembly of the synaptic ribbon, as shown by previous analyses of RIBEYE knockout and knockin mice and related investigations. How exactly the synaptic ribbon is assembled from RIBEYE subunits is not yet clear. To achieve further insights into the architecture of the synaptic ribbon, we performed analytical post-embedding immunogold-electron microscopy with direct gold-labelled primary antibodies against RIBEYE A-domain and RIBEYE B-domain for improved ultrastructural resolution. With direct gold-labelled monoclonal antibodies against RIBEYE A-domain and RIBEYE B-domain, we found that both domains show a very similar localization within the synaptic ribbon of mouse photoreceptor synapses, with no obvious differential gradient between the centre and surface of the synaptic ribbon. These data favour a model of the architecture of the synaptic ribbon in which the RIBEYE A-domain and RIBEYE B-domain are located similar distances from the midline of the synaptic ribbon.

Modeling of Ethiopian Beef Meat Marbling Score Using Image Processing for Rapid Meat Grading.

Meat characterized by a high marbling value is typically anticipated to display enhanced sensory attributes. This study aimed to predict the marbling scores of rib-eye, steaks sourced from the Longissimus dorsi muscle of different cattle types, namely Boran, Senga, and Sheko, by employing digital image processing and machine-learning algorithms. Marbling was analyzed using digital image processing coupled with an extreme gradient boosting (GBoost) machine learning algorithm. Meat texture was assessed using a universal texture analyzer. Sensory characteristics of beef were evaluated through quantitative descriptive analysis with a trained panel of twenty. Using selected image features from digital image processing, the marbling score was predicted with R2 (prediction) = 0.83. Boran cattle had the highest fat content in sirloin and chuck cuts (12.68% and 12.40%, respectively), followed by Senga (11.59% and 11.56%) and Sheko (11.40% and 11.17%). Tenderness scores for sirloin and chuck cuts differed among the three breeds: Boran (7.06 ± 2.75 and 3.81 ± 2.24, respectively), Senga (5.54 ± 1.90 and 5.25 ± 2.47), and Sheko (5.43 ± 2.76 and 6.33 ± 2.28 Nmm). Sheko and Senga had similar sensory attributes. Marbling scores were higher in Boran (4.28 ± 1.43 and 3.68 ± 1.21) and Senga (2.88 ± 0.69 and 2.83 ± 0.98) compared to Sheko (2.73 ± 1.28 and 2.90 ± 1.52). The study achieved a remarkable milestone in developing a digital tool for predicting marbling scores of Ethiopian beef breeds. Furthermore, the relationship between quality attributes and beef marbling score has been verified. After further validation, the output of this research can be utilized in the meat industry and quality control authorities.

A Novel Cre Recombinase Mouse Strain for Cell-Specific Deletion of Floxed Genes in Ribbon Synapse-Forming Retinal Neurons.

We generated a novel Cre mouse strain for cell-specific deletion of floxed genes in ribbon synapse-forming retinal neurons. Previous studies have shown that the RIBEYE promotor targets the expression of recombinant proteins such as fluorescently tagged RIBEYE to photoreceptors and retinal bipolar cells and generates fluorescent synaptic ribbons in situ in these neurons. Here, we used the same promotor to generate a novel transgenic mouse strain in which the RIBEYE promotor controls the expression of a Cre-ER(T2) recombinase (RIBEYE-Cre). To visualize Cre expression, the RIBEYE-Cre animals were crossed with ROSA26 tau-GFP (R26-τGFP) reporter mice. In the resulting RIBEYE-Cre/R26 τGFP animals, Cre-mediated removal of a transcriptional STOP cassette results in the expression of green fluorescent tau protein (tau-GFP) that binds to cellular microtubules. We detected robust tau-GFP expression in retinal bipolar cells. Surprisingly, we did not find fluorescent tau-GFP expression in mouse photoreceptors. The lack of tau-GFP reporter protein in these cells could be based on the previously reported absence of tau protein in mouse photoreceptors which could lead to the degradation of the recombinant tau protein. Consistent with this, we detected Cre and tau-GFP mRNA in mouse photoreceptor slices by RT-PCR. The transgenic RIBEYE-Cre mouse strain provides a new tool to study the deletion of floxed genes in ribbon synapse-forming neurons of the retina and will also allow for analyzing gene deletions that are lethal if globally deleted in neurons.

