Bovine Blood Research Articles

Elasticity of whole blood clots measured via Volume Controlled Cavity Expansion

Measuring and understanding the mechanical properties of blood clots can provide insights into disease progression and the effectiveness of potential treatments. However, several limitations hinder the use of standard mechanical testing methods to measure the response of soft biological tissues, like blood clots. These tissues can be difficult to mount, and are inhomogeneous, irregular in shape, scarce, and valuable. To remedy this, we employ in this work Volume Controlled Cavity Expansion (VCCE), a technique that was recently developed, to measure local mechanical properties of soft materials in their natural environment. Through highly controlled volume expansion of a water bubble at the tip of an injection needle, paired with simultaneous measurement of the resisting pressure, we obtain a local signature of whole blood clot mechanical response. Comparing this data with predictive theoretical models, we find that a 1-term Ogden model is sufficient to capture the nonlinear elastic response observed in our experiments and produces shear modulus values that are comparable to values reported in the literature. Moreover, we find that bovine whole blood stored at 4 °C for greater than 2 days exhibits a statistically significant shift in the shear modulus from 2.53 ± 0.44 kPa on day 2 (N = 13) to 1.23 ± 0.18 kPa on day 3 (N = 14). In contrast to previously reported results, our samples did not exhibit viscoelastic rate sensitivity within strain rates ranging from 0.22 – 21.1 s−1. By surveying existing data on whole blood clots for comparison, we show that this technique provides highly repeatable and reliable results, hence we propose the more widespread adoption of VCCE as a path forward to building a better understanding of the mechanics of soft biological materials.

Bioinspired Polycation Functionalization of the Polyurethane Surface for Enhanced Lubrication, Antibacterial Property, and Anticoagulation

Endowing the implanted biomaterial with a wide range of functions, such as lubrication and antibacterial and anticoagulation properties, is essential for their safe clinical use. In the present study, based on the superlubrication mechanism of articular cartilage, a branched polyelectrolyte polymer (PEI-PMPC) was successfully synthesized based on tert-butyl hydroperoxide-initiated grafting polymerization and then applied to the biocompatible polyurethane (PU) surface. Specifically, the PU sheet was pretreated by polydopamine (PDA), and the PEI-PMPC polymer was grafted onto the PDA-modified PU surface by Schiff base reaction to generate a solid PDA/PEI-PMPC coating. The unmodified and modified surfaces were characterized by Fourier transform infrared spectroscopy, water contact angle, X-ray photoelectron spectroscopy, and surface zeta potential. To compare the lubrication, antibacterial behavior, and hemocompatibility, the relevant properties of the modified surfaces were evaluated by the tribological test, plate count method, surface morphology, hemolysis rate, activated partial thromboplastin time, and thrombin time. The experimental results demonstrated that the adhesion of bovine serum albumin, Escherichia coli, Staphylococcus aureus, platelets, and red blood cells was significantly decreased on the PEI-PMPC-modified PU surface compared with that of the unmodified PU surface. The multi-functional properties of the coating were attributed to the hydration layer formed by zwitterionic phosphorylcholine groups, and additionally, the cationic PEI served to kill the bacteria adhering to the surface. In summary, the bioinspired coating proposed in this study could potentially be used as a promising surface functionalization strategy for biomedical implants.

Construction and comparison of Prussian blue analogs nanomaterials with tailorable coordination metal ions as novel adsorbents for efficient structure-selective protein adsorption

Efficient and selective purify histidine (His)-rich proteins from complex biological sample for disease initiation and progression diagnostic is crucial, but it is still a challenge. Herein, for the first time, nanomaterials composed of Prussian blue analogues (PBAs) with tailorable coordination metal ions were synthesized and operated as adsorbents for selective separation of His-rich proteins. Benefiting from the strong metal affinity forces between the His residual in protein and the immobilized Cu2+, Ni2+ and Co2+ in CuFe, NiFe and CoFe PBAs, the obtained adsorbents exhibit highly selective adsorption performance for structure-selective proteins toward bovine hemoglobin (BHb). As a results, the maximum adsorption capacities of CuFe, NiFe and CoFe PBAs for BHb are 3365.8 mg g−1, 2421.3 mg g−1 and 1791.2 mg g−1, respectively. Moreover, the high-performance CuFe, NiFe and CoFe PBAs can be easily regenerated and used to effectively separation of BHb from real bovine blood samples, permitting their excellent practicality in the complex biological sample pretreatment, and further expanding the application scope of the traditional classical coordination compound in biochemical analysis fields.

