Characterization of Removal Reasons for Nurse Sows and the Associated Removal Due to Their Extended Lactation Length in Hyperprolific Farrow-Wean Herds.

This study aimed to characterize and quantify reasons for the removal of nurse sows and identify the removal associated with their extended lactation length (ELL). A total of 100,756 removed nurse sows within a period of 2016-2022 from 53 sow herds in the Midwest USA were analyzed. Reproductive failure was the most common removal reason (χ2 = 8748.421, p < 0.001) affecting P1, P2, and P3 nurse sows. Failure to conceive and absence of estrus were the main causes of reproductive failure (χ2 = 352.480, p < 0.001) affecting P1 and P2 nurse sows and P1 and P5 nurse sows, respectively. When P2 and P6 nurse sows had an ELL of 0-7 d, they faced a high chance (χ2 = 13.312, p = 0.021) of removal due to conception failure and failure to return to heat, respectively. When P2 and P5 nurse sows had an ELL of 8-14 d, they were highly vulnerable (χ2 = 59.847, p < 0.001) to removal due to failure to conceive and showing heat, respectively. Finally, when ELL was at 15-21 days, P4 and P5 nurse sows were more likely (χ2 = 41.751, p < 0.001) to be removed due to failure to express heat, whereas at the same time, P2 and P3 nurse sows experienced the same removal threat due to failing to conceive. These results could help producers manage nurse sow systems.

Computational Studies on the Diastereospecific Lithium Variant of Oppenauer Oxidation of a Tofisopam Intermediate.

During the synthesis of tofisopam drug substance, an interesting diastereospecific lithium variant of Oppenauer oxidation was observed and investigated by density functional theory (DFT) calculations. The computations revealed energetic differences caused by steric differences between the diastereomers that might provide an explanation for the experimentally formed products. In addition, the trend in the measured NMR shifts was also in line with the computed values, which allowed the assignment of the absolute configuration of the diastereomers.

Splenectomy at early stage of autoimmune arthritis delayed inflammatory response and reduced joint deterioration in mice.

The spleen plays a role in innate and adaptive immunity, and autoimmune diseases like rheumatoid arthritis (RA). We investigated the effect of splenectomy in early and moderate stages of autoimmune arthritis in a mouse model. To induce recombinant human G1-induced arthritis (GIA), BALB/c mice were immunized intraperitoneally three times in 4-week intervals with the rhG1 antigen. Mice were splenectomized on day 7 (SPE1) or day 35 (SPE2) after the initiation of immunization; tested for clinical severity, joint radiological and histological changes, serum levels of inflammatory cytokines and autoantibodies, and rhG1-specific immune responses; and compared to those in control mice with spleen left intact. Circulating Tregs and T-helper subset ratios in the spleen and inguinal lymph nodes (LNs) were also examined using flow cytometry. The onset of severe inflammatory response was significantly delayed in SPE1 and SPE2 groups compared to control mice at early stages of GIA, which was associated with increased circulating Tregs. After the third immunization, as disease progressed, the severity scores were robustly increased in all mice. Nevertheless, in splenectomized mice, we observed reduced joint deterioration and cartilage damage, more Th2 cells in LNs, and reduced levels of pro-inflammatory cytokines and autoantibodies in their sera. Mesenteric LN cells of splenectomized mice exhibited weaker response in vitro against the rhG1 antigen compared to control mice spleen. In conclusion, splenectomy in the early stages of GIA delayed the inflammatory response, suggesting a protective effect against the development and progression of severe destructive arthritis.

Quantitative Analysis of NKX2-3 Expression in Human Colon: An Immunohistochemical Study.

In mice, Nkx2-3 homeodomain transcription factor defines the vascular specification of secondary and tertiary lymphoid tissues of the intestines. In human studies, polymorphisms in NKX2-3 have been identified as a susceptibility factor in inflammatory bowel diseases, whereas in mice, its absence is associated with protection against experimental colitis and enhanced intestinal epithelial proliferation. Here, we investigated the expression of NKX2-3 in normal, polyp, and adenocarcinoma human colon samples using immunohistochemistry and quantitative morphometry, correlating its expression with endothelial and mesenchymal stromal markers. Our results revealed that the expression of NKX2-3 is regionally confined to the lamina propria and lamina muscularis mucosae, and its production is restricted mostly to endothelial cells and smooth muscle cells with variable co-expression of CD34, alpha smooth muscle antigen (αSMA), and vascular adhesion protein-1 (VAP-1). The frequency of NKX2-3-positive cells and intensity of expression correlated inversely with aging. Furthermore, in most colorectal carcinoma samples, we observed a significant reduction of NKX2-3 expression. These findings indicate that the NKX2-3 transcription factor is produced by both endothelial and non-endothelial tissue constituents in the colon, and its expression changes during aging and in colorectal malignancies. (J Histochem Cytochem XX: XXX-XXX, XXXX).