Prediction models of carcass characteristics from non­castrated Nellore cattle finished in the feedlot system under tropical conditions.

Our objective was to use measures of intake and productive performance to adjust prediction models for the carcass traits of non-castrated Nellore cattle finished in a feedlot. Individual data from 168 non-castrated male Nellore steers finished in feedlot between the years 2016-2021 were used. Descriptive statistical analyzes and Pearson correlation coefficients were performed. The outliers were tested by evaluating the studentized residuals in relation to the values predicted by the equations. Residues that were outside the range of -2.5 to 2.5 were removed. The goodness of fit of the developed equations was evaluated by the coefficients of determination (R2) and root mean square error (RMSE). Models for carcass yield, subcutaneous fat thickness, ribeye area, and shear force were adjusted. Means of 53.5% carcass yield, 4.8 mm subcutaneous fat thickness, 73 cm2 loin eye area, and 8.1 kg shear force were observed. The observed average intakes were 9.9 kg/day of dry matter, 3.3 kg/day of neutral detergent fiber content, 1.5 kg/day of crude protein, and 7.1 kg/day of total digestible nutrients. The average confinement time was 113 days, the average total weight gain was 152.2 kg and the average daily gain was 1.35 kg/day. Intake measures significantly correlated with shear force and subcutaneous fat thickness and ribeye area. Carcass yield was significantly correlated with total weight gain, feedlot time, and hot carcass weight. Measures of nutrient intake, performance, and confinement time can be used as predictors of carcass yield, ribeye area, fat thickness, and shear force of non-castrated Nellore cattle finished in a feedlot. The prediction equations for ribeye area, carcass yield, subcutaneous fat thickness, and shear force showed sufficient precision and accuracy for non-castrated Nellore cattle finished in confinement systems under tropical conditions. All equations can be used with caution to estimate carcass traits of cattle finished in a feedlot using measures of intake and productive performance.

Differential expressions of FASN, SCD, and FABP4 genes in the ribeye muscle of omega-3 oil-supplemented Tattykeel Australian White lambs.

BACKGROUND: The concept of the functional nutritional value of health-beneficial omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) is becoming a phenomenon among red meat consumers globally. This study examined the expressions of three lipogenic genes (fatty acid binding protein 4, FABP4, fatty acid synthase, FASN; and stearoyl-CoA desaturase, SCD) in the ribeye (Longissimus thoracis et lumborum) muscle of Tattykeel Australian White (TAW) lambs fed fortified omega-3 diets and correlations with fatty acids. To answer the research question, "are there differences in the expression of lipogenic genes between control, MSM whole grain and omega-3 supplemented lambs?", we tested the hypothesis that fortification of lamb diets with omega-3 will lead to a down-regulation of lipogenic genes. Seventy-five six-month old TAW lambs were randomly allocated to the (1) omega-3 oil-fortified grain pellets, (2) unfortified grain pellets (control) or (3) unfortified MSM whole grain pellets diet supplements to generate three treatments of 25 lambs each. The feeding trial lasted 47 days. RESULTS: From the Kruskal-Wallis test, the results showed a striking disparity in lipogenic gene expression between the three dietary treatments in which the FABP4 gene was significantly up-regulated by 3-folds in the muscles of lambs fed MSM Milling (MSM) whole grain diet compared to the omega-3 and control diets. A negative correlation was observed between FASN gene expression and intramuscular fat (IMF), eicosapentaenoic acid (EPA), total polyunsaturated fatty acids (PUFA), omega-6 polyunsaturated fatty acids (n-6 PUFA) and monounsaturated fatty acids (MUFA). The FABP4 gene expression was positively correlated (P < 0.05) with EPA and docosahexaenoic acid (DHA). CONCLUSION: Taken together, this study's results suggest that FABP4 and FASN genes perform an important role in the biosynthesis of fatty acids in the ribeye muscle of TAW lambs, and supplementary diet composition is an important factor influencing their expressions.

Effects of Shear Stress Waves on Meat Tenderness: Ultrasonoporation.

