Mining and engineering activity in catalytic amyloids.

This chapter describes how to test different amyloid preparations for catalytic properties. We describe how to express, purify, prepare and test two types of pathological amyloid (tau and α-synuclein) and two functional amyloid proteins, namely CsgA from Escherichia coli and FapC from Pseudomonas. We therefore preface the methods section with an introduction to these two examples of functional amyloid and their remarkable structural and kinetic properties and high physical stability, which renders them very attractive for a range of nanotechnological designs, both for structural, medical and catalytic purposes. The simplicity and high surface exposure of the CsgA amyloid is particularly useful for the introduction of new functional properties and we therefore provide a computational protocol to graft active sites from an enzyme of interest into the amyloid structure. We hope that the methods described will inspire other researchers to explore the remarkable opportunities provided by bacterial functional amyloid in biotechnology.

Do Herbal Supplements and Probiotics Complement Antibiotics and Diet in the Management of SIBO? A Randomized Clinical Trial.

Small intestinal bacterial overgrowth (SIBO) arises from dysbiosis in the small intestine, manifesting with abdominal symptoms. This study aims to assess the efficacy of combined antibiotic therapy, herbal supplements, probiotics, and dietary modifications in SIBO management. A total of 179 SIBO-diagnosed patients underwent clinical evaluation and breath testing. Patients were categorized into hydrogen (H2-SIBO) and methane (CH4-SIBO) groups. The control group received standard antibiotic therapy and a low-FODMAP diet, while the intervention group received additional herbal antibiotics, probiotics, and prebiotics. After treatment, both groups exhibited reduced gas levels, particularly in CH4-SIBO. Clinical remission rates were higher in the intervention group, especially in CH4-SIBO cases. Logistic regression analysis showed gas concentrations at diagnosis as significant predictors of treatment success. In conclusion, adjunctive herbal supplements and probiotics did not significantly impact gas levels, but showed potential for clinical improvement, especially in CH4-SIBO.

Functional Amyloids: The Biomaterials of Tomorrow?

Functional amyloid (FAs), particularly the bacterial proteins CsgA and FapC, have many useful properties as biomaterials: high stability, efficient, and controllable formation of a single type of amyloid, easy availability as extracellular material in bacterial biofilm and flexible engineering to introduce new properties. CsgA in particular has already demonstrated its worth in hydrogels for stable gastrointestinal colonization and regenerative tissue engineering, cell-specific drug release, water-purification filters, and different biosensors. It also holds promise as catalytic amyloid; existing weak and unspecific activity can undoubtedly be improved by targeted engineering and benefit from the repetitive display of active sites on a surface. Unfortunately, FapC remains largely unexplored and no application is described so far. Since FapC shares many common features with CsgA, this opens the window to its development as a functional scaffold. The multiple imperfect repeats in CsgA and FapC form a platform to introduce novel properties, e.g., in connecting linkers of variable lengths. While exploitation of this potential is still at an early stage, particularly for FapC, a thorough understanding of their molecular properties will pave the way for multifunctional fibrils which can contribute toward solving many different societal challenges, ranging from CO2 fixation to hydrolysis of plastic nanoparticles.

Development of Small Molecules Targeting α-Synuclein Aggregation: A Promising Strategy to Treat Parkinson's Disease.

Parkinson's disease, the second most common neurodegenerative disorder worldwide, is characterized by the accumulation of protein deposits in the dopaminergic neurons. These deposits are primarily composed of aggregated forms of α-Synuclein (α-Syn). Despite the extensive research on this disease, only symptomatic treatments are currently available. However, in recent years, several compounds, mainly of an aromatic character, targeting α-Syn self-assembly and amyloid formation have been identified. These compounds, discovered by different approaches, are chemically diverse and exhibit a plethora of mechanisms of action. This work aims to provide a historical overview of the physiopathology and molecular aspects associated with Parkinson's disease and the current trends in small compound development to target α-Syn aggregation. Although these molecules are still under development, they constitute an important step toward discovering effective anti-aggregational therapies for Parkinson's disease.

Depression in Major Neurodegenerative Diseases and Strokes: A Critical Review of Similarities and Differences among Neurological Disorders.

