Hemodynamic Characteristics of a Tortuous Microvessel Using High-Fidelity Red Blood Cell Resolved Simulations.

OBJECTIVE: Tortuous microvessels are characteristic of microvascular remodeling associated with numerous physiological and pathological scenarios. Three-dimensional (3D) hemodynamics in tortuous microvessels influenced by red blood cells (RBCs), however, are largely unknown, and important questions remain. Is blood viscosity influenced by vessel tortuosity? How do RBC dynamics affect wall shear stress (WSS) patterns and the near-wall cell-free layer (CFL) over a range of conditions? The objective of this work was to parameterize hemodynamic characteristics unique to a tortuous microvessel. METHODS: RBC-resolved simulations were performed using an immersed boundary method-based 3D fluid dynamics solver. A representative tortuous microvessel was selected from a stimulated angiogenic network obtained from imaging of the rat mesentery and digitally reconstructed for the simulations. The representative microvessel was a venule with a diameter of approximately 20 µm. The model assumes a constant diameter along the vessel length and does not consider variations due to endothelial cell shapes or the endothelial surface layer. RESULTS: Microvessel tortuosity was observed to increase blood apparent viscosity compared to a straight tube by up to 26%. WSS spatial variations in high curvature regions reached 23.6 dyne/cm2 over the vessel cross-section. The magnitudes of WSS and CFL thickness variations due to tortuosity were strongly influenced by shear rate and negligibly influenced by tube hematocrit levels. CONCLUSIONS: New findings from this work reveal unique tortuosity-dependent hemodynamic characteristics over a range of conditions. The results provide new thought-provoking information to better understand the contribution of tortuous vessels in physiological and pathological processes and help improve reduced-order models.

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.

Site-Selective Antibody Conjugation with Dibromopyrazines.

In recent years, antibody conjugates have evolved as state-of-the-art options for diagnostic and therapeutic applications. During site-selective antibody conjugation, incomplete rebridging of antibody chains limits the homogeneity of conjugates and calls for the development of new rebridging agents. Herein, we report a dibromopyrazine derivative optimized to reach highly homogeneous conjugates rapidly and with high conversion on rebridging of trastuzumab, even providing a feasible route for antibody modification in acidic conditions. Furthermore, coupling a fluorescent dye and a cytotoxic drug resulted in effective antibody conjugates with excellent serum stability and in vitro selectivity, demonstrating the utility of the dibromopyrazine rebridging agent to produce on-demand future antibody conjugates for diagnostic or therapeutic applications.

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.

Age-Associated B Cell Features of the Murine High-Grade B Cell Lymphoma Bc.DLFL1 and Its Extranodal Expansion in Abdominal Adipose Tissues.

Diffuse large B cell lymphoma comprises a heterogeneous group of B cell-derived tumors, with different degrees of aggressiveness, as defined by their cellular origin and tissue microenvironment. Using the spontaneous Bc.DLFL1 lymphoma originating from a BALB/c mouse as a diffuse large B cell lymphoma model, in this study we demonstrate that the lymphoma cells display surface phenotype, IgH V-region somatic mutations, transcription factor characteristics and in vivo location to splenic extrafollicular regions of age-associated B cells (ABCs), corresponding to T-bet+ and Blimp-1+/CD138- plasmablasts derivation. The expansion of lymphoma cells within lymphoid tissues took place in a close arrangement with CD11c+ dendritic cells, whereas the extranodal infiltration occurred selectively in the mesentery and omentum containing resident gp38/podoplanin+ fibroblastic reticular cells. Antagonizing BAFF-R activity by mBR3-Fc soluble receptor fusion protein led to a significant delay of disease progression. The extranodal expansion of Bc.DLFL1 lymphoma within the omental and mesenteric adipose tissues was coupled with a significant change of the tissue cytokine landscape, including both shared alterations and tissue-specific variations. Our findings indicate that while Bc.DLFL1 cells of ABC origin retain the positioning pattern within lymphoid tissues of their physiological counterpart, they also expand in non-lymphoid tissues in a BAFF-dependent manner, where they may alter the adipose tissue microenvironment to support their extranodal growth.