Intralipid as a matrix additive for evaluating hyperlipidemic postmortem blood.

Postmortem whole blood samples can differ greatly in quality where hyperlipemia is a frequent variable that can influence the results of analytical methods. The aim of this study was to investigate the influence of lipemia on postmortem analysis as well as demonstrate the usage of Intralipid in comparison to pooled postmortem lipids as matrix additives for meaningful evaluation and validation of hyperlipidemic postmortem samples. Hyperlipidemic blood samples were simulated by adding different concentrations of Intralipid or pooled authentic postmortem lipids to bovine whole blood. The hyperlipidemic blood samples were spiked with 14 benzodiazepines and five sedative and antianxiety drugs (alprazolam, clonazepam, 7-aminoclonazepam, diazepam, flunitrazepam, 7-aminoflunitrazepam, hydroxyzine, lorazepam, midazolam, nitrazepam, 7-aminonitrazepam, nordazepam, oxazepam, propiomazine, dihydropropiomazine, temazepam, triazolam, zolpidem and zopiclone). Samples were prepared with liquid-liquid extraction followed by ultra-high performance liquid chromatography-mass spectrometry. The effects of lipemia on the recovery of analytes and internal standards (ISs) were evaluated to determine the effect of, and any differences between, the two additives. Lipemia was found to cause major interference when quantifying the analytes. For most analytes, the ISs could compensate for analyte losses. However, the most hydrophilic analytes (7-amino metabolites), together with the most lipophilic analytes (propiomazine and dihydropropiomazine), were greatly affected by lipemia (<50% recovery), and the IS could not compensate for analyte losses. In general, lower analyte recoveries were observed for samples with Intralipid as a lipemic additive in comparison to those containing pooled postmortem lipids. Both Intralipid and pooled postmortem lipids showed marked effects on the analytical results. Intralipid gave a good indication of the effects of lipemia and could be a useful tool for making a meaningful evaluation of hyperlipidemic postmortem samples during the method development and validation.

Development and Validation of a Repeatable Exsanguination Simulator Using Live Tissue (RESULT)

Introduction:Proper packing technique is crucial to minimize blood loss and successfully stop fatal junctional (axilla, groin, and neck) bleedings. Several medical simulators and mannequins are used to teach techniques for manual pressure and wound packing. Live tissue training (LTT) using animal models represents a high-fidelity simulator, but the number of times massive hemorrhage can be practiced is limited due to cumulative blood loss of the animal. Moreover, the animal’s potent coagulation limits the reuse of injuries. The study aimed at creating and validating a reproducible and repeatable exsanguination simulator to be used for high-volume training.Method:This study was approved by the regional animal ethics committee (Dnr 17953-2020). All animals were fully anesthetized throughout the duration of experiments. A repeatable exsanguination simulator using live tissue (RESULT) was created using commonly available materials to add reproducible junctional bleedings in an LTT context. A canister of porcine or bovine blood is connected to a standard gravity infusion set with roll clamp and pump chamber removed and added to a 100 cm 3-way stopcock connected to a 60 ml syringe. The free end of tubing is surgically inserted into the hind leg of a pig and placed inside a 5 cm long and 5 cm deep wound cavity. The simulator was evaluated with instructors controlling the rate of bleeding using the syringe while training participants packed the wound.Results:According to the instructors, participants benefited from the additional training made possible by using RESULT. Instructors received direct feedback on applied manual pressure and packing through the resistance of the syringe plunger. Moreover, participants found the increased number of repetitions beneficial to their training. The animals had no change in status from the multiple bleeding interventions.Conclusion:Both participants and instructors found the novel bleeding model useful for high-volume training in stopping massive junctional bleeding.

Use of Blood Powder (Ground and Irradiated) for the Manufacture of Chocolate Agar.