Electrophilic MiniFrags Revealed Unprecedented Binding Sites for Covalent HDAC8 Inhibitors.

Screening of ultra-low-molecular weight ligands (MiniFrags) successfully identified viable chemical starting points for a variety of drug targets. Here we report the electrophilic analogues of MiniFrags that allow the mapping of potential binding sites for covalent inhibitors by biochemical screening and mass spectrometry. Small electrophilic heterocycles and their N-quaternized analogues were first characterized in the glutathione assay to analyze their electrophilic reactivity. Next, the library was used for systematic mapping of potential covalent binding sites available in human histone deacetylase 8 (HDAC8). The covalent labeling of HDAC8 cysteines has been proven by tandem mass spectrometry measurements, and the observations were explained by mutating HDAC8 cysteines. As a result, screening of electrophilic MiniFrags identified three potential binding sites suitable for the development of allosteric covalent HDAC8 inhibitors. One of the hit fragments was merged with a known HDAC8 inhibitor fragment using different linkers, and the linker length was optimized to result in a lead-like covalent inhibitor.

Enhancing Adaptive Physics Refinement Simulations Through the Addition of Realistic Red Blood Cell Counts.

Simulations of cancer cell transport require accurately modeling mm-scale and longer trajectories through a circulatory system containing trillions of deformable red blood cells, whose intercellular interactions require submicron fidelity. Using a hybrid CPU-GPU approach, we extend the advanced physics refinement (APR) method to couple a finely-resolved region of explicitly-modeled red blood cells to a coarsely-resolved bulk fluid domain. We further develop algorithms that: capture the dynamics at the interface of differing viscosities, maintain hematocrit within the cell-filled volume, and move the finely-resolved region and encapsulated cells while tracking an individual cancer cell. Comparison to a fully-resolved fluid-structure interaction model is presented for verification. Finally, we use the advanced APR method to simulate cancer cell transport over a mm-scale distance while maintaining a local region of RBCs, using a fraction of the computational power required to run a fully-resolved model.

Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

Estimation of shear stress heterogeneity along capillary segments in angiogenic rat mesenteric microvascular networks.

OBJECTIVE: Fluid shear stress is thought to be a regulator of endothelial cell behavior during angiogenesis. The link, however, requires an understanding of stress values at the capillary level in angiogenic microvascular networks. Critical questions remain. What are the stresses? Do capillaries experience similar stress magnitudes? Can variations explain vessel-specific behavior? The objective of this study was to estimate segment-specific shear stresses in angiogenic networks. METHODS: Images of angiogenic networks characterized by increased vascular density were obtained from rat mesenteric tissues stimulated by compound 48/80-induced mast cell degranulation. Vessels were identified by perfusion of a 40 kDa fixable dextran prior to harvesting and immunolabeling for PECAM. Using a network flow-based segment model with physiologically relevant parameters, stresses were computed per vessel for regions across multiple networks. RESULTS: Stresses ranged from 0.003 to 2328.1 dyne/cm2 and varied dramatically at the capillary level. For all regions, the maximum segmental shear stresses were for capillary segments. Stresses along proximal capillaries branching from arteriole inlets were increased compared to stresses along capillaries in more distal regions. CONCLUSIONS: The results highlight the variability of shear stresses along angiogenic capillaries and motivate new discussions on how endothelial cells may respond in vivo to segment-specific microenvironment during angiogenesis.

Angiogenic Microvascular Wall Shear Stress Patterns Revealed Through Three-dimensional Red Blood Cell Resolved Modeling.

