Intramammary administration of lipopolysaccharides at parturition does not affect the transfer of passive immunity in goat kids.

This study evaluated the effect of feeding colostrum obtained from an intramammary administration (IA) of LPS from Escherichia coli (O55:B5) to dairy goats at parturition, on goat kids performance, biochemical parameters (i.e., calcium, LDH, glucose, total proteins, albumin, and urea) and immune status (i.e., IgG and IgM) during the first month of life. At birth, goat kids were weighted (d 0) and immediately allocated into either the LPS group (n = 15) or the CON group (n = 21) based on the experimental group of the dam. At parturition, 20 multiparous dairy goats were allocated in one of the 2 experimental groups (LPS vs. CON). The LPS group received an IA of saline solution (2 mL) containing 50 µg of LPS in each half udder whereas goats in the CON group received an IA of saline solution (2 mL) without LPS. Goat kids were bottle-fed dam colostrum equivalent to 10% of the birth BW divided in 2 meals (i.e., at 3 and 12 h relative to birth), and then fed twice daily with milk replacer ad libitum. Individual milk intake (MI) and BW were recorded on d 7, 15, 21 and 30 of life. Blood samples were collected on d 0, 1, 2, 4, 7, 15, 21 and 30 after birth. Data was analyzed using the MIXED procedure of SAS (9.4). The model included IA, time (T) and the interaction (IA x T) as fixed effects and sex and litter size as random effects. Both groups showed similar MI, except on d 7 relative to birth as the LPS group showed higher MI than the CON group (910.5 ± 69.77 and 683.9 ± 59.64 mL, respectively). No differences in BW or rectal temperature were observed between groups, neither in plasma IgG nor IgM concentrations. Despite the IA did not affect calcium, glucose, LDH, total protein, and albumin concentrations an interaction between the IA and T was observed for urea concentration, showing the LPS group higher urea concentrations than the CON group on d 0 (20.1 ± 1.34 and 20.0 ± 1.25 mg/dL, respectively). In conclusion, feeding colostrum from goats that received an IA of LPS at parturition does not affect goat kid performance, plasma immunoglobulin concentrations and serum metabolites during the first month of life.

Characteristics and type of strabismus associated to macular diplopia. Treatment outcomes.

OBJECTIVE: To study the clinical characteristics of macular diplopia, treatment, and outcome. METHODS: Retrospective descriptive study of cases referred to the ocular motility section of a tertiary hospital with diplopia, diagnosed with macular diplopia between 2022-23. The etiology of the macular pathology and the type of associated strabismus were recorded. The result was considered good if the diplopia improved or was eliminated with the medical or surgical treatment. Follow-up time from the onset of diplopia until data collection was recorded. RESULTS: a total of 19 cases comprised the sample (63.2% women), mean age: 67.16 years. Amblyopia (21.1%), high myopia (47.4%), epirretinal membrane (ERM) (36.8%), neovascular membrane (26.3%), macular hole (10.5%), and lamellar (15.8%), and age macular degeneration (5.3%) were registered. The 47.4% had vertical diplopia, horizontal: 5.3 and 47.4% mixed. The mean horizontal deviation was: 7.3 PD (prism diopters) and vertical: 6.22 PD. Ocular extorsion was observed in 26.3%, and intorsion: 5.3%. Torticollis was present in 15.8%. The treatment consisted of strabismus surgery + Botox (15.8%), strabismus surgery (47.4%), medical treatment with Fresnel prims or Scotch cellophane (36.8%). A 68.4% presented a good result at the end of the study. The mean follow-up was 55.58 months. CONCLUSIONS: Misregistration of macular photoreceptors is the most common cause of binocular diplopia in patients with ERM or other macular pathologies. Most complains of vertical or mixed diplopia. Sensorimotor evaluation of these patients should be thorough. Early diagnosis prevents unnecessary prescription of prism glasses. Surgical and/or medical treatment achieves good results in most cases.

Intramammary administration of lipopolysaccharides at parturition enhances immunoglobulin concentration in goat colostrum.