Meat is an important part of the food pyramid in Mexico, to such an extent that it is included in the basic food basket. In recent years, there has been great interest in the application of so-called emerging technologies, such as high-intensity ultrasound (HIU), to modify the characteristics of meat and meat products. The advantages of the HIU in meat such as pH, increased water-holding capacity, and antimicrobial activity are well documented and conclusive. However, in terms of meat tenderization, the results are confusing and contradictory, mainly when they focus on three HIU parameters: acoustic intensity, frequency, and application time. This study explores via a texturometer the effect of HIU-generated acoustic cavitation and ultrasonoporation in beef (m. Longissimus dorsi). Loin-steak was ultrasonicated with the following parameters: time tHIU = 30 min/each side; frequency fHIU = 37 kHz; acoustic intensity IHIU = ~6, 7, 16, 28, and 90 W/cm2. The results showed that acoustic cavitation has a chaotic effect on the loin-steak surface and thickness of the rib-eye due to Bjerknes force, generating shear stress waves, and acoustic radiation transmittance via the internal structure of the meat and the modification of the myofibrils, in addition to the collateral effect in which the collagen and pH generated ultrasonoporation. This means that HIU can be beneficial for the tenderization of meat.

Muscle mineral profile of water buffaloes (Bubalus bubalis) reared in different production systems of the Brazilian Eastern Amazon.

Healthy food must have an adequate balance of macroelements, such as calcium or phosphorus and, microelements, such as iron, copper. This study aimed to evaluate the influence of three extensive systems, during the dry and rainy seasons, and an intensive (feedlot) system in the Eastern Amazon, on the muscle mineral profile of water buffaloes. In total, 12 male buffaloes, aged between 24 and 36 months, slaughtered in commercial slaughterhouses, were used in each of the systems considered: Marajó island, Santarém, Nova Timboteua, and a feedlot. Approximately 5 g of muscle was collected, stored, and frozen, until freeze-dried. The samples were analyzed for the mineral profile using inductively coupled plasma-optical emission spectrometry (ICP-OES). There were significant differences (P < 0.05) for concentrations of sodium (Na), magnesium (Mg), phosphorus (P), sulfur (S), copper (Cu), zinc (Zn), and iron (Fe). Extensive and intensive systems showed significant differences (P < 0.05) for Na, Ca, S, Cu, and Fe concentrations. The season also influenced (P < 0.05) K, Ca, P, S, Zn, and Fe concentrations. The location and season of the year had a significant interaction (P < 0.05) for K, Mg, P, Zn, and Fe concentrations. The study showed that the different Amazonian production systems and the year season influenced the levels of minerals present in buffalo muscle. The values obtained were, in general, higher in extensive production systems, and Marajó Island stood out with higher mineral values in the dry season. Therefore, the meat from animals reared in these systems is a good mineral source for daily human needs.

Embryonic Hyperglycemia Delays the Development of Retinal Synapses in a Zebrafish Model.

Embryonic hyperglycemia negatively impacts retinal development, leading to abnormal visual behavior, altered timing of retinal progenitor differentiation, decreased numbers of retinal ganglion cells and Müller glia, and vascular leakage. Because synaptic disorganization is a prominent feature of many neurological diseases, the goal of the current work was to study the potential impact of hyperglycemia on retinal ribbon synapses during embryonic development. Our approach utilized reverse transcription quantitative PCR (RT-qPCR) and immunofluorescence labeling to compare the transcription of synaptic proteins and their localization in hyperglycemic zebrafish embryos, respectively. Our data revealed that the maturity of synaptic ribbons was compromised in hyperglycemic zebrafish larvae, where altered ribeye expression coincided with the delay in establishing retinal ribbon synapses and an increase in the immature synaptic ribbons. Our results suggested that embryonic hyperglycemia disrupts retinal synapses by altering the development of the synaptic ribbon, which can lead to visual defects. Future studies using zebrafish models of hyperglycemia will allow us to study the underlying mechanisms of retinal synapse development.

Carcass Traits of Growing Meat Goats Fed Different Levels of Hempseed Meal.

Hempseed meal (HSM) is the byproduct of hemp seeds and is rich in crude protein and fiber, making it an ideal candidate as a feedstuff for ruminants. The objective of the present study is to evaluate the effects of feeding different levels of HSM on the carcass traits of crossbred Boer goats. Forty castrated goat kids (approximately six months, 25.63 ± 0.33 kg) were assigned to one of four treatments (n = 10) in a completely randomized design. Goats were fed pelleted diets (50% forage and 50% concentrate) with additional supplementation of HSM: control with 0%, 10%, 20%, and 30% of the total diets. Goats were harvested and processed after a 60-day feeding trial. There were no significant differences (p > 0.05) in the mean values of dressing percentages, carcass weights, body wall thickness, and ribeye area among treatments. Marbling scores and percentages of moisture, fats, proteins, and collagen in the muscles showed no significant differences (p > 0.05) among the treatments. Results suggest that including up to 30% of HSM in the diet of growing meat goats does not affect their carcass traits.

