Surgical approach and etiopathogenetic considerations to the umbilical tumefactions in cattle: Case review in twenty years (2000/2020)

B S T R A C T Objective: Our investigation was conducted to evaluate the incidence of umbilical pathologies and the result of related surgical interventions in Sicilian cattle. Study design: 320 (214 females, 106 males) cattle with umbilical lesions were collected, anesthetized, operated on and follow-up recorded. We evaluated the injury incidence rate. Population: The animals considered were: high productivity dairy cattle (Holstein Friesian and Brown Swiss); meat breeds (Charolaise and Limousine) and some crosses. Results: The highest injury rate was simple omphalocele, followed by purulent omphalitis. A high percentage was found in young cattle. Friesians are the most prone to navel diseases. The dairy breeds, compared to the meat breeds, and double aptitude and crosses, were the most affected. The most common was simple omphalocele, with a small hernial port (1 to 3 cm), while the rest was greater than 3 cm, with severe organs or complications. Methods: the most commonly used surgery was herniorraphy with autologous reinforcement, while classic su- tures and mesh sutures were used in the remaining cases. Follow-up demonstrated healing in most of the treated subjects. Conclusions: The study aimed to estimate some surgical clinical cases in Sicilian cattle. Most injuries are lower abdomen injuries, mostly simple or complicated umbilical hernias. Young dairy females were the most affected. Meaning/Impact: Radical surgery is the treatment of choice in the vast majority of symptomatic or asymptomatic umbilical diseases; moreover, the correct management and the choice of an appropriate surgical approach allow to obtain an effective treatment of the lesions. Simple summary: In cattle farm, the correct management of births is a fundamental step for the future of the breeding. In fact, there are numerous critical factors in this phase: calving area management, umbilical disin- fection, correct administration of colostrum, management of individual pens. Generally, after a few hours from birth, the calf is transferred in individual pens, the walls of this box allow visual and tactile contact but avoid the possibility of mutual sucking of the umbilical region, a risk factor, in that area, of infections and hernias. In the study have been collected umbilical interventions carried out over twenty years by a team operating in south- eastern Sicily: Ragusa. Out of a total of 320 cases, have been reported: the prevalence of types of umbilical tumefactions, anesthetic techniques and surgical techniques performed. The purpose of the work was to highlight any predispositions of race, sex, age regarding the lesion considered and above all, the effectiveness of the surgical therap

Surface functionalization of extracellular vesicle nanoparticles with antibodies: a first study on the protein corona "variable"

To be profitably exploited in medicine, nanosized systems must be endowed with biocompatibility, targeting capability, the ability to evade the immune system, and resistance to clearance. Currently, biogenic nanoparticles, such as extracellular vesicles (EVs), are intensively investigated as the platform that naturally recapitulates these highly needed characteristics. EV native targeting properties and pharmacokinetics can be further augmented by decorating the EV surface with specific target ligands as antibodies. However, to date, studies dealing with the functionalization of the EV surface with proteins have never considered the protein corona "variable", namely the fact that extrinsic proteins may spontaneously adsorb on the EV surface, contributing to determine the surface, and in turn the biological identity of the EV. In this work, we explore and compare the two edge cases of EVs modified with the antibody Cetuximab (CTX) by chemisorption of CTX (through covalent binding via biorthogonal click-chemistry) and by formation of a physisorbed CTX corona. The results indicate that (i) no differences exist between the two formulations in terms of binding affinity imparted by molecular recognition of CTX versus its natural binding partner (epidermal growth factor receptor, EGFR), but (ii) significant differences emerge at the cellular level, where CTX-EVs prepared by click chemistry display superior binding and uptake toward target cells, very likely due to the higher robustness of the CTX anchorage

Assessing the effect of protein corona formation in the process of EV surface engineering

To be profitably exploited in medicine, nanosized systems must be endowed with biocompatibility, targeting capability, the ability to evade the immune system, and resistance to clearance. Currently, biogenic nanoparticles, such as Extracellular Vesicles (EVs), are intensively investigated as the platform that naturally recapitulates these highly needed characteristics. EV native targeting properties and pharmacokinetics can be further augmented by decorating the EV surface with specific target ligands as antibodies. However, up to date, works dealing with the functionalization of EV surface with proteins have never considered the biomolecular corona (BC) “variable”, namely the fact that extrinsic biomolecules, mainly proteins, may spontaneously adsorb on the EV surface in biofluids, contributing to determine the biological identity of the EV. In this work, we explore and compare EVs modified with the antibody Cetuximab (CTX) by chemisorption (covalent binding of CTX via biorthogonal click-chemistry) and by physisorption (formation of a CTX corona). Results (surprisingly) indicate that (i) no differences exist between the two formulations in terms of binding affinity imparted by molecular recognition of CTX versus its natural binding partner (epidermal growth factor receptor, EGFR), but (ii) significative differences emerge at the cellular level, where CTX-EVs prepared by click chemistry display superior binding and uptake toward target cells in Fetal Bovine Serum (FBS)-supplied culture medium, very likely due to the higher robustness of the CTX anchorage that resists to the formation of a BC due to interaction with the FBS proteins

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators' safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators’ safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (&lt;150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis

Comparing digital detection platforms in high sensitivity immune‐phenotyping of extracellular vesicles