The wall shear stress (WSS) exerted by blood flowing through microvascular capillaries is an established driver of new blood vessel growth, or angiogenesis. Such adaptations are central to many physiological processes in both health and disease, yet three-dimensional (3D) WSS characteristics in real angiogenic microvascular networks are largely unknown. This marks a major knowledge gap because angiogenesis, naturally, is a 3D process. To advance current understanding, we model 3D red blood cells (RBCs) flowing through rat angiogenic microvascular networks using state-of-the-art simulation. The high-resolution fluid dynamics reveal 3D WSS patterns occurring at sub-endothelial cell (EC) scales that derive from distinct angiogenic morphologies, including microvascular loops and vessel tortuosity. We identify the existence of WSS hot and cold spots caused by angiogenic surface shapes and RBCs, and notably enhancement of low WSS regions by RBCs. Spatiotemporal characteristics further reveal how fluctuations follow timescales of RBC "footprints." Altogether, this work provides a new conceptual framework for understanding how shear stress might regulate EC dynamics in vivo.

Covalent fragment approaches targeting non-cysteine residues.

Covalent fragment approaches combine advantages of covalent binders and fragment-based drug discovery (FBDD) for target identification and validation. Although early applications focused mostly on cysteine labeling, the chemistries of available warheads that target other orthosteric and allosteric protein nucleophiles has recently been extended. The range of different warheads and labeling chemistries provide unique opportunities for screening and optimizing warheads necessary for targeting non-cysteine residues. In this review, we discuss these recently developed amino-acid-specific and promiscuous warheads, as well as emerging labeling chemistries, which includes novel transition metal catalyzed, photoactive, electroactive, and noncatalytic methodologies. We also highlight recent applications of covalent fragments for the development of molecular glues and proteolysis-targeting chimeras (PROTACs), and their utility in chemical proteomics-based target identification and validation.

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging.

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

Natural and Pathological Autoantibodies Show Age-Related Changes in a Spontaneous Autoimmune Mouse (NZB) Model.

The natural autoantibody (natAAb) network is thought to play a role in immune regulation. These IgM antibodies react with evolutionary conserved antigens; however, they do not lead to pathological tissue destruction as opposed to pathological autoantibodies (pathAAb). The exact relation between the natAAbs and pathAAbs is still not completely understood; therefore, in the present study, we set out to measure nat- and pathAAb levels against three conserved antigens in a spontaneous autoimmune disease model: the NZB mouse strain which develops autoimmune hemolytic anemia (AIHA) from six months of age. There was an age dependent increase in the natAAb levels in the serum against Hsp60, Hsp70, and the mitochondrial citrate synthase until 6-9 months of age, followed by a gradual decrease. The pathological autoantibodies appeared after six months of age, which corresponded with the appearance of the autoimmune disease. The changes in nat/pathAAb levels were coupled with decreasing B1- and increasing plasma cell and memory B cell percentages. Based on this, we propose that there is a switch from natAAbs towards pathAAbs in aged NZB mice.

Absence of Nkx2-3 induces ectopic lymphatic endothelial differentiation associated with impaired extramedullary stress hematopoiesis in the spleen.

The red and white pulps as two main parts of the spleen are arranged around distinct types of vasculature, and perform significantly different functions in both humans and mice. Previous observations indicated a profound alteration of the local vessel specialization in mice lacking Nkx2-3 homeodomain transcription factor, including contradictory results suggesting presence of an ectopic lymphatic vascular structure. Furthermore, how the absence of Nkx2-3 and the consequential changes in endothelial components affect the extramedullary hematopoietic activity restricted to the splenic red pulp is unknown. In this work, we investigated the role of Nkx2-3 homeodomain transcription factor as a major morphogenic determinant for vascular specification, and its effect in the extramedullary hematopoiesis following acute blood loss and pharmacological stimulation of megakaryocyte differentiation after treatment with thrombopoietin-receptor mimetic Romiplostim. We found that, in mice lacking Nkx2-3, Prox1-positive lymphatic capillaries containing gp38/CD31 double positive lymphatic endothelial cells develop, arranged into an extensive meshwork, while the Clever1-positive venous segments of red pulp blood vasculature are absent. This lymphatic endothelial shift is coupled with a severely compromised splenic erythropoiesis and a significantly reduced splenic megakaryocyte colony formation following Romiplostim treatment in mice lacking Nkx2-3. These findings indicate that the shift of microvascular patterning in the absence of Nkx2-3 includes the emergence of ectopic Prox1-positive lymphatic vessels, and that this pivoting towards lymph node-like vascular patterning is associated with an impaired reserve hematopoietic capacity of the splenic red pulp.