RIBEYE B-Domain Is Essential for RIBEYE A-Domain Stability and Assembly of Synaptic Ribbons.

Synaptic ribbons are presynaptic specializations that define eponymous ribbon synapses. Synaptic ribbons are largely composed of RIBEYE, a protein containing an N-terminal A-domain and a carboxyterminal B-domain that is identical with CtBP2, a NAD(H)-binding transcriptional co-repressor. Previously we showed that synaptic ribbons are completely absent in RIBEYE knockout mice in which the RIBEYE A-domain-encoding exon had been deleted, but CtBP2 is still made, demonstrating that the A-domain is required for synaptic ribbon assembly. In the present study, we asked whether the RIBEYE B-domain also has an essential role in the assembly of synaptic ribbons. For this purpose, we made use of RIBEYE knockin mice in which the RIBEYE B-domain was replaced by a fluorescent protein domain, whereas the RIBEYE A-domain was retained unchanged. We found that replacing the RIBEYE B-domain with a fluorescent protein module destabilizes the resulting hybrid protein and causes a complete loss of synaptic ribbons. Our results thus demonstrate an essential role of the RIBEYE B-domain in enabling RIBEYE assembly into synaptic ribbons, reinforcing the notion that RIBEYE is the central organizer of synaptic ribbons.

Carcass and Primal Composition Predictions Using Camera Vision Systems (CVS) and Dual-Energy X-ray Absorptiometry (DXA) Technologies on Mature Cows.

This study determined the potential of computer vision systems, namely the whole-side carcass camera (HCC) compared to the rib-eye camera (CCC) and dual energy X-ray absorptiometry (DXA) technology to predict primal and carcass composition of cull cows. The predictability (R2) of the HCC was similar to the CCC for total fat, but higher for lean (24.0%) and bone (61.6%). Subcutaneous fat (SQ), body cavity fat, and retail cut yield (RCY) estimations showed a difference of 6.2% between both CVS. The total lean meat yield (LMY) estimate was 22.4% better for CCC than for HCC. The combination of HCC and CCC resulted in a similar prediction of total fat, SQ, and intermuscular fat, and improved predictions of total lean and bone compared to HCC/CCC. Furthermore, a 25.3% improvement was observed for LMY and RCY estimations. DXA predictions showed improvements in R2 values of 26.0% and 25.6% compared to the HCC alone or the HCC + CCC combined, respectively. These results suggest the feasibility of using HCC for predicting primal and carcass composition. This is an important finding for slaughter systems, such as those used for mature cattle in North America that do not routinely knife rib carcasses, which prevents the use of CCC.

Genetic parameters for performance, feed efficiency, and carcass traits in Senepol heifers.

Improving feed efficiency is a key breeding goal in the beef cattle industry. In this study, we estimated the genetic parameters for feed efficiency and carcass traits in Senepol cattle raised in tropical regions. Various indicators of feed efficiency [gain to feed ratio (G:F), feed conversion ratio (FCR), residual weight gain (RG), residual intake and body weight gain (RIG), and residual feed intake (RFI)] as well as growth [final BW, average daily gain (ADG), and DM intake (DMI)], and carcass [rib-eye area (REA), backfat thickness (BF), intramuscular fat score, and carcass conformation score] traits were included in the study. After data editing, records from 1 393 heifers obtained between 2009 and 2018 were used for the analyses. We fitted an animal model that included contemporary group (animals from the same farm that were evaluated in the same test season) as the fixed effect, and a linear effect of animal age at the beginning of the test as a covariate; in addition to random direct additive genetic and residual effects. The (co)variance components were estimated by Bayesian inference in uni- and bivariate analyses. Our results showed that feed efficiency indicators derived from residual variables such as RG, RIG, and RFI can be improved through genetic selection (h2 = 0.14 ± 0.06, 0.13 ± 0.06, and 0.20 ± 0.08, respectively). Variables calculated as ratios such as G:F and FCR were more influenced by environmental factors (h2 = 0.08 ± 0.05 and 0.09 ± 0.05), and were, therefore, less suitable for use in breeding programs. The traits with the greatest and impact on genetic progress in feed efficiency were ADG, REA, and BF. The traits with the greatest and least impact on growth and carcass traits were RG and RFI, respectively. Selection for feed efficiency will result in distinct overall effects on the growth and carcass traits of Senepol heifers. Direct selection for lower RFI may reduce DMI and increase carcass fatness at the finishing stage, but it might also result in reduced growth and muscle deposition. Residual BW gain is associated with the highest weight gain and zero impact on REA and BF, however, it is linked to higher feed consumption. Thus, the most suitable feed efficiency indicator was RIG, as it promoted the greatest decrease in feed intake concomitant with faster growth, with a similar impact on carcass traits when compared to the other feed efficiency indicators.

