Tendon tissues connect muscle to bone allowing the transmission of forces resulting in joint movement. Tendon injuries are prevalent in society and the impact on public health is of utmost concern. Thus, clinical options for tendon treatments are in demand, and tissue engineering aims to provide reliable and successful long-term regenerative solutions. Moreover, the possibility of regulating cell fate by triggering intracellular pathways is a current challenge in regenerative medicine. In the last decade, the use of magnetic nanoparticles as nano-instructive tools has led to great advances in diagnostics and therapeutics. Recent advances using magnetic nanomaterials for regenerative medicine applications include the incorporation of magnetic biomaterials within 3D scaffolds resulting in mechanoresponsive systems with unprecedented properties and the use of nanomagnetic actuators to control cell signaling. Mechano-responsive scaffolds and nanomagnetic systems can act as mechanostimulation platforms to apply forces directly to single cells and multicellular biological tissues. As transmitters of forces in a localized manner, the approaches enable the downstream activation of key tenogenic signaling pathways. In this minireview, we provide a brief outlook on the tenogenic signaling pathways which are most associated with the conversion of mechanical input into biochemical signals, the novel bio-magnetic approaches which can activate these pathways, and the efforts to translate magnetic biomaterials into regenerative platforms for tendon repair. This review identifies the latest advances and potential of magnetically actuated biomaterials within the scope of tendon tissue engineering. The overuse of tendons is a significant problem in individuals who perform repetitive activities, both in sports and at work,4 and it is estimated that 30–50% of all injuries related to sports medicine involve tendinopathy.5,6 In fact, this musculoskeletal disease has a significant impact on health care system expenditure making the investigation of molecular mechanisms involved in tendon repair essential to develop novel treatment therapies.Tendons are transmitters of forces generated by muscle to the bone. Tendons are one of the tissues exposed to the most extreme mechanical forces in the body.7 Mechanobiology has revealed that such environmental cues and cellular mechanotransduction can be pivotal in a variety of responses, such as apoptosis, division, migration, and differentiation. Thus, given the recognition of the importance of biomechanical cues for mechanotransduction events, biomechano-responsive materials have emerged as promising platforms to realize biomedical functions.8,9 In the tendon tissue engineering field, the appropriate combination of teno-inductive cues such as appropriate cells, stimuli-responsive biomaterials, and mechanical stimuli is of key importance to boost tenogenic differentiation.10–14 Overall, biomechanical stimuli generated by either endogenous forces  or exogenous forces (ultrasound and magnetic forces), can be exploited as triggers for mechanoresponsive materials to be interfaced with biological systems.8Presently, tissue engineering is an emergent field that could become a real therapeutic option in the treatment of tendon injuries. As transmitters of forces and as mechanoresponsive tissues, the delivery of stimuli is of utmost importance in tissue engineering approaches aimed at tendon regeneration. Moreover, cells within tissues perceive a complex microenvironment in terms of extracellular signals, chemical compounds, and metabolic precursors and intermediates, or even physical properties of their surroundings.in vitro and in vivo, due to the feasibility of remote non-invasive actuation, post transplantation. Additionally, magnetic forces induced by a magnetic field can remotely and noninvasively activate the magneto-responsive components embedded in the scaffold matrix or attached to the cells. In this review, we briefly overview the tendon structure and the importance of mechanical stimulation to maintain tendon homeostasis, summarizing the signaling cascades involved in mechanotransduction. Finally, some insights are given into tackling tendon regeneration through magnetically assisted tissue engineering tools and magnetic biomaterials serving as mediators of mechanotransduction.Magnetically responsive biomaterials and magnetotherapy are potential actuators that may enable cell stimulation both 1.1.15,16 The mechanical properties of tendon tissue derive from type I collagen fibers that are arranged in dense parallel arrays.17 Tropocollagen is a triple-helix type I collagen molecule which is synthesized by tendon fibroblasts or tenocytes.18 A myofibril is five tropocollagen molecules stacked in a quarter-stage array17 and, in turn, neighboring microfibrils interdigitate and form a fibril which is the smallest tendon structural unit with a 10–500 nm diameter depending on species, age and location.19 Fibers are composed of collagen fibrils having a diameter between 3 and 7 μm, which are bound by the endotenon, a thin layer that contains blood vessels, lymphatics and nerves.19 An ensemble of fibers forms fascicles that are bundled together through a fascicular membrane designated epitenon,17 which is a fine, loose connective tissue containing vascular, lymphatic and nerve supply to the tendon.20 The fascicles, closely packed and arranged in parallel, form the tendon. Surrounding the epitenon, there is a layer of loose areolar connective tissue consisting of type I and type III collagen fibrils organized in a perpendicular direction called the paratenon.18 The set of these two layers that surround the tendon is called the peritendon, and it has the function of reducing friction with adjacent tissues.The tendon presents highly intricately organized structure that supports forces with large magnitudes between the muscles and bones during daily activities. This structure depends on the interaction between local cell