Activation-Free Sulfonyl Fluoride Probes for Fragment Screening.

SuFEx chemistry is based on the unique reactivity of the sulfonyl fluoride group with a range of nucleophiles. Accordingly, sulfonyl fluorides label multiple nucleophilic amino acid residues, making these reagents popular in both chemical biology and medicinal chemistry applications. The reactivity of sulfonyl fluorides nominates this warhead chemotype as a candidate for an external, activation-free general labelling tag. Here, we report the synthesis and characterization of a small sulfonyl fluoride library that yielded the 3-carboxybenzenesulfonyl fluoride warhead for tagging tractable targets at nucleophilic residues. Based on these results, we propose that coupling diverse fragments to this warhead would result in a library of sulfonyl fluoride bits (SuFBits), available for screening against protein targets. SuFBits will label the target if it binds to the core fragment, which facilitates the identification of weak fragments by mass spectrometry.

The miR-20a/miR-92b Profile Is Associated with Circulating γδ T-Cell Perturbations in Mild Psoriasis.

Psoriasis vulgaris (PV) is an autoinflammatory dermatosis of unknown etiology. Current evidence suggests a pathogenic role of γδT cells, but the growing complexity of this population has made the offending subset difficult to pinpoint. The work on γδTCRint and γδTCRhi subsets, which express intermediate and high levels of γδTCR at their surface, respectively, is particularly scarce, leaving their inner workings in PV essentially unresolved. We have shown here that the γδTCRint/γδTCRhi cell composition and their transcriptom are related to the differential miRNA expression by performing a targeted miRNA and mRNA quantification (RT-qPCR) in multiplexed, flow-sorted γδ blood T cells from healthy controls (n = 14) and patients with PV (n = 13). A significant loss of miR-20a in bulk γδT cells (~fourfold decrease, PV vs. controls) largely mirrored increasing Vδ1-Vδ2- and γδintVδ1-Vδ2- cell densities in the bloodstream, culminating in a relative excess of γδintVδ1-Vδ2- cells for PV. Transcripts encoding DNA-binding factors (ZBTB16), cytokine receptors (IL18R1), and cell adhesion molecules (SELPLG) were depleted in the process, closely tracking miR-20a availability in bulk γδ T-cell RNA. Compared to controls, PV was also associated with enhanced miR-92b expression (~13-fold) in bulk γδT cells that lacked association with the γδT cell composition. The miR-29a and let-7c expressions remained unaltered in case-control comparisons. Overall, our data expand the current landscape of the peripheral γδT cell composition, underlining changes in its mRNA/miRNA transcriptional circuits that may inform PV pathogenesis.

Covalent fragment mapping of KRasG12C revealed novel chemotypes with in vivo potency.

G12C mutant KRas is considered druggable by allele-specific covalent inhibitors due to the nucleophilic character of the oncogenic mutant cysteine at position 12. Discovery of these inhibitors requires the optimization of both covalent and noncovalent interactions. Here, we report covalent fragment screening of our electrophilic fragment library of diverse non-covalent scaffolds equipped with 40 different electrophilic functionalities to identify fragments as suitable starting points targeting Cys12. Screening the library against KRasG12C using Ellman's free thiol assay, followed by protein NMR and cell viability assays, resulted in two potential inhibitor chemotypes. Characterization of these scaffolds in in vitro cellular- and in vivo xenograft models revealed them as promising starting points for covalent drug discovery programs.

Examination of Preferences for COVID-19 Vaccines in Hungary Based on Their Properties-Examining the Impact of Pandemic Awareness with a Hybrid Choice Approach.