Temporal and Isoform-Specific Expression of CTBP2 Is Evolutionarily Conserved Between the Developing Chick and Human Retina.

Complex transcriptional gene regulation allows for multifaceted isoform production during retinogenesis, and novel isoforms transcribed from a single locus can have unlimited potential to code for diverse proteins with different functions. In this study, we explored the CTBP2/RIBEYE gene locus and its unique repertoire of transcripts that are conserved among vertebrates. We studied the transcriptional coregulator (CTBP2) and ribbon synapse-specific structural protein (RIBEYE) in the chicken retina by performing comprehensive histochemical and sequencing analyses to pinpoint cell and developmental stage-specific expression of CTBP2/RIBEYE in the developing chicken retina. We demonstrated that CTBP2 is widely expressed in retinal progenitors beginning in early retinogenesis but becomes limited to GABAergic amacrine cells in the mature retina. Inversely, RIBEYE is initially epigenetically silenced in progenitors and later expressed in photoreceptor and bipolar cells where they localize to ribbon synapses. Finally, we compared CTBP2/RIBEYE regulation in the developing human retina using a pluripotent stem cell derived retinal organoid culture system. These analyses demonstrate that similar regulation of the CTBP2/RIBEYE locus during chick and human retinal development is regulated by different members of the K50 homeodomain transcription factor family.

Effects of marbling on physical and sensory characteristics of ribeye steaks from four different cattle breeds.

OBJECTIVE: Marbling or intramuscular fat (IMF) has been widely reported to directly impact the sensory acceptance of meat. This study was carried out to determine the physical and sensory characteristics of ribeye, Longissimus dorsi steaks obtained from four different cattle breeds namely Wagyu, Angus, Brahman, and Malaysian local beef, the Kedah-Kelantan (KK). METHODS: The degree of marbling was determined by using an established combined camera-image analysis technique while instrumental texture determination was carried out by using Warner-Bratzler shear force analysis. Sensory evaluation of the beef steaks was performed following a quantitative descriptive analysis incorporating 10 trained consumer panelists. RESULTS: Wagyu was found to possess the highest (p<0.05) percentage of IMF at 33.90% and the lowest shear force (raw = 5.61 N/mm2; cooked = 14.72 N/mm2) followed by Angus (20.87%), Brahman (12.17%), and KK (p<0.05, 6.86%). The difference in sensory properties of the four steaks was evident, with Wagyu appearing to be highly correlated with most sensory attributes measured namely sustained buttery, tooth-packing, chewiness, juiciness, tenderness, mouthfeel, oiliness, and overall acceptability. The Malaysian local beef, KK was found to be less acceptable (p<0.05), although most of its sensory attributes were found similar (p>0.05) in appearance, aroma, texture, juiciness, and flavour to the cooked steak from Angus and Brahman. CONCLUSION: This present study demonstrated the role of IMF in determining the quality and sensory acceptance of beef from different cattle breeds. These data have provided new information and further understanding on the physical and sensory quality of Malaysian local beef.

Palatability assessments of beef strip loin steaks portioned by weight or by thickness sourced from various carcass weight/ribeye area size combinations.