Depression and anxiety are highly prevalent in most neurological disorders and can have a major impact on the patient's disability and quality of life. However, mostly due to the heterogeneity of symptoms and the complexity of the underlying comorbidities, depression can be difficult to diagnose, resulting in limited recognition and in undertreatment. The early detection and treatment of depression simultaneously with the neurological disorder is key to avoiding deterioration and further disability. Although the neurologist should be able to identify and treat depression initially, a neuropsychiatry team should be available for severe cases and those who are unresponsive to treatment. Neurologists should be also aware that in neurodegenerative diseases, such as Alzheimer's or Parkinson's, different depression symptoms could develop at different stages of the disease. The treatment options for depression in neurological diseases include drugs, cognitive-behavioral therapy, and somatic interventions, among others, but often, the evidence-based efficacy is limited and the results are highly variable. Here, we review recent research on the diagnosis and treatment of depression in the context of Alzheimer's disease, Parkinson's disease, and strokes, with the aim of identifying common approaches and solutions for its initial management by the neurologist.

Ligand-Based Discovery of a Small Molecule as Inhibitor of α-Synuclein Amyloid Formation.

α-Synuclein (α-Syn) aggregates are implicated in Parkinson's disease (PD), so inhibitors of α-Syn aggregation have been intensively explored. It has been demonstrated that small molecules might be able to reduce α-Syn aggregation in fibrils, thus exerting neuroprotective effects in models of PD. To expand our knowledge about the structural requirements for blocking the recognition process into the oligomeric assembly of α-Syn aggregates, we performed a ligand-based virtual screening procedure using two well-known α-Syn aggregation inhibitors, SynuClean-D and ZPD-2, as query compounds. A collection of thirty-four compounds bearing distinct chemical functionalities and mutual chemical features were studied in a Th-T fluorescence test, thus identifying 5-(2,6-dinitro-4-(trifluoromethyl)benzyl)-1-methyl-1H-tetrazole (named MeSC-04) as a potent α-Syn amyloid formation inhibitor that demonstrated similar behavior when compared to SynuClean-D in the thioflavin-T-monitored kinetic assays, with both molecules reducing the number and size of amyloid fibrils, as evidenced by electron microscopy. Molecular modeling studies suggested the binding mode of MeSC-04 through the identification of putative druggable pockets on α-syn fibrils and a subsequent consensus docking methodology. Overall, this work could furnish new insights in the development of α-Syn amyloid inhibitors from synthetic sources.

The small aromatic compound SynuClean-D inhibits the aggregation and seeded polymerization of multiple α-synuclein strains.

Parkinson's disease is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, as well as the accumulation of intraneuronal proteinaceous inclusions known as Lewy bodies and Lewy neurites. The major protein component of Lewy inclusions is the intrinsically disordered protein α-synuclein (α-Syn), which can adopt diverse amyloid structures. Different conformational strains of α-Syn have been proposed to be related to the onset of distinct synucleinopathies; however, how specific amyloid fibrils cause distinctive pathological traits is not clear. Here, we generated three different α-Syn amyloid conformations at different pH and salt concentrations and analyzed the activity of SynuClean-D (SC-D), a small aromatic molecule, on these strains. We show that incubation of α-Syn with SC-D reduced the formation of aggregates and the seeded polymerization of α-Syn in all cases. Moreover, we found that SC-D exhibited a general fibril disaggregation activity. Finally, we demonstrate that treatment with SC-D also reduced strain-specific intracellular accumulation of phosphorylated α-Syn inclusions. Taken together, we conclude that SC-D may be a promising hit compound to inhibit polymorphic α-Syn aggregation.

Discovery of Neuroprotective Agents Based on a 5-(4-Pyridinyl)-1,2,4-triazole Scaffold.