Target-templated Construction of Functional Proteomimetics Using Photo-foldamer Libraries.

Current methods for proteomimetic engineering rely on structure-based design. Here we describe a design strategy that allows the construction of proteomimetics against challenging targets without a priori characterization of the target surface. Our approach relies on (i) a 100-membered photoreactive foldamer library, the members of which act as local surface mimetics, and (ii) the subsequent affinity maturation of the primary hits using systems chemistry. Two surface-oriented proteinogenic side chains drove the interactions between the short helical foldamer fragments and the proteins. Diazirine-based photo-crosslinking was applied to sensitively detected and localize binding even to shallow and dynamic patches on representatively difficult targets. Photo-foldamers identified functionally relevant protein interfaces, allosteric and previously unexplored targetable regions on the surface of STAT3 and an oncogenic K-Ras variant. Target-templated dynamic linking of foldamer hits resulted in two orders of magnitude affinity improvement in a single step. The dimeric K-Ras ligand mimicked protein-like catalytic functions. The photo-foldamer approach thus enables the highly efficient mapping of protein-protein interaction sites and provides a viable starting point for proteomimetic ligand development without a priori structural hypotheses.

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.

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.

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.

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.

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.

Splenectomy modulates the immune response but does not prevent joint inflammation in a mouse model of RA.

The spleen is the largest secondary lymphoid organ which is involved in the development of B cells and also in systemic (auto)immune responses. Using the recombinant human G1 domain-induced arthritis (GIA) model in splenectomized and control BALB/c mice, we investigated the role of the spleen in the induction and pathogenesis of autoimmune arthritis. Splenectomized mice developed GIA with a similar clinical picture to the control group. However, we observed significant alterations in the humoral and cellular immune responses in splenectomized mice. In the sera of the splenectomized mice, we found lower pro-inflammatory cytokine and anti-rhG1 IgM levels, but higher IL-4, anti-rhG1 IgG1 and anti-CCP and RF antibodies. The arthritis induction in the splenectomized group was associated with a significant expansion of activated helper T cells and an increase in the proportion of the circulating B1 and marginal zone B cell subsets. Importantly, immunization of the splenectomized mice with rhG1 induced the formation of germinal centers in the inguinal- and mesenteric lymph nodes (i/mLNs) which showed an active immune response to rhG1. Finally, both B and T cells from the mLNs of the splenectomized mice showed decreased intracellular Ca2+ signaling than those of the control group. Collectively, these findings indicate that the presence of the spleen is not critical for the induction of GIA, and in its absence the autoimmune arthritis is most likely promoted through the compensatory activity of the i/mLNs. However, our data implies the immunological role of the spleen in arthritis which could be further assessed in human RA.

Estimation of shear stress values along endothelial tip cells past the lumen of capillary sprouts.

Shear stress is recognized as a regulator of angiogenesis. However, the shear stress experienced by the endothelial cells of capillary sprouts remains unknown. The objective of this study was to estimate shear stress due to local interstitial flow along endothelial tip cells at the end of the capillary sprout lumen. Computational fluid dynamics were used to model flow within a blind-ended vessel, transendothelial flow across the vessel wall, and flow within the surrounding perivascular/interstitial space. Shear stress along the wall of the tip cells was calculated while varying sprout length, perivascular space channel width, and vessel wall hydraulic conductivity. Increasing sprout length, increasing wall hydraulic conductivity, and decreasing perivascular space width increased shear stress magnitude. Wall shear stress magnitude within the lumen ranged from 0.015 to 0.55 dyne/cm2 at the sprout entrance and linearly decreased to near zero at the base of the tip cells. Tip cell wall shear stress magnitude due to interstitial flow ranged from 0.009 to 4.65 dyne/cm2. In 3 out of 8 cases, shear stress magnitude was above 1 dyne/cm2 and considered physiologically relevant. The results provide a framework for discussing the role of local mechanical cues in regulating endothelial cell dynamics involved in angiogenesis. Mainly, interstitial flows may generate physiologically relevant shear stresses on tip cells in certain scenarios. This source of tip cell shear stress has not been previously considered or modeled.