types and regulation of extracellular matrix (ECM) remodeling.21,22 These fibroblast-like cells are capable of producing collagen type I and ECM components and can be found between collagen fibers and the endotenon.15,21,23 These cells are organized in linear arrays along the long axis of the tendon and interspersed between the collagen fibers.24 TSPCs are another type of tendon cell, recently discovered, which exhibit regenerative capacity and have an important role in tendon maintenance and repair.16,25,26 Directly surrounding the cells is the pericellular specialized matrix27 that may have an important role in mechanobiological mechanisms since it is composed of type VI collagen, elastin and fibrillin-1 which maintain the structural and biomechanical integrity of the tendon.27The conservation of the highly organized structure of this tissue is carried out by cells and it is crucial for maintaining mechanical properties and preventing injury. The three main cellular types present in tendons are tenoblasts, tenocytes and tendon stem/progenitor cells (TPCs/TSPCs). Tenoblasts can be stimulated to differentiate into tenocytes in response to various stimuli, such as exercise and trauma, in order to induce proliferation and matrix remodeling.28 Several of these proteins have the ability to regulate fibrillogenesis in terms of fibril diameter, alignment and stability.29–31 Elastin represents 1–2% of the tendon dry weight32 and provides flexibility for distension during unidirectional elongation.15 Furthermore, this protein has the ability to recover the configuration of fibers after mechanical loading.21 Among the most important glycoproteins are tenascin C and fibronectin, which enhance mechanical stability, allowing tendons to return to their prestretched lengths after physiological loading.33 Thrombospondin-4 (TSP4) is a glycoprotein abundant in mature tenocytes and is associated with fibrillar structures due to regulation of collagen assembly, organization, and ECM remodeling.34,35 Small leucine-rich proteoglycans (SLRPs) are abundant proteoglycans present in the ECM and function to regulate collagen fibril self-assembly;36 fibromodulin is one of the most expressed in tendons and crucial for the organization of the collagen fibril structure.37 Furthermore, other proteins are important for tendon development such as tenomodulin (TNMD), highly expressed in developing and mature tendons being a key marker for differentiated tenocytes.38 Also, TNMD was recently reported as a mechanosensitive gene required for proper tendon function since its expression in human TSPCs rapidly decreases in static cultures but is restored upon axial stretching, indicating the mechanosensitivity of the Tnmd gene.39In addition to collagen, the ECM is composed of other components, such as elastin, glycoproteins, proteoglycans and other molecules like collagen oligomeric matrix protein (COMP) and lubrican.2.40 Moreover, tendons present high mechanical strength and good flexibility and viscoelasticity which make this tissue more deformable at low strain rates and less deformable at high strain rates. However, at low strain rates tendons absorb more mechanical energy but are less effective in transmitting loads. At higher strain rates, tendons become stiffer and more effective in transmitting high muscular loads to bones.19,33 In addition to this mechanical behavior, each tendon has different mechanical properties depending on the function it performs in the body. For example, the human patellar tendon exhibits a Young's modulus of 660 ± 226 MPa  and tissue inhibitors of metalloproteinases (TIMPs).44As discussed above, tendon mechanical properties derive largely from type I collagen fibers which are arranged in dense parallel arrays. This arrangement results in a resilient tissue with high tensile stiffness in the direction of fiber orientation. 226 MPa  whereas 33 resulting in changes in growth factor or cytokine expression.17 In this context, tenocyte sensitivity to their environment has been well studied in vivo and in vitro.45–49 There are four mechanisms by which cells respond to mechanical forces such as activation of ion channels, release of ATP, changes in cytoplasmic filament organization and composition, alteration of protein expression and secretion of MMPs.50 It is suggested that mechanical loading induces biochemical changes which, in turn, increase repair and remodeling by tenocyte activity stimulation. However, overloading of the tissue leads to injuries, altering its structure and mechanical properties, and increases the production of inflammatory mediators.51,52 On the other hand, insufficient mechanical loading leads to changes in the cellular shape, number of cells and collagen fiber alignment, which culminate in degeneration of the tissue. Moreover, the absence of mechanical forces leads to tissue atrophy and, consequently, loss of its weight, stiffness, and the capacity to support tensile forces without being damaged.19,33Tendons are mechanosensitive tissues that respond to load in an adaptive manner, which means that the tendon alters its biological structure and its mechanical behavior in response to various mechanical stimuli2.1.53 Mechanotransduction is the ability of cells to respond to mechanical stimuli through biochemical signals.50 These stimuli are transduced by cells to stimulate biochemical pathways and effective cellular processes such as differentiation, proliferation, tissue development and skeletal maintenance.54The coordination of cell growth and proliferation with the production of the ECM is responsible for tissue homeostasis. This is achieved by cell signaling, in which a cell secretes a cytokine acting on that same cell (autocrine activity) or on another cell (paracrine activity), which regulates tissue remodeling.50Cells can perceive external mechanical stimuli through integrins, cadherins, catenins, stretch-activated ion-channels, and growth factor receptors. Integrins are transmembrane heterodimer proteins composed