The COVID-19 pandemic has posed a huge challenge to the world in recent years. The development of vaccines that are as effective as possible and accessible to society offers a promising alternative for addressing the problems caused by this situation as soon as possible and to restore the pre-epidemic system. The present study investigated the preferences of residents in Hungary's second-largest city (Debrecen) for the COVID-19 vaccine. To achieve this aim, a discrete choice experiment was conducted with 1011 participants, and the vaccine characteristics included in the design of the experiment were determined by qualitative methods and a pilot survey: (1) country of origin; (2) efficiency; (3) side effect; and (4) duration of protection. During the data collection at three vaccination sites, respondents were asked to choose between three vaccine alternatives and one "no choice" option in eight decision situations. Discrete choice model estimations were performed using a random parameter logit (RPL) specification with the final model extended to include a latent variable measuring pandemic awareness. The results showed that the vaccine with a Chinese country of origin is the least preferred among the respondents, while the Hungarian and the European vaccines are the most preferred. Furthermore, the increase in the vaccine efficiency level increased the respondents' sense of utility for the vaccine; the short-term side effect was preferred to the long-term one; and the increase in the duration of protection provided by the vaccine increased the respondents' sense of utility for the vaccine. Based on the parameter estimated for the latent variable, it can be concluded that as the level of pandemic awareness (which is more positive among people with chronic diseases and less important among health workers) increases, the choice of a vaccine option becomes more preferred among respondents compared to the "no choice". The results of our investigation could contribute towards increasing compliance in the case of the vaccination-rejecting population, not only for COVID-19, but for any kind of vaccination procedure.

An NKX-COUP-TFII morphogenetic code directs mucosal endothelial addressin expression.

Immunoglobulin family and carbohydrate vascular addressins encoded by Madcam1 and St6gal1 control lymphocyte homing into intestinal tissues, regulating immunity and inflammation. The addressins are developmentally programmed to decorate endothelial cells lining gut post-capillary and high endothelial venules (HEV), providing a prototypical example of organ- and segment-specific endothelial specialization. We identify conserved NKX-COUP-TFII composite elements (NCCE) in regulatory regions of Madcam1 and St6gal1 that bind intestinal homeodomain protein NKX2-3 cooperatively with venous nuclear receptor COUP-TFII to activate transcription. The Madcam1 element also integrates repressive signals from arterial/capillary Notch effectors. Pan-endothelial COUP-TFII overexpression induces ectopic addressin expression in NKX2-3+ capillaries, while NKX2-3 deficiency abrogates expression by HEV. Phylogenetically conserved NCCE are enriched in genes involved in neuron migration and morphogenesis of the heart, kidney, pancreas and other organs. Our results define an NKX-COUP-TFII morphogenetic code that targets expression of mucosal vascular addressins.

Next-Generation Heterocyclic Electrophiles as Small-Molecule Covalent MurA Inhibitors.

Heterocyclic electrophiles as small covalent fragments showed promising inhibitory activity on the antibacterial target MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase, EC:2.5.1.7). Here, we report the second generation of heterocyclic electrophiles: the quaternized analogue of the heterocyclic covalent fragment library with improved reactivity and MurA inhibitory potency. Quantum chemical reaction barrier calculations, GSH (L-glutathione) reactivity assay, and thrombin counter screen were also used to demonstrate and explain the improved reactivity and selectivity of the N-methylated heterocycles and to compare the two generations of heterocyclic electrophiles.

Global alteration of colonic microRNAome landscape associated with inflammatory bowel disease.

Inflammatory Bowel Disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract that associates with, among others, increased risk of colorectal cancer. There is a growing evidence that miRNAs have important roles in pathological processes, such as inflammation or carcinogenesis. Understanding the molecular mechanisms such as alterations in microRNAome upon chronic intestinal inflammation is critical for understanding the exact pathomechanism of IBD. Hence, we conducted a genome wide microRNAome analysis by applying miRNA-Seq in a rat model of experimental colitis, validated the data by QPCR, examined the expression of a selection of precursor and mature miRNAs, performed in depth biological interpretation using Ingenuity Pathway Analysis and tested the obtained results on samples derived from human patients. We identified specific, interdependent expression pattern of activator/repressor transcription factors, miRNAs and their direct targets in the inflamed colon samples. Particularly, decreased expression of the miR-200 family members (miR-200a/b/c,-141, and -429) and miR-27b correlates with the reduced level of their enhancers (HNF1B, E2F1), elevated expression of their repressors (ZEB2, NFKB1) and increased expression of their target genes (ZEB2, RUNX1). Moreover, the marked upregulation of six miR-27b target genes (IFI16, GCA, CYP1B1, RUNX1, MEF2C and MMP13) in the inflamed colon tissues is a possible direct consequence of the lack of repression due to the downregulated miRNA-27b expression. Our data indicate that changes in microRNAome are associated with the pathophysiology of IBD, consequently, microRNAs offer potential targets for the diagnosis, prognosis and treatment of IBD.