Parkinson's disease (PD) is characterized by the death of dopaminergic neurons. The common histopathological hallmark in PD patients is the formation of intracellular proteinaceous accumulations. The main constituent of these inclusions is alpha-synuclein (α-syn), an intrinsically disordered protein that in pathological conditions creates amyloid aggregates that lead to neurotoxicity and neurodegeneration. The main goal of our study was to optimize our previously identified α-syn aggregation inhibitors of 5-(4-pyridinyl)-1,2,4-triazole chemotype in terms of in vivo efficacy. Our efforts resulted in the identification of ethyl 2-((4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetate (15), which displayed the ability to prevent 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridine-induced bradykinesia as well as to affect the levels of PD markers after the administration of the same neurotoxin. In addition to the in vivo evaluation, for the 5-(4-pyridinyl)-1,2,4-triazole-based compounds, we measured the prevention of the fibrillization process using light scattering and a ThT binding assay; these compounds have been shown to slightly reduce the α-syn aggregation.

DisProt in 2022: improved quality and accessibility of protein intrinsic disorder annotation.

The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure.

One ring is sufficient to inhibit α-synuclein aggregation.

Parkinson's disease, the second most prevalent neurodegenerative disorder worldwide, is characterized by a progressive loss of dopaminergic neurons in substantia nigra pars compacta, causing motor symptoms. This disorder's main hallmark is the formation of intraneuronal protein inclusions, named Lewy bodies and neurites. The major component of these arrangements is α-synuclein, an intrinsically disordered and soluble protein that, in pathological conditions, can form toxic and cell-to-cell transmissible amyloid structures. Preventing α-synuclein aggregation has attracted significant effort in the search for a disease-modifying therapy for Parkinson's disease. Small molecules like SynuClean-D, epigallocatechin gallate, trodusquemine, or anle138b exemplify this therapeutic potential. Here, we describe a subset of compounds containing a single aromatic ring, like dopamine, ZPDm, gallic acid, or entacapone, which act as molecular chaperones against α-synuclein aggregation. The simplicity of their structures contrasts with the complexity of the aggregation process, yet the block efficiently α-synuclein assembly into amyloid fibrils, in many cases, redirecting the reaction towards the formation of non-toxic off-pathway oligomers. Moreover, some of these compounds can disentangle mature α-synuclein amyloid fibrils. Their simple structures allow structure-activity relationship analysis to elucidate the role of different functional groups in the inhibition of α-synuclein aggregation and fibril dismantling, making them informative lead scaffolds for the rational development of efficient drugs.

Design, synthesis and structure-activity evaluation of novel 2-pyridone-based inhibitors of α-synuclein aggregation with potentially improved BBB permeability.

The treatment of Parkinson's disease (PD), the second most common neurodegenerative human disorder, continues to be symptomatic. Development of drugs able to stop or at least slowdown PD progression would benefit several million people worldwide. SynuClean-D is a low molecular weight 2-pyridone-based promising drug candidate that inhibits the aggregation of α-synuclein in human cultured cells and prevents degeneration of dopaminergic neurons in a Caenorhabditis elegans model of PD. Improving SynuClean-D pharmacokinetic/pharmacodynamic properties, performing structure/activity studies and testing its efficacy in mammalian models of PD requires the use of gr-amounts of the compound. However, not enough compound is on sale, and no synthetic route has been reported until now, which hampers the molecule progress towards clinical trials. To circumvent those problems, we describe here an efficient and economical route that enables the synthesis of SynuClean-D with good yields as well as the synthesis of SynuClean-D derivatives. Structure-activity comparison of the new compounds with SynuClean-D reveals the functional groups of the molecule that can be disposed of without activity loss and those that are crucial to interfere with α-synuclein aggregation. Several of the derivatives obtained retain the parent's compound excellent in vitro anti-aggregative activity, without compromising its low toxicity. Computational predictions and preliminary testing indicate that the blood brain barrier (BBB) permeability of SynuClean-D is low. Importantly, several of the newly designed and obtained active derivatives are predicted to display good BBB permeability. The synthetic route developed here will facilitate their synthesis for BBB permeability determination and for efficacy testing in mammalian models of PD.

α-Helical peptidic scaffolds to target α-synuclein toxic species with nanomolar affinity.