A stepwise one-pot synthesis of aliphatic thiols and their derivatives from acrylamides and sulfur.

Elemental sulfur enables the convenient formation of C-S bonds and the direct incoporation of S-S bonds. The reactivity of easily accessible electron deficient alkenes towards sulfur, however, is barely disclosed. Herein, we investigated the reactivity of acrylamides with sulfur and eventually developed a new pseudo-multicomponent reaction for the preparation of polysulfides. Sequential one-pot reduction led to diversely substituted thiols. Additional third stage one-pot modifications provided thioethers, unsymmetric disulfide and thioester.

Experimental and computational study of BF3-catalyzed transformations of ortho-(pivaloylaminomethyl)benzaldehydes: an unexpected difference from TFA catalysis.

Previously, we have studied the trifluoroacetic acid (TFA)-catalyzed rearrangements of unsubstituted and alkoxy-substituted ortho-(pivaloylaminomethyl)benzaldehydes and revealed the formation of rearranged, regioisomeric aldehydes along with dimer-like products ("TFA dimers"). In the present study, related reactions of ortho-(pivaloylaminomethyl)benzaldehydes are described with the difference that boron trifluoride diethyl etherate (BF3·OEt2) is used as the catalyst. Although in these reactions the formation of the same "TFA dimers" can be observed after a couple of hours reaction time, during further stirring these are transformed into a new dimer-like keto compound ("BF3 dimer") that gradually becomes the main product. Apart from this, an oxoindene-type by-product is also formed. The new products are characterized by detailed NMR studies and two of them also by single-crystal X-ray diffraction. DFT calculations support the mechanism proposed for the transformations and explain the differences observed in the product distribution.

Foliate Lymphoid Aggregates as Novel Forms of Serous Lymphocyte Entry Sites of Peritoneal B Cells and High-Grade B Cell Lymphomas.

The cellular homeostasis of lymphoid tissues is determined by the continuous interactions of mobile hematopoietic cells within specialized microenvironments created by sessile stromal cells. In contrast to the lymph nodes and mucosal lymphoid tissues with well-defined entry and exit routes, the movement of leukocytes in the peritoneal cavity is largely unknown. In this study, we report that, in addition to the omental milky spots and fat-associated lymphoid clusters, in mice, the serous surface of the mesenteric adipose streaks contains lymphocyte-rich organoids comprised of a highly compacted leaf-like part connected to the adipose tissue that can also efficiently bind B cells and high-grade B cell lymphoma (diffuse large B cell lymphoma) cells. Denoted as foliate lymphoid aggregates (FLAgs), these structures show incomplete T/B segregation and a partially differentiated stromal architecture. LYVE-1-positive macrophages covering FLAgs efficiently bind i.p. injected normal B cells as well as different types of diffuse large B cell lymphoma cells. Within FLAgs, the lymphocytes compartmentalize according to their chemokine receptor pattern and subsequently migrate toward the mesenteric lymph nodes via the mesenteric lymphatic capillaries. The blood supply of FLAgs includes short vascular segments displaying peripheral lymph node addressin, and the extravasation of lymphocytes to the omental and mesenteric adipose tissues is partly mediated by L-selectin. The appearance of i.p. injected cells in mesenteric lymph nodes suggests that the mesentery-associated lymphatics may also collect leukocytes from the fat-associated lymphoid clusters and FLAgs, thus combining the mucosal and serous exit of mobile leukocytes and increasing the range of drainage sites for the peritoneal expansion of lymphoid malignancies.