Chocolate agar (CA) is an enriched medium for the isolation and identification of fastidious bacteria. Defibrinated blood is used to manufacture CA, but this expensive product is not always affordable for companies in developing countries. Blood powder (BP) is potentially a cheaper alternative, although its pre-treatment using autoclaving can impair the quality of the media. Therefore, optimization of BP as a substitute for defibrinated blood for CA manufacture deserves further research. CA was manufactured with irradiated BP (dehydrated bovine blood powder) and its physical and microbiological characteristics were compared with those of conventional CA and CA prepared with autoclaved BP. Each medium was seeded with 20-200 CFU of target bacteria using the spiral pouring method. Finally, another medium was prepared using BP pre-treated by grinding and gamma irradiation and its performance assessed. Compared to conventional CA, the medium containing ground and irradiated BP provided a similar CFU count for both fastidious (Neisseria, Haemophilus, Campylobacter, and Streptococcus) and non-fastidious (Moraxella, Staphylococcus, Enterococcus, Klebsiella, and Pseudomonas) species, unlike the medium prepared with BP subjected only to irradiation, which provided a lower growth of fastidious species. Morphology and characteristics of all bacterial colonies were very similar in conventional CA and the new medium, the number of Pseudomonas CFU being higher in the latter. The medium prepared with ground plus irradiated vs. irradiated BP more closely resembled conventional CA, having a browner background. The new CA medium prepared with ground and gamma irradiation-sterilized BP has comparable productivity properties to conventional CA. Therefore, it could be a more practical and economical methodology to facilitate large-scale CA manufacture.

A disposable edoxaban sensor chip using carbon paste electrode grafted with molecularly imprinted polymer.

Although direct oral anticoagulants (DOACs) are generally safe and TDM is not required, blood levels of the drug are important information for response decisions in emergency care. In this study, an attempt was made to develop a disposable sensor chip for the rapid detection of edoxaban in blood, a type of DOAC. Molecularly imprinted polymers with edoxaban tosilate as a template and sodium p-styrene sulfonate as a functional monomer were grafted onto the surface of graphite particles, mixed with silicon oil dissolved in ferrocene to form a paste, and filled onto a substrate made of plastic film. Sensor chips were fabricated. The current obtained from this sensor by voltammetry within 150s depended on the edoxaban concentration. Sensitivity to edoxaban was also confirmed in bovine whole blood. The potential of disposable sensors to rapidly detect edoxaban in whole blood was demonstrated in this study, although selectivity, reproducibility, and sensitivity need to be improved for practical use.

Potential for Introduction of African Swine Fever Virus into High-Biosecurity Pig Farms by Flying Hematophagous Insects

Background. DNA of African swine fever virus (ASFV) has previously been detected in hematophagous insects on ASF outbreak farms. However, it remains unclear whether the viral DNA derived from blood meals that originated from pigs on the outbreak farm or was introduced from infected domestic pigs or wild boar sources located outside of the outbreak farm. Methods. We caught 644 hematophagous insects on the windows at two non-outbreak high-biosecurity pig farms (i.e., without ASFV-infected pigs) using plastic meshes coated with sticky glue, as well as 3576 hematophagous insects using H-traps on or around the farms. Using PCR analyses, we identified which insects were present, whether these hematophagous insects carried blood from an external (exogenous) source and if the blood contained ASFV DNA. Results. We found blood meals with ASFV DNA in one pool of five Haematopota spp. in the H-traps. From the window traps, we found 0–2.7% of Haematopota spp., Stomoxys calcitrans, and Aedes spp. carrying blood meals from exogenous sources into the farms. Some insects carried bovine blood; the closest registered source for this was 2500m from the pig farm. Conclusion. Hematophagous insects carrying ASFV-positive blood meals or blood meals from exogenous sources seem to be attracted to high-biosecurity pig farms and attempt to enter them through their windows. Despite the small percentage of insects carrying blood and the small amounts carried by each insect, the large numbers of insects result in a sufficient volume of exogenous blood, potentially containing ASFV, to constitute a non-negligible risk for ASFV-introduction into unprotected pig stables. This study is the first to provide quantitative data on the number of hematophagous insects trying to enter high-biosecurity pig farms. It is also the first to provide information about the origin of their blood meals, indicating that insect-borne introduction of blood containing ASFV into high-biosecurity pig farms is possible and, therefore, could potentially be responsible for some of the outbreaks observed during the summer peaks of infection.