Beef carcasses (n = 90; U.S. Choice) met a 3 ribeye area (REA - Small, Medium, Large) × 3 carcass weight (CW - Light, Intermediate, Heavy) scheme to assess palatability on steaks cut by portion thickness (PT- 3.18 cm) and weight (PW -340 g). Significant interactions revealed trends for steaks from the Small REA, regardless of CW, to have among the lowest shear-force values. For PT steaks, significant interaction for overall liking revealed no differences for Small and Medium REA across all CW categories, but steaks from Large REA from Light CW differed (P < 0.05) from the other two CW categories. For PT steaks, overall liking and tenderness liking scores were higher (P < 0.05) for Small REA compared to other categories, whereas CW did not influence any palatability trait. REA and CW do impact beef steak palatability, though steaks from all combinations were "very tender" and highly acceptable from a palatability standpoint.

LKB1 coordinates neurite remodeling to drive synapse layer emergence in the outer retina.

Structural changes in pre and postsynaptic neurons that accompany synapse formation often temporally and spatially overlap. Thus, it has been difficult to resolve which processes drive patterned connectivity. To overcome this, we use the laminated outer murine retina. We identify the serine/threonine kinase LKB1 as a key driver of synapse layer emergence. The absence of LKB1 in the retina caused a marked mislocalization and delay in synapse layer formation. In parallel, LKB1 modulated postsynaptic horizontal cell refinement and presynaptic photoreceptor axon growth. Mislocalized horizontal cell processes contacted aberrant cone axons in LKB1 mutants. These defects coincided with altered synapse protein organization, and horizontal cell neurites were misdirected to ectopic synapse protein regions. Together, these data suggest that LKB1 instructs the timing and location of connectivity in the outer retina via coordinate regulation of pre and postsynaptic neuron structure and the localization of synapse-associated proteins.

Deformation patterns in WorldSID 50th percentile dummy instrumented with IR-TRACC and RibEye™ in different loading configurations.

Objective: The purpose of this study was to investigate the effect of different loading configurations on the WorldSID 50th percentile male dummy instrumented either with the Infra-Red Telescoping Rod for the Assessment of Chest Compression (IR-TRACC) or the RibEye™ rib deflection measurement system. Methods: The optical sensors of the RibEye system were used to capture the multipoint deformation of the dummy at frontal and rearward off-center locations in addition to the center of the rib location. The experimental setup consisted of 2 types of loadings: Low severity and high severity. Low-severity loading was performed by deploying a fixture-mounted side airbag on the dummy and high-severity loading was achieved by deploying a driver front airbag mounted in a similar fashion. The low-severity condition aimed at deforming the dummy's ribs locally at off-center locations where the RibEye light emitting diodes (LEDs) were positioned to capture the deformations at those locations. The high-severity condition aimed at loading the dummy at high speed in lateral and oblique directions similar to what is experienced by dummies in side impacts. Results: In the low-severity tests, the peak deflections, in terms of length change, were approximately 15-20 mm, whereas for the high-severity cases the peak deflections were in the range of 30-40 mm for both IR-TRACC and RibEye cases. Conclusions: For similar physical insults, dummies with the IR-TRACC and RibEye systems showed varying results for both length changes and the shoulder forces depending on the severity and direction of loading. Under purely lateral loading, the mid-length changes with the RibEye and the 1D IR-TRACC were comparable. In the oblique loading conditions, more differences were seen with the 2 systems depending on the impact direction. The shoulder forces consistently differed between the 2 systems. In the frontal oblique low-severity cases, the ribs pivoted along the spine end and the length change was not found to be a suitable parameter to quantify rib deformation in such loading scenarios.

A Multiple Piccolino-RIBEYE Interaction Supports Plate-Shaped Synaptic Ribbons in Retinal Neurons.