In newborn ruminants, transfer of passive immunity is essential to obtain protection against pathogens. This study aimed to increase the permeability of the blood-milk barrier using intramammary lipopolysaccharides (LPS) in goats at parturition to modulate colostrum composition. Twenty multiparous Majorera dairy goats were randomly allocated in one of the two experimental groups. The LPS group (n = 10) received an intramammary administration (IA) of saline (2 mL) containing 50 µg of LPS from Escherichia coli (O55:B5) in each half udder at parturition. The control group (n = 10) received an IA of saline (2 mL). Rectal temperature (RT) was recorded, and a blood sample was collected at parturition (before IA). In addition, RT was measured, and blood and colostrum/milk samples were collected on day (d) 0.125 (3 hours), 0.5 (12 hours), 1, 2, 4, 7, 15 and 30 relative to the IA. Goat plasma immunoglobulin G (IgG) and M (IgM) and serum ß-hydroxybutyrate, glucose, calcium, free fatty acids, lactate dehydrogenase and total protein concentrations were determined. Colostrum and milk yields as well as chemical composition, somatic cell count (SCC), IgG and IgM concentrations were measured. The MIXED procedure (SAS 9.4) was used, and the model included the IA, time, and the interaction between both fixed effects. Statistical significance was set as P < 0.05. Goats from the LPS group showed higher RT on d 0.125, 0.5 and 4 relative to the IA compared to the control group (PIA×Time = 0.007). Goat serum biochemical variables and plasma IgG and IgM concentrations were not affected by the IA. Colostrum and milk yield as well as chemical composition were not affected by the IA, except for milk lactose percentage that was lower in the LPS group compared to the control group (4.3 ± 0.08 and 4.6 ± 0.08%, respectively PIA = 0.026). Colostrum SCC was higher in the LPS group than in the control group (3.5 ± 0.09 and 3.1 ± 0.09 cells × 106/mL, respectively; PIA = 0.011). Similarly, milk SCC increased in the LPS group compared to the control group (PIA = 0.004). The LPS group showed higher IgG (PIA = 0.044) and IgM (PIA = 0.037) concentrations on colostrum than the control group (31.9 ± 4.8 and 19.0 ± 4.8 mg/mL, 0.8 ± 0.08 and 0.5 ± 0.08 mg/mL, respectively). No differences in milk IgG and IgM concentrations between groups were observed. In conclusion, the IA of LPS at parturition increases RT, SCC and IgG and IgM concentrations in colostrum without affecting either yield or chemical composition.

Chemoproteomic Profiling of 8-Oxoguanosine-Sensitive RNA-Protein Interactions.

Cellular nucleic acids are subject to assault by endogenous and exogenous agents that can perturb the flow of genetic information. Oxidative stress leads to the accumulation of 8-oxoguanine (8OG) in DNA and RNA. 8OG lesions on mRNA negatively impact translation, but their effect on global RNA-protein interactions is largely unknown. Here, we apply an RNA chemical proteomics approach to investigate the effect of 8OG on RNA-protein binding. We find proteins that bind preferentially to 8OG-modified RNA, including IGF2BP1-3 and hnRNPD, and proteins that are repelled by 8OG such as RBM4. We characterize these interactions using biochemical and biophysical assays to quantify the effect of 8OG on binding and show that a single 8OG abolishes the binding of RBM4 to its preferred CGG-containing substrate. Taken together, our work establishes the molecular consequences of 8OG on cellular RNA-protein binding and provides a framework for interrogating the role of RNA oxidation in biological systems.

Scattering and transport properties of the three classical Wigner-Dyson ensembles at the Anderson transition.

An extensive numerical analysis of the scattering and transport properties of the power-law banded random matrix model (PBRM) at criticality in the presence of orthogonal, unitary, and symplectic symmetries is presented. Our results show a good agreement with existing analytical expressions in the metallic regime and with heuristic relations widely used in studies of the PBRM model in the presence of orthogonal and unitary symmetries. Moreover, our results confirm that the multifractal behavior of disordered systems at criticality can be probed by measuring scattering and transport properties, which is of paramount importance from the experimental point of view. Thus, a full picture of the scattering and transport properties of the PBRM model at criticality corresponding to the three classical Wigner-Dyson ensembles is provided.