Specifics of the use of bovine blood serum in the technology of cultivation of avian metapneumovirus

When obtaining virus-containing material of avian metapneumovirus infection (MPVI) on Vero cell culture, special attention should be paid to the composition of growth and supporting nutrient media, since their components directly affect the proliferation and viability of the cell culture, and hence the quality of the resulting target product.For Vero cell culture, the main nutrient medium is the DMEM medium, into which various additional components are added. It is customary to add up to 10% of fetal bovine serum (FBS) to the growth medium, as for the supporting medium, the question of the need to add blood serum of cattle (cattle) to it does not have an unambiguous solution. Since the financial costs of nutrient media have a significant impact on the cost of the final product, it is necessary to analyze the economic feasibility of using one or another component, in connection with which we have studied the role of blood serum of cattle (FBS and blood serum of adult cattle) in the technology of obtaining the MPVI virus.As a result of the conducted studies, it was revealed that in the growth medium for Vero cell culture, it is possible to replace FBS with blood serum of adult cattle with no more than two consecutive passages. However, if the Vero cell culture is replanted more than twice, then such a replacement is impossible, since already in the 3-4 passage there are signs of a decrease in the rate of cell division and cell culture degeneration. A large number of voids and rounded cells are noted in the cell layer, which makes it impossible to use it further for the cultivation of viruses or further replanting.It was also found that when cultivating the MPVI virus on a Vero cell culture, the DMEM maintenance medium containing 2% of cattle blood serum is optimal for use, since it ensures the production of virus-containing material with higher infectious activity compared to the DMEM medium, which does not contain or contains 10% of cattle blood serum at all.

A small-angle X-ray scattering study of red blood cells in continuous flow.

Owing to their large penetration depth and high resolution, X-rays are ideally suited to study structures and structural changes within intact biological cells. For this reason, X-ray-based techniques have been used to investigate adhesive cells on solid supports. However, these techniques cannot easily be transferred to the investigation of suspended cells in flow. Here, an X-ray compatible microfluidic device that serves as a sample delivery system and measurement environment for such studies is presented. As a proof of concept, the microfluidic device is applied to investigate chemically fixed bovine red blood cells by small-angle X-ray scattering (SAXS). A very good agreement is found between in-flow and static SAXS data. Moreover, the data are fitted with a hard-sphere model and screened Coulomb interactions to obtain the radius of the protein hemoglobin within the cells. Thus, the utility of this device for studying suspended cells with SAXS in continuous flow is demonstrated.

In vitro evaluation of Lactiplantibacillus plantarum HOKKAIDO strain, effective lactic acid bacteria for calf diarrhea.

Calf diarrhea adversely affects growth and sometimes results in mortality, leading to severe economic losses to the cattle industry. Antibiotics are useful in the treatment against bacterial diarrhea, but not against viral, protozoan, and antibiotic-resistant bacterial diarrhea. Therefore, there are growing requirements for a novel control method for calf diarrhea. Probiotics have been considered promising candidates for preventive and supportive therapy for calf diarrhea for many years. A recent study has revealed that Lactiplantibacillus plantarum HOKKAIDO strain (Lp-HKD) reduces intestinal pathology and the severity of diarrhea in bovine rotavirus (BRV)-infected calves. Lp-HKD is known to enhance the function of human immune cells and expected to be used as probiotics for humans. Therefore, it is hypothesized that Lp-HKD modulates antiviral immune response in cattle and provide the clinical benefits in BRV-infected calves. However, the detailed mechanism of Lp-HKD-induced immunomodulation remains unknown. Thus, this study aimed to elucidate the immunomodulatory and antiviral effects of Lp-HKD in cattle. Cultivation assay of bovine peripheral blood mononuclear cells (PBMCs) showed that live and heat-killed Lp-HKD stimulates the production of interleukin-1β (IL-1β), IL-6, IL-10, and interferon-γ (IFN-γ) from PBMCs. Stimulation by heat-killed Lp-HKD yielded stronger cytokine production than stimulation by the live Lp-HKD. Additionally, CD14+ monocytes were identified as major producers of IL-1β, IL-6, and IL-10 under Lp-HKD stimulation; however, IFN-γ was mainly produced from immune cells other than CD14+ monocytes. Depletion of CD14+ monocytes from the PBMCs cultivation strongly decreased cytokine production induced by heat-killed Lp-HKD. The inhibition of toll-like receptor (TLR) 2/4 signaling decreased IL-1β and IL-6 production induced by live Lp-HKD and IL-1β, IL-6, and IFN-γ production induced by heat-killed Lp-HKD. Furthermore, live or heat-killed Lp-HKD also activated T cells and their production of IFN-γ and tumor necrosis factor-α. Then, culture supernatants of bovine PBMCs treated with heat-killed Lp-HKD demonstrated antiviral effects against BRV in vitro. In conclusion, this study demonstrated that Lp-HKD activates the functions of bovine immune cells via TLR2/4 signaling and exerts an antiviral effect against BRV through the induction of antiviral cytokines. Lp-HKD could be useful for the prevention and treatment of calf diarrhea through its immune activating effect.