Abstract Despite their clinical potential, Extracellular Vesicles (EVs) struggle to take the scene as a preeminent source of biomarkers in liquid biopsy. Limitations in the use of EVs origin from their inherent complexity and heterogeneity and from the sensitivity demand in detecting low to very low abundant disease‐specific sub‐populations. Such need can be met by digital detection, namely capable to reach the single‐molecule sensitivity. Here we set to compare, side by side, two digital detection platforms that have recently gained increasing importance in the field of EVs. The platforms, both commercially available, are based on the principles of the Single Particle Interferometric Reflectance Imaging Sensing (SP‐IRIS) and the Single Molecule Array technology (SiMoA) respectively. Sensitivity in immune‐phenotyping of a well characterized EV sample is reported, discussing possible applicative implications and rationales for alternative or complementary use of the two platforms in biomarker discovery or validation

Composite Peptide-Agarose Hydrogels for Robust and High-Sensitivity 3D Immunoassays

Canonical immunoassays rely on highly sensitive and specific capturing of circulating biomarkers by interacting biomolecular baits. In this frame, bioprobe immobilization in spatially discrete three-dimensional (3D) spots onto analytical surfaces by hydrogel encapsulation was shown to provide relevant advantages over conventional two-dimensional (2D) platforms. Yet, the broad application of 3D systems is still hampered by hurdles in matching their straightforward fabrication with optimal functional properties. Herein, we report on a composite hydrogel obtained by combining a self-assembling peptide (namely, Q3 peptide) with low-temperature gelling agarose that is proved to have simple and robust application in the fabrication of microdroplet arrays, overcoming hurdles and limitations commonly associated with 3D hydrogel assays. We demonstrate the real-case scenario feasibility of our 3D system in the profiling of Covid-19 patients' serum IgG immunoreactivity, which showed remarkably improved signal-to-noise ratio over canonical assays in the 2D format and exquisite specificity. Overall, the new two-component hydrogel widens the perspectives of hydrogel-based arrays and represents a step forward towards their routine use in analytical practices

Structure, Immunoreactivity, and In Silico Epitope Determination of SmSPI S. mansoni Serpin for Immunodiagnostic Application

The human parasitic disease Schistosomiasis is caused by the Schistosoma trematode flatworm that infects freshwaters in tropical regions of the world, particularly in Sub-Saharan Africa, South America, and the Far-East. It has also been observed as an emerging disease in Europe, due to increased immigration. In addition to improved therapeutic strategies, it is imperative to develop novel, rapid, and sensitive diagnostic tests that can detect the Schistosoma parasite, allowing timely treatment. Present diagnosis is difficult and involves microscopy-based detection of Schistosoma eggs in the feces. In this context, we present the 3.22 Å resolution crystal structure of the circulating antigen Serine protease inhibitor from S. mansoni (SmSPI), and we describe it as a potential serodiagnostic marker. Moreover, we identify three potential immunoreactive epitopes using in silico-based epitope mapping methods. Here, we confirm effective immune sera reactivity of the recombinant antigen, suggesting the further investigation of the protein and/or its predicted epitopes as serodiagnostic Schistosomiasis biomarkers

Structure, Immunoreactivity, and In Silico Epitope Determination of SmSPI S. mansoni Serpin for Immunodiagnostic Application

The human parasitic disease Schistosomiasis is caused by the Schistosoma trematode flatworm that infects freshwaters in tropical regions of the world, particularly in Sub-Saharan Africa, South America, and the Far-East. It has also been observed as an emerging disease in Europe, due to increased immigration. In addition to improved therapeutic strategies, it is imperative to develop novel, rapid, and sensitive diagnostic tests that can detect the Schistosoma parasite, allowing timely treatment. Present diagnosis is difficult and involves microscopy-based detection of Schistosoma eggs in the feces. In this context, we present the 3.22 angstrom resolution crystal structure of the circulating antigen Serine protease inhibitor from S. mansoni (SmSPI), and we describe it as a potential serodiagnostic marker. Moreover, we identify three potential immunoreactive epitopes using in silico-based epitope mapping methods. Here, we confirm effective immune sera reactivity of the recombinant antigen, suggesting the further investigation of the protein and/or its predicted epitopes as serodiagnostic Schistosomiasis biomarkers

Elucidating the 3D Structure of a Surface Membrane Antigen from Trypanosoma cruzi as a Serodiagnostic Biomarker of Chagas Disease

Chagas disease (CD) is a vector-borne parasitosis, caused by the protozoan parasite Trypanosoma cruzi, that affects millions of people worldwide. Although endemic in South America, CD is emerging throughout the world due to climate change and increased immigratory flux of infected people to non-endemic regions. Containing of the diffusion of CD is challenged by the asymptomatic nature of the disease in early infection stages and by the lack of a rapid and effective diagnostic test. With the aim of designing new serodiagnostic molecules to be implemented in a microarray-based diagnostic set-up for early screening of CD, herein, we report the recombinant production of the extracellular domain of a surface membrane antigen from T. cruzi (TcSMP) and confirm its ability to detect plasma antibodies from infected patients. Moreover, we describe its high-resolution (1.62 angstrom) crystal structure, to which in silico epitope predictions were applied in order to locate the most immunoreactive regions of TcSMP in order to guide the design of epitopes that may be used as an alternative to the full-length antigen for CD diagnosis. Two putative, linear epitopes, belonging to the same immunogenic region, were synthesized as free peptides, and their immunological properties were tested in vitro. Although both peptides were shown to adopt a structural conformation that allowed their recognition by polyclonal antibodies raised against the recombinant protein, they were not serodiagnostic for T. cruzi infections. Nevertheless, they represent good starting points for further iterative structure-based (re)design cycles