Active zones at chemical synapses are highly specialized sites for the regulated release of neurotransmitters. Despite a high degree of active zone protein conservation in vertebrates, every type of chemical synapse expresses a given set of protein isoforms and splice variants adapted to the demands on neurotransmitter release. So far, we know little about how specific active zone proteins contribute to the structural and functional diversity of active zones. In this study, we explored the nanodomain organization of ribbon-type active zones by addressing the significance of Piccolino, the ribbon synapse-specific splice variant of Piccolo, for shaping the ribbon structure. We followed up on previous results, which indicated that rod photoreceptor synaptic ribbons lose their structural integrity in a knockdown of Piccolino. Here, we demonstrate an interaction between Piccolino and the major ribbon component RIBEYE that supports plate-shaped synaptic ribbons in retinal neurons. In a detailed ultrastructural analysis of three different types of retinal ribbon synapses in Piccolo/Piccolino-deficient male and female rats, we show that the absence of Piccolino destabilizes the superstructure of plate-shaped synaptic ribbons, although with variable manifestation in the cell types examined. Our analysis illustrates how the expression of a specific active zone protein splice variant (e.g., Piccolino) contributes to structural diversity of vertebrate active zones.SIGNIFICANCE STATEMENT Retinal ribbon synapses are a specialized type of chemical synapse adapted for the regulated fast and tonic release of neurotransmitter. The hallmark of retinal ribbon synapses is the plate-shaped synaptic ribbon, which extends from the release site into the terminals' cytoplasm and tethers hundreds of synaptic vesicles. Here, we show that Piccolino, the synaptic ribbon specific splice variant of Piccolo, interacts with RIBEYE, the main component of synaptic ribbons. This interaction occurs via several PxDLS-like motifs located at the C terminus of Piccolino, which can connect multiple RIBEYE molecules. Loss of Piccolino disrupts the characteristic plate-shaped structure of synaptic ribbons, indicating a role of Piccolino in synaptic ribbon assembly.

Gene co-expression networks associated with carcass traits reveal new pathways for muscle and fat deposition in Nelore cattle.

BACKGROUND: Positively correlated with carcass weight and animal growth, the ribeye area (REA) and the backfat thickness (BFT) are economic important carcass traits, which impact directly on producer's payment. The selection of these traits has not been satisfactory since they are expressed later in the animal's life and multigene regulated. So, next-generation technologies have been applied in this area to improve animal's selection and better understand the molecular mechanisms involved in the development of these traits. Correlation network analysis, performed by tools like WGCNA (Weighted Correlation Network Analysis), has been used to explore gene-gene interactions and gene-phenotype correlations. Thus, this study aimed to identify putative candidate genes and metabolic pathways that regulate REA and BFT by constructing a gene co-expression network using WGCNA and RNA sequencing data, to better understand genetic and molecular variations behind these complex traits in Nelore cattle. RESULTS: The gene co-expression network analysis, using WGCNA, were built using RNA-sequencing data normalized by transcript per million (TPM) from 43 Nelore steers. Forty-six gene clusters were constructed, between them, three were positively correlated (p-value< 0.1) to the BFT (Green Yellow, Ivory, and Light Yellow modules) and, one cluster was negatively correlated (p-value< 0.1) with REA (Salmon module). The enrichment analysis performed by DAVID and WebGestalt (FDR 5%) identified eight Gene Ontology (GO) terms and three KEGG pathways in the Green Yellow module, mostly associated with immune response and inflammatory mechanisms. The enrichment of the Salmon module demonstrated 19 GO terms and 21 KEGG pathways, related to muscle energy metabolism, lipid metabolism, muscle degradation, and oxidative stress diseases. The Ivory and Light yellow modules have not shown significant results in the enrichment analysis. CONCLUSION: With this study, we verified that inflammation and immune response pathways modulate the BFT trait. Energy and lipid metabolism pathways, highlighting fatty acid metabolism, were the central pathways associated with REA. Some genes, as RSAD2, EIF2AK2, ACAT1, and ACSL1 were considered as putative candidate related to these traits. Altogether these results allow us to a better comprehension of the molecular mechanisms that lead to muscle and fat deposition in bovine.

Early auto-immune targeting of photoreceptor ribbon synapses in mouse models of multiple sclerosis.

Optic neuritis is one of the first manifestations of multiple sclerosis. Its pathogenesis is incompletely understood, but considered to be initiated by an auto-immune response directed against myelin sheaths of the optic nerve. Here, we demonstrate in two frequently used and well-validated mouse models of optic neuritis that ribbon synapses in the myelin-free retina are targeted by an auto-reactive immune system even before alterations in the optic nerve have developed. The auto-immune response is directed against two adhesion proteins (CASPR1/CNTN1) that are present both in the paranodal region of myelinated nerves as well as at retinal ribbon synapses. This occurs in parallel with altered synaptic vesicle cycling in retinal ribbon synapses and altered visual behavior before the onset of optic nerve demyelination. These findings indicate that early synaptic dysfunctions in the retina contribute to the pathology of optic neuritis in multiple sclerosis.