Live-Cell RNA Imaging with Metabolically Incorporated Fluorescent Nucleosides.

Fluorescence imaging is a powerful method for probing macromolecular dynamics in biological systems; however, approaches for cellular RNA imaging are limited to the investigation of individual RNA constructs or bulk RNA labeling methods compatible primarily with fixed samples. Here, we develop a platform for fluorescence imaging of bulk RNA dynamics in living cells. We show that fluorescent bicyclic and tricyclic cytidine analogues can be metabolically incorporated into cellular RNA by overexpression of uridine-cytidine kinase 2. In particular, metabolic feeding with the tricyclic cytidine-derived nucleoside tC combined with confocal imaging enables the investigation of RNA synthesis, degradation, and trafficking at single-cell resolution. We apply our imaging modality to study RNA metabolism and localization during the oxidative stress response and find that bulk RNA turnover is greatly accelerated upon NaAsO2 treatment. Furthermore, we identify cytoplasmic RNA granules containing RNA transcripts generated during oxidative stress that are distinct from canonical stress granules and P-bodies and co-localize with the RNA helicase DDX6. Taken together, our work provides a powerful approach for live-cell RNA imaging and reveals how cells reshape RNA transcriptome dynamics in response to oxidative stress.

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases.

Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m5C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC). We profile m5C dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hm5C- and f5C-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent m5C oxidation in a variety of tRNAs and mRNAs and analyze ALKBH1 substrate specificity in vitro. We also apply targeted pyridine borane-mediated sequencing to measure f5C sites on select tRNA. Finally, we show that f5C at the wobble position of tRNA-Leu-CAA plays a role in decoding Leu codons under stress. Our work provides powerful chemical approaches for studying RNA m5C dioxygenases and mapping oxidative m5C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.

Chemical Method to Sequence 5-Formylcytosine on RNA.

Epitranscriptomic RNA modifications can regulate biological processes, but there remains a major gap in our ability to identify and measure individual modifications at nucleotide resolution. Here we present Mal-Seq, a chemical method for sequencing 5-formylcytosine (f5C) modifications on RNA based on the selective and efficient malononitrile-mediated labeling of f5C residues to generate adducts that are read as C-to-T mutations upon reverse transcription and polymerase chain reaction amplification. We apply Mal-Seq to characterize the prevalence of f5C at the wobble position of mt-tRNA(Met) in different organisms and tissue types and find that high-level f5C modification is present in mammals but lacking in lower eukaryotes. Our work sheds light on mitochondrial tRNA modifications throughout eukaryotic evolution and provides a general platform for characterizing the f5C epitranscriptome.

Microwave graph analogs for the voltage drop in three-terminal devices with orthogonal, unitary, and symplectic symmetry.

Transmission measurements through three-port microwave graphs are performed, in analogy to three-terminal voltage drop devices with orthogonal, unitary, and symplectic symmetry. The terminal used as a probe is symmetrically located between two chaotic subgraphs, and each graph is connected to one port, the input and the output, respectively. The analysis of the experimental data clearly exhibits the weak localization and antilocalization phenomena. We find a good agreement with theoretical predictions, provided that the effects of dissipation and imperfect coupling to the ports are taken into account.

Rapid identification, capsular typing and molecular characterization of Streptococcus pneumoniae by using whole genome nanopore sequencing.