Blood from Horses and Cows In Vitro Exposed to Quaternium-15 and Thiacloprid: Haematology and Erythrocyte Osmotic Fragility Alterations

Blood cells are constantly exposed to several pollutants, including xenobiotics, and they can be considered a useful target for pollution exposition of the animal. The aim of this study was to investigate the effects of two xenobiotics (Quaternium-15, a preservative used in personal care products, and Thiacloprid, a neonicotinoid pesticide) on the haematological profile and erythrocyte osmotic fragility (EOF) of equine and bovine blood samples. Ten blood samples from horses and cows were exposed for 24 h to Quaternium-15 at a concentration of 0.1 and 1 mg/L and to Thiacloprid at a concentration of 30 and 60 µg/mL. A decrease in the values of the red blood cells, white blood cells, haematocrit, haemoglobin, mean corpuscular volume, and platelets, and an increase of EOF were found in blood samples exposed to xenobiotics compared to the control. According to the results gathered in the current study, the two xenobiotic compounds herein tested negatively affect the haematological indices causing haemolysis both in cattle and horse blood. This study, despite being preliminary, emphasizes the concept that blood cells are an excellent target for evaluating the effects of xenobiotics.

Assessment of an In Vitro Tick Feeding System for the Successful Feeding of Adult Rhipicephalus appendiculatus Ticks

This study assessed the efficiency of a new in vitro tick feeding system for the adult Rhipicephalus appendiculatus tick and compared the impact of different blood anticoagulating factors on their feeding process. A total of 10 feeders were each seeded with 30 or 60 R. appendiculatus adults. Bovine blood was added into each unit and changed every 12 h for 4 to 10 days during which tick attachment and engorgement was assessed. The tick attachment observed 4 days after feeding was 80.0% (48/60), 75.8% (182/240), and 70.8% (170/240) for lithium heparin, citrate phosphate dextrose, and defibrinated blood, respectively, with no significant difference (p &gt; 0.05) between the anticoagulants used. However, the ticks fed on heparinized and defibrinated blood reached repletion status. The in vitro tick feeding system was successfully used to feed adult R. appendiculatus ticks until repletion. This system could be used to facilitate studies on tick-pathogen interactions, such as those involved in the East Coast fever disease.

Identification of target genes and pathways related to heat tolerance in Chinese Holstein cows

Heat stress has detrimental effects on the production and reproduction of dairy cows. The present work reveals the role of differentially expressed (DE) miRNAs on the heat stress tolerance of bovine blood using RNA sequencing. Twelve dairy cows were divided into heat-resistant and heat-stressed groups according to rectal temperature and reduction of milk yield. Using the ACGT101-miR program, 1262 known miRNAs and 719 novel miRNAs were identified. Of the 719 novel miRNAs, 144 significantly DE miRNAs (67 up-regulated and 77 down-regulated) (P < 0.05) were screened between heat-resistant and heat-stressed cows using edgeR software. Eleven high DE miRNAs (bta-miR-455–3p, bta-miR-455–5p, mmu-mir-6240, bta-miR-106b, bta-miR-11,971, bta-miR-29a, bta-miR-142–5p, bta-miR-15b, bta-miR-342, bta-miR-32, and bta-miR-6524) were verified by qRT-PCR, an outcome which was consistent with the sequencing results. The target genes related to heat tolerance of the heat shock protein (HSP) family, malonyl CoA-acyl carrier protein transacylase (MACT), and prolactin receptor (PRLR), were predicted using the TargetScan and miRanda software. Furthermore, KEGG analyses with the R package OmicStudio and on the basis of DE miRNAs revealed an enriched MAPK signaling pathway, endocytosis, Th17 cell differentiation, cellular senescence, peroxisome, and Fc gamma R-mediated phagocytosis. These findings could help in screening for heat tolerance genes for improved dairy cow heat tolerance.