α-Synuclein aggregation is a key driver of neurodegeneration in Parkinson's disease and related syndromes. Accordingly, obtaining a molecule that targets α-synuclein toxic assemblies with high affinity is a long-pursued objective. Here, we exploit the biophysical properties of toxic oligomers and amyloid fibrils to identify a family of α-helical peptides that bind to these α-synuclein species with low nanomolar affinity, without interfering with the monomeric functional protein. This activity is translated into a high anti-aggregation potency and the ability to abrogate oligomer-induced cell damage. Using a structure-guided search we identify a human peptide expressed in the brain and the gastrointestinal tract with analogous binding, anti-aggregation, and detoxifying properties. The chemical entities we describe here may represent a therapeutic avenue for the synucleinopathies and are promising tools to assist diagnosis by discriminating between native and toxic α-synuclein species.

Epilepsy, status epilepticus, and hemiplegic migraine coexisting with a novel SLC4A4 mutation.

BACKGROUND: Recessive mutations in the SLC4A4 gene cause a syndrome characterised by proximal renal tubular acidosis (pRTA), mental retardation, dental and ocular abnormalities, and hemiplegic migraine. Rare cases involving the development of epilepsy or its severe complication-status epilepticus-have been described. METHODS: The clinical and genetic status of four affected members in a Spanish family was studied. The SLC4A4 gene mutation was detected with a next-generation sequencing (NGS) panel in the proband, and Sanger confirmed the putative mutations in affected relatives. In silico analysis was performed to elucidate the putative effect of mutation on the splicing process. RESULTS: A novel mutation, c.2562+2T>G, was identified in the homozygous state in all diseased members of the family. This mutation affected a canonical splice site and is predicted to abolish the wild-type donor site, which predicts a premature truncated NBCe1 protein with cotransport activity. The resulting protein lacks the 190 amino acids of the carboxyl-terminus, and the effect is likely to be a loss of function. All patients suffered from severe pRTA and ocular abnormalities, and the adults also suffered from neurological complications, such as hemiplegic migraine and/or epilepsy. Two developed life-threatening status epilepticus, although they fully recovered and remained free of seizures with valproate. CONCLUSION: These results expand the clinical and mutational spectra of SLC4A4-related disease and have implications for understanding the potential role of NBCe1 in the pathophysiologic processes of hemiplegic migraine and epilepsy/status epilepticus associated with the mutation.

Inhibition of α-Synuclein Aggregation and Mature Fibril Disassembling With a Minimalistic Compound, ZPDm.

Synucleinopathies are a group of disorders characterized by the accumulation of α-Synuclein amyloid inclusions in the brain. Preventing α-Synuclein aggregation is challenging because of the disordered nature of the protein and the stochastic nature of fibrillogenesis, but, at the same time, it is a promising approach for therapeutic intervention in these pathologies. A high-throughput screening initiative allowed us to discover ZPDm, the smallest active molecule in a library of more than 14.000 compounds. Although the ZPDm structure is highly related to that of the previously described ZPD-2 aggregation inhibitor, we show here that their mechanisms of action are entirely different. ZPDm inhibits the aggregation of wild-type, A30P, and H50Q α-Synuclein variants in vitro and interferes with α-Synuclein seeded aggregation in protein misfolding cyclic amplification assays. However, ZPDm distinctive feature is its strong potency to dismantle preformed α-Synuclein amyloid fibrils. Studies in a Caenorhabditis elegans model of Parkinson's Disease, prove that these in vitro properties are translated into a significant reduction in the accumulation of α-Synuclein inclusions in ZPDm treated animals. Together with previous data, the present work illustrates how different chemical groups on top of a common molecular scaffold can result in divergent but complementary anti-amyloid activities.

Rational design of small molecules able to inhibit α-synuclein amyloid aggregation for the treatment of Parkinson's disease.

Parkinson's disease is one of the most common neurodegenerative disorders in elderly age. One of the mechanisms involved in the neurodegeneration appears related to the aggregation of the presynaptic protein alpha synuclein (α-syn) into toxic oligomers and fibrils. To date, no highly effective treatment is currently available; therefore, there is an increasing interest in the search of new therapeutic tools. The modulation of α-syn aggregation represents an emergent and promising disease-modifying strategy for reducing or blocking the neurodegenerative process. Herein, by combining in silico and in vitro screenings we initially identified 3-(cinnamylsulfanyl)-5-(4-pyridinyl)-1,2,4-triazol-4-amine (3) as α-syn aggregation inhibitor that was then considered a promising hit for the further design of a new series of small molecules. Therefore, we rationally designed new hit-derivatives that were synthesised and evaluated by biological assays. Lastly, the binding mode of the newer inhibitors was predicted by docking studies.