BACKGROUND: Whole genome sequencing has emerged as a useful tool for identification and molecular characterization of pathogens. MinION (Oxford Nanopore) is a real-time third generation sequencer whose portability, affordability and speed in data production make of it an attractive device for whole genome sequencing. The objective of this study is to evaluate MinION sequencer for pathogen identification and molecular characterization of Streptococcus pneumoniae isolated at a children's Hospital. Whole genome sequencing of 32 Streptococcus pneumoniae invasive isolates, previously characterized by standard methods (Quellung reaction, Multiplex PCR and Sanger-MLST), were performed. DNA was extracted using ZymoBIOMICS DNA Microprep kit. Quantification and purity of DNA was assessed by Qubit and Nanodrop, respectively. Library preparation was performed using the Rapid Barcoding Kit. Real-time workflow EPI2ME platform "What's it in my pot" was used for species identification. Fast5 sequences were converted into FASTQ by Albacore software. Reads were assembled using CANU software. PathogenWatch, genomic epidemiology and pubmlst online tools were used for capsular typing and/or whole genome-MLST profile. RESULTS: Rapid identification of Streptococcus pneumoniae was achieved by "What's in my pot". Capsular typing was correctly assigned with PathogenWatch in all 32 isolates at serogroup level and 24 at serotype level. Whole genome-MLST results obtained by genomic epidemiology and pubmlst were consistent with double locus variant clonal complex obtained by Sanger-MLST in 31 isolates. CONCLUSION: MinION sequencer provides a rapid, cost-effective and promising pathway for performing WGS by a pocked-sized device for epidemiological purposes but improving its sequencing accuracy will make it more appealing to be used in clinical microbiology laboratories.

Short communication: Red deer (Cervus elaphus) colostrum during its transition to milk.

We studied changes in chemical composition, somatic cell count, and immunoglobulin G (IgG) and M (IgM) content in red deer (Cervus elaphus) colostrum during the transition to milk at different times after parturition (<5 h, 24 h, 48 h, 2 wk, and 4 wk). The production level was higher at 2 and 4 wk of lactation than during the first day after parturition, with intermediate values at 48 h postpartum. Fat content did not vary during the study period. However, total protein and casein contents were particularly high in the initial 5 h after parturition, decreasing to approximately 50% after 24 h postpartum. Conversely, lactose concentration was low in the beginning (<5 h), increasing gradually throughout the study. Similarly, dry matter dropped during the first 24 h and then remained constant throughout the study. Urea content decreased during the study, showing a slight recovery at 4 wk. Somatic cell count was higher during the first hours after parturition and gradually decreased throughout the study period. The IgG content was higher before 5 h postpartum than at 24 h postpartum. After 5 h, the level of IgG decreased progressively until it reached 0.18 mg/mL at 4 wk of lactation. We observed a similar pattern for IgM content, but it decreased more quickly than IgG and was not detected after 2 wk. In the case of deer, milk should be considered transitional from 24 to 48 h after parturition, and samples collected after 2 wk can be considered mature milk.

Use of glycerol and propylene glycol as additives in heat-treated goat colostrum.

This experiment aimed to evaluate the suitability of glycerol and propylene glycol to reduce microbial count and preserve immune properties in heat-treated goat colostrum. Colostrum samples from 11 goats were each divided into 9 aliquots. Different concentrations (2, 6, 10, and 14%; vol/vol) of either glycerol or propylene glycol were added to the aliquots. Phosphate buffer solution was added to one aliquot, which was set as the control (CG). After the respective additions, all colostrum samples were heat treated at 56°C for 1 h. After cooling, aerobic mesophilic bacteria were cultured. The samples were frozen until free fatty acid, IgG, IgA, and IgM concentrations and chitotriosidase activity were measured. No differences were found in aerobic mesophilic bacteria counts between either 10 or 14% glycerol and propylene glycol additives. These additions reduced bacterial count to a greater extent than CG, and 2 or 6% additions. Colostrum IgG concentration was not affected by either of the additives or their concentrations. The propylene glycol additive reduced IgA and IgM concentrations and chitotriosidase activity, compared with CG. Conversely, glycerol did not affect any of the studied immune variables. In conclusion, glycerol addition to goat colostrum before heat treatment is suitable to enhance bacterial reduction, whereas colostrum immune properties were not affected.

Phenomenological approach to transport through three-terminal disordered wires.