The connection between manufacturer and private label brands and brand loyalty in chocolate bar buying decisions - A hybrid choice approach.

With the increase in health awareness more and more attention is paid to how manufacturers can respond to ever-changing consumer needs. This is especially true for the market of popular consumer goods such as chocolate. In order to understand Hungarian consumers' preferences in chocolate bars, we used the stated choice experiment method in our research. The attributes of our experiment included brand (manufacturer and private label), type (milk, dark, and white), a health claim (sugar free), as well as price, and our model estimations were done using the multinomial logit specification. In order to increase the explained rate of utility perceived by respondents, we also estimated a hybrid model containing a latent variable (representing consumers' brand loyalty). Our results reveal that the respondents showed a clear preference for manufacturer brands compared to private label brands. Regarding the type of chocolate, we found that milk chocolate received the most positive evaluation, which was followed by dark and white chocolate, respectively; we also demonstrated that sugar free products have a negative rating. In line with our preliminary expectations, a rise in the price of the product has a negative impact on utility as perceived by consumers. Brand loyalty is most characteristic of young and highly educated respondents, and a rise in brand loyalty lead to an increase in the preference towards manufacturer brand products compared to private label brand products.

Association between sociodemographic, obstetric, and lifestyle factors among Hungarian pregnant women-A cross-sectional study.

AIM: To learn the association between sociodemographic and obstetric factors and lifestyle characteristics of pregnant women, and to identify factors that can influence pregnant women's health consciousness. METHODS: A cross-sectional, questionnaire-based study was performed among women who gave birth in Szeged in 2014-2015. Data collection was based on a self-administered questionnaire and health documentations. Overall maternal health promoting behavior (MHPB) index was defined by summarizing the scores obtained from diet, physical activity, smoking status, and alcohol consumption. RESULTS: The final analysis included 1548 mothers; 41.3% (n = 602) of the sample had healthy diet, 9.0% (n = 134) were physically active and attended special pregnancy exercise classes, 84.4% (n = 1279) did not drink alcohol, and 93.5% (n = 1447) were nonsmokers. Regarding the MHPB index, 0.8% (n = 11) of the women reached the maximum score (20), while the average was 14.8 (SD = 2.58). Advanced maternal age (p < 0.001), having a spouse or partner (p < 0.001), higher educational level (p < 0.001), planned pregnancy (p < 0.001), and early visit at pregnancy care (p = 0.046) were significantly associated with higher MHPB index. CONCLUSION: The lifestyle of pregnant women can have a great impact on the developing fetus, either in a positive or negative way. In order to evaluate maternal lifestyle, overall health behavior should be considered. Lifestyle of the included women was not satisfactory, an improvement in health consciousness is needed at every social level; however, the differences between the various social classes may suggest the importance of further promotion and improvement of pregnancy planning and pregnancy care among younger and lower educated women.

A Study on the Direct Esterification of Monoalkylphosphates and Dialkylphosphates; The Conversion of the Latter Species to Trialkylphosphates by Alkylating Esterification.

The microwave (MW)-assisted direct esterification of certain P-acids is a green method. Quantum chemical calculations revealed that the activation enthalpy (ΔH#) for the exothermic monoalkylphosphate → dialkylphosphate transformation was on the average 156.6 kJ mol-1, while ΔH# for the dialkylphosphate → trialkylphosphate conversion was somewhat higher, 171.2 kJ mol-1, and the energetics of the elemental steps of this esterification was less favorable. The direct monoesterification may be performed on MW irradiation in the presence of a suitable ionic liquid additive. However, the second step, with the less favorable energetics as a whole, could not be promoted by MWs. Hence, dialkylphosphates had to be converted to triesters by another method that was alkylation. In this way, it was also possible to synthesize triesters with different alkyl groups. Eventually a green, P-chloride free MW-promoted two-step method was elaborated for the synthesis of phosphate triesters.

Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments.

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.