Chemical Chaperones as Novel Drugs for Parkinson's Disease.

Parkinson's disease (PD) is characterized by progressive loss of dopaminergic neurons and the accumulation of deposits of α-synuclein (α-syn) in the brain. The pivotal role of α-syn aggregation in PD makes it an attractive target for potential disease-modifying therapies. However, the disordered nature of the protein, its multistep aggregation mechanism, and the lack of structural information on intermediate species complicate the discovery of modulators of α-syn amyloid deposition. Despite these difficulties, small molecules have been shown to block the misfolding and aggregation of α-syn, and can even disentangle mature α-syn amyloid fibrils. In this review we provide an updated overview of these leading small compounds and discuss how these chemical chaperones hold great promise to alter the course of PD progression.

Improvement of the Efficiency and Completeness of Neuro-Oncology Patient Referrals to a Tertiary Center Through the Implementation of an Electronic Referral System: Retrospective Cohort Study.

BACKGROUND: Quality referrals to specialist care are key for prompt, optimal decisions about the management of patients with brain tumors. OBJECTIVE: This study aimed to determine the impact of introducing a Web-based, electronic referral (eReferral) system to a specialized neuro-oncology center, using a service-developed proforma, in terms of waiting times and information completeness. METHODS: We carried out a retrospective cohort study based on the review of medical records of referred adult patients, excluding follow-ups. Primary outcome measures were durations of three key phases within the referral pathway and completion rates of six referral fields. RESULTS: A total of 248 patients were referred to the specialist center during the study period. Median (IQR) diagnostic imaging to referral intervals were 3 (1-5) days with eReferrals, and 9 (4-19), 19 (14-49), and 8 (4-23) days with paper proforma, paper letter, and internal referrals, respectively (P<.001). Median (IQR) referral to multidisciplinary team decision intervals were 3 (2-7), 2 (1-3), 8 (2-24), and 3 (2-6) days respectively (P=.01). For patients having surgery, median (IQR) diagnostic imaging to surgery intervals were 28 (21-41), 34 (27-51), 104 (69-143), and 32 (15-89) days, respectively (P<.001). Proportions of complete fields differed significantly by referral type in all study fields (all with Ps <.001) except for details of presentation, which were present in all referrals. All study fields were always present in eReferrals, as these are compulsory for referral submission. Depending on the data field, level of completeness in the remaining referral types ranged within 69% (65/94) to 87% (82/94), 15% (3/20) to 65% (13/20), and 22% (8/41) to 63% (26/41) in paper proforma, paper letter, and internal referrals, respectively. CONCLUSIONS: An electronic, Web-based, service-developed specific proforma for neuro-oncology referrals performs significantly better, with shorter waiting times and greater completeness of information than other referral types. A wider application of eReferrals is an important first step to streamlining specialist care pathways and providing excellent care. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/10.2196/15002.

Canola oil/candelilla wax oleogel improves texture, retards staling and reduces in vitro starch digestibility of maize tortillas.

BACKGROUND: Maize tortilla staling is a major drawback that affects its commercialization and consumption, and so novel methods for retarding staling are continuously being explored. The present study evaluated the effect of adding a canola oil/candelilla wax oleogel (CWO; 0. 2, 4 and 6 g 100 g-1 ) to a basic masa formulation (water, 60 g 100 g-1 ; nixtamalized maize flour, 40 g 100 g-1 ) on the texture, staling and in vitro starch digestibility of maize tortillas made using a hot plate (200 °C). RESULTS: Textural analysis showed that CWO reduced hardness and increased the tensile strength of tortillas. Fourier transform infrared analysis indicated that the retrogradation of starch chains, quantified in terms of the intensity ratio 1047/1022, was reduced by oleogel incorporation. In vitro starch digestibility tests showed that tortillas containing CWO had lower readily digestible and slowly digestible starch fractions compared to the control tortilla without oleogel. CONCLUSION: The formation of amylose-lipid inclusion complexes and the formation of an oily physical barrier around starch granules were postulated as mechanisms underlying the reduced starch digestibility. © 2019 Society of Chemical Industry.