We study the voltage drop along three-terminal disordered wires in all transport regimes, from the ballistic to the localized regime. This is performed by measuring the voltage drop on one side of a one-dimensional disordered wire in a three-terminal setup as a function of disorder. Two models of disorder in the wire are considered: (i) the one-dimensional Anderson model with diagonal disorder and (ii) finite-width bulk-disordered waveguides. Based on the known ß dependence of the voltage drop distribution of the three-terminal chaotic case, ß being the Dyson symmetry index (ß=1, 2, and 4 for orthogonal, unitary, and symplectic symmetries, respectively), the analysis is extended to a continuous parameter ß>0 and uses the corresponding expression as a phenomenological one to reach the disordered phase. We show that our proposal encompasses all the transport regimes with ß depending linearly on the disorder strength.

In Vitro Selection with a Site-Specifically Modified RNA Library Reveals the Binding Preferences of N6-Methyladenosine Reader Proteins.

Epitranscriptomic RNA modifications can serve as recognition elements for the recruitment of effector proteins (i.e., "readers") to modified transcripts. While these interactions play an important role in mRNA regulation, there is a major gap in our understanding of the sequence determinants critical for the binding of readers to modified sequence motifs. Here, we develop a high-throughput platform, relying upon in vitro selection with a site-specifically modified random sequence RNA library and next-generation sequencing, to profile the binding specificity of RNA modification reader proteins. We apply our approach to interrogate the effect of sequence context on the interactions of YTH-domain proteins with N6-methyladenosine (m6A)-modified RNA. We find that while the in vitro binding preferences of YTHDC1 strongly overlap with the well-characterized DR(m6A)CH motif, the related YTH-domain proteins YTHDF1 and YTHDF2 can bind tightly to noncanonical m6A-containing sequences. Our results reveal the principles underlying substrate selection by m6A reader proteins and provide a powerful approach for investigating protein-modified RNA interactions in an unbiased manner.

Short communication: Differences in distribution of serotonin receptor subtypes in the mammary gland of sheep, goats, and cows during lactation and involution.

Serotonin receptors (5-HTR) are present in the mammary tissue of mouse, humans, cows, and rats. In these species, serotonin is important for the mammary gland function and lactation performance. The mammary gland expression of 5-HTR in small dairy ruminants has yet to be described. In the present study, primer sequences were developed to amplify 5-HTR (1A, 1D, 1E,1B, 1F, 2A, 2B, 2C, 3a, 4, 5a, 6, and 7) using real-time quantitative PCR for the detection of mRNA expression in mammary tissue of dairy sheep, goats, and cows. The distribution of commonly expressed 5-HTR between the 3 species (1B, 1E, 2A, 2B, 4, and 7) was analyzed in the mammary tissue of late-lactation and dried-off sheep, goats, and cows using immunohistochemical staining. Real-time quantitative PCR analysis showed that the 3 studied species expressed receptors 5-HTR1B, 1E, 2A, 2B, 4, and 7. Goats and sheep expressed 5-HTR1D and 5a; 5-HTR1A and 1F were expressed only in sheep. The mammary epithelial cells were positively stained for all the studied receptors by immunohistochemistry (5-HTR1B, 1E, 2A, 2B, 4, and 7). The endothelial cells of blood vessels were positively stained for 5-HTR1B, 2A, 2B, and 7 in all the species. Additionally, 5-HTR1E was present in cow endothelium. The myoepithelial cells stained positively for 5-HTR1E in all the species, and 5-HTR4 myoepithelial staining was present only in cows and sheep. Between the lactating and dried-off mammary glands, the location of 5-HTR in the epithelial cells changed from a cytoplasmic reaction in lactating udders to a reaction in the apical region in dry udders. These results showed that the distribution of 5-HTR subtypes in the mammary gland of dairy ruminants vary among species, tissue type, and stage of gland development. These findings warrant future studies aimed at understanding whether the differences in 5-HTR subtype expression and location accounts for the differences in milk secretion and lactocyte activity among cows, goats, and sheep.

A Photocrosslinking-Based RNA Chemical Proteomics Approach to Profile m6 A-Regulated Protein-RNA Interactions.