Disposable Sensor Chips with Molecularly Imprinted Carbon Paste Electrodes for Monitoring Anti-Epileptic Drugs.

Epilepsy is a neurological disorder that affects millions of people worldwide. Anti-epileptic drugs (AEDs) are critical for their management. However, the therapeutic window is narrow, and traditional laboratory-based therapeutic drug monitoring (TDM) methods can be time consuming and unsuitable for point-of-care testing. To address this issue, we developed a disposable sensor chip based on molecularly imprinted polymer-modified carbon paste electrodes (MIP-CPs) for the TDM of AEDs such as phenobarbital (PB), carbamazepine (CBZ), and levetiracetam (LEV). In this work, functional monomers (methacrylic acid) and crosslinking monomers (methylene bisacrylamide and ethylene glycol dimethacrylate) were copolymerized in the presence of the AED template and grafted on the graphite particles by simple radical photopolymerization. The grafted particles were mixed with silicon oil, dissolving ferrocene as a redox marker to make the MIP-carbon paste (CP). Disposable sensor chips were fabricated by packing the MIP-CP into the base made of poly (ethylene glycol terephthalate) (PET) film. The sensor's sensitivity was determined using differential pulse voltammetry (DPV), carried out on a single sensor chip for each operation. Linearity was obtained from 0-60 μg/mL in PB and LEV and 0-12 μg/mL in CBZ, covering their respective therapeutic range. The time taken for each measurement was around 2 min. The experiment using whole bovine blood and bovine plasma indicated that the existence of species that interfered had a negligible effect on the test's sensitivity. This disposable MIP sensor provides a promising approach for point-of-care testing and facilitating the management of epilepsy. Compared with existing tests, this sensor offers a faster and more accurate way to monitor AEDs, which is crucial for optimizing therapy and improving patient outcomes. Overall, the proposed disposable sensor chip based on MIP-CPs represents a significant advancement in AED monitoring, with the potential for rapid, accurate, and convenient point-of-care testing.

Artificial feeding of Ornithodoros rostratus using a silicone membrane system.

The in vitro feeding of ticks facilitates the conduction of studies involving the intrinsic vector-pathogen relationship, susceptibility tests, and resistance to acaricides, in addition to mimicking the use of experimental hosts. The objective of this study was to establish an in vitro feeding system using silicone membranes to supply various diets to the species Ornithodoros rostratus. Each experimental group included 130 first-instar O. rostratus nymphs. The groups were divided according to the diet provided: citrated rabbit blood, citrated bovine blood, bovine blood with antibiotics, and defibrinated bovine blood. The control group was fed directly on rabbits. Ticks were weighed before and after the feeding and monitored individually according to their biological parameters. The results of the experiment demonstrated that the proposed system was efficient in terms of fixation stimulus and satisfactory in terms of tick engorgement, which would allow the maintenance of O. rostratus colonies by using artificial feeding through silicone membranes. All diets provided were efficient for the maintenance of colonies, but the ticks that received citrated rabbit blood displayed similar biological parameters to those observed under in vivo feeding conditions.

Application of Multiparametric Flow Cytometry Panels to Study Lymphocyte Subpopulations in Tuberculin-Positive Cattle.

Flow cytometry (FC) is widely used in microbiology, immunology, hematology, and oncology. In the veterinary field, FC enabled the study of the immune response in cattle infected with different pathogens, as well as vaccine testing. However, few fluorochrome-conjugated antibodies recognize bovine antigens, limiting the possible benefits of FC and the implementation of multiparametric analysis for more complex studies. Two cytometry panels with five colors each were designed and implemented for the study and identification of populations and subpopulations of T cells derived from the peripheral blood mononuclear cells of dairy heifers. Both panels detected differences in T cell subpopulations between heifers positively and negatively tested for tuberculin; they detected overexpression of CD25+ and CD45RO+ in tuberculin-positive heifers after stimulation with a culture filtrate protein extract (CFPE) from Mycobacterium bovis (M. bovis). We identified subpopulations of T cells from peripheral blood mononuclear cells using two multicolor panels. These panels could be used to analyze total bovine blood in immunopathogenic studies and vaccine development. The same strategy could be implemented in other species of veterinary interest.