Uso de la vía intra-rectal en ratones CF1 para la administración de una combinación de acepromazina y midazolam como premedicación anestésica / Use of intra-rectal via in CF1 mice for the administration of a combination of acepromazin and midazolam as anesthetic premedication

En el ratón la vía de administración de drogas anestésicas es generalmente intraperitoneal, la cual requiere de una sujeción física particular que genera estrés. El objetivo del presente trabajo fue evaluar la vía intra-rectal (IR) para administrar una combinación de fármacos alternativa que permitiera, con bajas dosis de droga, una cómoda manipulación del animal; y además evaluar el impacto que produce la administración repetida de acepromacina/midazolam IR en ratones CF1. En un primer ensayo se emplearon 12 ratones CF1 a los que se le administró acepromazina maleato y midazolam clorhidrato por vía IR, previo vaciamiento manual de la ampolla rectal. En cada caso se registró latencia y duración del efecto. En todos los animales el tiempo de latencia fue de 3 a 5 minutos y la duración del efecto fue entre 30 y 60 minutos. En un segundo ensayo se utilizaron 10 ratones CF1 hembras. El grupo tratado fue de 7 animales a los que se les administró la misma dosis que al grupo anterior y por la misma vía, utilizando los mismos materiales y la misma técnica. El grupo control fueron 3 animales a los que se les administró igual volumen de solución fisiológica. La maniobra se repitió diariamente durante 12 días consecutivos. Al finalizar el ensayo se realizó eutanasia y necropsia de los animales tomándose muestras de recto y colon que fueron procesadas según técnicas de rutina. En los animales tratados no se observaron lesiones a nivel epitelial, reacción inflamatoria, ni congestión y edema vascular. No se observaron diferencias estadísticas significativas entre los animales tratados y los controles. La administración IR es una opción sencilla y rápida para el operador y segura para el animal. Asimismo, el uso de la vía IR para la administración repetida de esta combinación pre-anestésica no ha presentado efectos secundarios que deban ser considerados al momento de su utilización, constituyendo una alternativa que ofrece un excelente margen de seguridad y contribuyendo, por ser menos invasiva, al bienestar animal.

Intraperitoneal is the route of administration for anesthetic drugs generally used in mice. It requires a particular physical restraint that generates stress. The aim of the present work is to evaluate the intra-rectal (IR) route to administer an alternative drug combination that would allow, with low doses of drug, a comfortable handling of the animal and also to evaluate the impact produced by the repeated administration of acepromazine/midazolam IR in CF1 mice. At the initial stage, 12 CF1 mice were used. Each animal was administered acepromazine maleate and midazolam hydrochloride by IR using an intravenous Teflon catheter, previous manual emptying of the rectal ampoule, registering latency and duration of the effect in each case. In all the animals, the latency time recorded was 3 to 5 minutes and the duration of the effect was between 30 and 60 minutes. At the second stage, 10 female CF1 mice were used. The treated group was composed of 7 animals to which were administered the same dose as the previous group and by the same route, using the same materials and the same technique. The control group consisted of 3 animals that were administered the same volume of physiological solution. The maneuver was repeated daily for 12 consecutive days. At the end of the trial, the animals were euthanized. Necropsy examinations were made, taking samples of the rectum and colon that were processed according to routine techniques. There were no lesions at the epithelial level or inflammatory reaction, nor congestion and vascular edema in the treated animals. No significant statistical differences were observed between the treated animals and controls. The IR administration is a simple and fast option for the operator and it is safe for the animal. Additionally, the IR route for repeated administration of this pre-anesthetic combination has not presented side effects that should be considered at the time of use. It constitutes an alternative that offers an excellent margin of safety and contributes to animal welfare, as it is less invasive.