Post-transcriptional modifications play an important role in RNA biology. In particular, the addition of small chemical groups to the nucleobases of mRNA can affect how modified transcripts are processed in the cell, thereby impacting gene expression programs. In order to study the molecular mechanisms underlying these modifications, it is necessary to characterize their 'readers', that is, proteins that directly bind to these modifications to mediate their functional consequences; this is a major challenge because we lack approaches to precisely manipulate RNA chemistry in the cell and because protein-modified RNA interactions can be low affinity. In this unit, we describe in detail a photocrosslinking-based RNA chemical proteomics approach to profile the protein-modified RNA interactome modulated by N6 -methyladenosine (m6 A), the most abundant internal modification in eukaryotic mRNA. First, we present protocols for the synthesis and characterization of short, diazirine-containing synthetic RNA probes, followed by a description of their use in mass spectrometry-based proteomics with HeLa cell lysate and a short commentary on data analysis and result interpretation. © 2018 by John Wiley & Sons, Inc.

Blood immune transcriptome analysis of artificially fed dairy calves and naturally suckled beef calves from birth to 7 days of age.

Neonatal calves possess a very immature and naïve immune system and are reliant on the intake of maternal colostrum for passive transfer of immunoglobulins. Variation in colostrum management of beef and dairy calves is thought to affect early immune development. Therefore, the objective of this study was to examine changes in gene expression and investigate molecular pathways involved in the immune-competence development of neonatal Holstein dairy calves and naturally suckled beef calves using next generation RNA-sequencing during the first week of life. Jugular whole blood samples were collected from Holstein (H) dairy calves (n = 8) artificially fed 5% B.W. colostrum, and from beef calves which were the progenies of Charolais-Limousin (CL; n = 7) and Limousin-Friesian beef suckler cows (LF; n = 7), for subsequent RNA isolation. In dairy calves, there was a surge in pro-inflammatory cytokine gene expression possibly due to the stress of separation from the dam. LF calves exhibited early signs of humoral immune development with observed increases in the expression genes coding for Ig receptors, which was not evident in the other breeds by 7 days of age. Immune and health related DEGs identified as upregulated in beef calves are prospective contender genes for the classification of biomarkers for immune-competence development, and will contribute towards a greater understanding of the development of an immune response in neonatal calves.

A DNA-conjugated small molecule catalyst enzyme mimic for site-selective ester hydrolysis.

The challenge of site-selectivity must be overcome in many chemical research contexts, including selective functionalization in complex natural products and labeling of one biomolecule in a living system. Synthetic catalysts incorporating molecular recognition domains can mimic naturally-occurring enzymes to direct a chemical reaction to a particular instance of a functional group. We propose that DNA-conjugated small molecule catalysts (DCats), prepared by tethering a small molecule catalyst to a DNA aptamer, are a promising class of reagents for site-selective transformations. Specifically, a DNA-imidazole conjugate able to increase the rate of ester hydrolysis in a target ester by >100-fold compared with equimolar untethered imidazole was developed. Other esters are unaffected. Furthermore, DCat-catalyzed hydrolysis follows enzyme-like kinetics and a stimuli-responsive variant of the DCat enables programmable "turn on" of the desired reaction.

RNA Chemical Proteomics Reveals the N6-Methyladenosine (m6A)-Regulated Protein-RNA Interactome.

Epitranscriptomic RNA modifications can regulate mRNA function; however, there is a major gap in our understanding of the biochemical mechanisms mediating their effects. Here, we develop a chemical proteomics approach relying upon photo-cross-linking with synthetic diazirine-containing RNA probes and quantitative proteomics to profile RNA-protein interactions regulated by N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic RNA. In addition to identifying YTH domain-containing proteins and ALKBH5, known interactors of this modification, we find that FMR1 and LRPPRC, two proteins associated with human disease, "read" this modification. Surprisingly, we also find that m6A disrupts RNA binding by the stress granule proteins G3BP1/2, USP10, CAPRIN1, and RBM42. Our work provides a general strategy for interrogating the interactome of RNA modifications and reveals the biochemical mechanisms underlying m6A function in the cell.