Molecularly Imprinted Polymers with Shape-Memorable Imprint Cavities for Efficient Separation of Hemoglobin from Blood.

Efficient separation and purification of hemoglobin from blood and other complicated biological fluids still remains a big challenge. Molecularly imprinted polymers (MIPs) of hemoglobin are potential choices; however, they suffer from severe problems including difficult template removal and low imprinting efficiency like other protein-imprinted polymers. Herein, a novel MIP of bovine hemoglobin (BHb) was designed in which a peptide crosslinker (PC), instead of the commonly used crosslinkers, was used. The PC, a random copolymer of lysine and alanine, adopts an α-helical conformation at pH 10 but transits to a random coil conformation at pH 5. The introduction of alanine residues lowers the pH range at which the PC undergoes helix-coil transition. The imprint cavities in the polymers are shape-memorable due to the reversible and precise helix-coil transition of the peptide segments in the polymers. They can be enlarged by lowering pH from 10 to 5, thus allowing complete removal of the template protein under mild conditions. When the pH is adjusted back to 10, their original size and shape will be recovered. Therefore, the MIP binds the template protein BHb with high affinity. Compared with the MIP crosslinked with the commonly used crosslinker, the imprinting efficiency of the PC-crosslinked MIP is significantly improved. In addition, both the maximum adsorption capacity (641.9 mg/g) and imprinting factor (7.2) are much higher than the BHb MIPs reported previously. The new BHb MIP also exhibits high selectivity toward BHb and good reusability. Thanks to the high adsorption capacity and high selectivity of the MIP, when it was applied to extract BHb from bovine blood, BHb in the blood sample was extracted almost completely, and high purity product was obtained.

The effect of crystal composition and environment on the color Doppler ultrasound twinkling artifact

Objective. Pathological mineralizations form throughout the body and can be difficult to detect using conventional imaging methods. Color Doppler ultrasound twinkling highlights ∼60% of kidney stones with a rapid color shift and is theorized to arise from crevice microbubbles as twinkling disappears on kidney stones at elevated pressures and scratched acrylic balls in ethanol. Twinkling also sometimes appears on other pathological mineralizations; however, it is unclear whether the etiology of twinkling is the same as for kidney stones. Approach. In this study, five cholesterol, calcium phosphate, and uric acid crystals were grown in vitro and imaged in Doppler mode with a research ultrasound system and L7-4 transducer in water. To evaluate the influence of pressure on twinkling, the same crystals were imaged in a high-pressure chamber. Then, the effect of surface tension on twinkling was evaluated by imaging crystals in different concentrations of surfactant (1%, 2%, 3%, 4%) and ethanol (10%, 30%, 50%, 70%), artificial urine, bovine blood, and a tissue-mimicking phantom. Main results. Results showed that all crystals twinkled in water, with cholesterol twinkling significantly more than calcium phosphate and uric acid. When the ambient pressure was increased, twinkling disappeared for all tested crystals when pressures reached 7 MPa (absolute) and reappeared when returned to ambient pressure (0.1 MPa). Similarly, twinkling across all crystals decreased with surface tension when imaged in the surfactant and ethanol (statistically significant when surface tension <22 mN m−1) and decreased in blood (surface tension = 52.7 mN m−1) but was unaffected by artificial urine (similar surface tension to water). In the tissue-mimicking phantom, twinkling increased for cholesterol and calcium phosphate crystals with no change observed in uric acid crystals. Significance. Overall, these results support the theory that bubbles are present on crystals and cause twinkling, which could be leveraged to improve twinkling for the detection of other pathological mineralizations.

Bovine blood and milk T-cell subsets in distinct states of activation and differentiation during subclinical Staphylococcus aureus mastitis

T-lymphocytes are key mediators of adaptive cellular immunity and knowledge about distinct subsets of these cells in healthy and infected mammary gland secretions remains limited. In this study, we used a multiplex cytometry panel to show that staphylococcal mastitis causes the activation of CD4+, CD8+ and γδ T-cells found in bovine milk. We also highlight remarkable differences in the proportions of naïve and memory T-cells subsets found in blood and milk. These observations will contribute to a better understanding of cell-mediated immune mechanisms in the udder and to the development of new therapeutic and preventive strategies targeting mastitis.