of α and β subunits that physically couple the ECM to the cytoskeleton through linker proteins, conveying forces between the inside and outside of the cell.55 and in hMSCs.56,57 Particularly in tendons, collagen I-binding integrins, α1, α2 and α11, were strongly upregulated and the integrin downstream kinases p38 and ERK1/2 were activated in mechanically loaded TSPCs.58The manipulation of integrin attached magnetic particles and internalized particles has been shown to induce intracellular calcium signalling in human osteoblasts59 with the main growth factors involved in vertebrate tendon development being transforming growth factor (TGF)-β and fibroblast growth factor (FGF), which are transduced via SMAD2/3 and ERK/MAPK cascades, respectively.60 Moreover, the bone morphogenetic protein (BMP) related members of the TGF-β family are elevated in early tendon healing processes and are transduced via the BMP/SMAD1/5/8 signaling pathway.61,62 The family of TGF-β ligands includes TGF-βs, activins, NODAL, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and the anti-Müllerian hormone (AMH),63 and the mechanism of signaling constitutes a cascade of phosphorylation events to transduce the signal to the nucleus and consequently regulate gene expression , the homeobox protein Mohawk (Mkx), and the zinc finger transcription factor early growth response 1 (Egr1). Scx is an early tendon specific marker, associated with ECM organization and development of functional 3.72,73 The magnetic fields applied in these strategies can be either static magnetic fields (SMFs) or electromagnetic fields (EMFs): the SMFs are constant fields that exert an attractive force on metallic objects, so magnets are commonly used for this purpose; the EMFs result from a combination of electric and magnetic fields, that is, the magnetic field is produced by the movement of electrically charged particles. The most commonly used EMF is a pulsed electromagnetic field (PEMF) since it is FDA approved, non-invasive,74 can be applied directly to the treatment site, and also enables varying signal configurations to modulate the cells' response at the molecular level, acting as a mediator of inflammation in the treatment of tendinopathic disorders or to prevent post-operative re-tears.75,76Mechanical loads generated by gravity and locomotion stimulate tendon remodeling to maintain the optimal mechanical performance of the tissue. Magnetic mechano-actuation is an interesting approach to remotely deliver mechanical stimulation directly to individual cells, as magnetic nanoparticles (MNPs) attached to the cell membrane can be manipulated using an oscillating gradient of external magnetic fields thus applying forces in the pN range to the particles and activating the receptors.11,77,78 and custom-designed systems can be developed according to the purpose of the application in the field.79–83 Combination strategies which include magnetic stimulation and magnetic biomaterials may boost cell signal transduction by means of remote activation of mechanotransduction pathways.Commercial devices are available which are capable of generating magnetic fields with different patterns3.1.3O4) or maghemite (Fe2O3) and are often polymer coated84 to improve their biocompatibility and structural and colloidal stability, while providing functional groups for bioactive molecule and/or ligand conjugation for targeting cells or tissues.85 Polymeric composites incorporating superparamagnetic iron oxide particles is one approach for remotely actuate biomaterials8 using magnetic fields as exogenous mechanical triggers to exert forces over seeded layers of cells. Thus, the incorporation of MNPs within a 3D scaffold or hydrogel and/or the association of MNPs with stem cells result in magnetically responsive systems suitable for tissue engineering applications.85–87Over the past few years, there has been considerable interest in magnetic biomaterials in biomedicine, in particular because the properties of these materials can be controlled in a remote fashion enabling non-invasive (noncontact) forms of actuation. The use of MNPs, especially superparamagnetic iron oxide nanoparticles (SPIONs), in biomedical and tissue engineering applications has exceeded expectations, mainly because of their superparamagnetic behavior which makes them desirable as a magnetic-targeting tool for medical applications. SPIONs are composed of a magnetic core of magnetite  or a combination of translational and torque forces, which are transmitted directly to the cell membrane or the cytoskeleton, and can be varied in three dimensions within a scaffold.89Sapir-Lekhovitser and co-workers hypothesized that magnetic fields coupled with magnetizable nanoparticles embedded within 3D scaffold structures remotely create transient physical forces that can be transferrable to cells present in close proximity to the nanoparticles.11 Moreover, and to understand the effect of magnetically actuated biomaterials in modulating the inflammatory process of tendons, a magnetically actuated SPCL membrane was produced and implanted subcutaneously in rats.77 The results showed that the magnetic membranes under PEMF stimulation maintain metabolic activity, proliferation and reactive oxygen species production by hASCs as well as preventing the formation of scar tissue by decreasing the presence of profibrotic inflammatory cells surrounding the explanted biomaterials.77 More recently, Tomás et al. used the setup proposed in a previous study90 to produce yarns of continuous and aligned electrospun threads of PCL and cellulose nanocrystals (CNCs) coated with iron oxide magnetic nanoparticles, resulting in magnetically responsive fibrous scaffolds.91 Cell studies revealed that magneto-mechanical stimulation of hASCs promotes higher degrees of cell cytoskeleton anisotropic organization, increased expression of tendon-related markers, and an anti-inflammatory gene profile. This work suggests a synergistic effect of nanotopography and magneto-mechanical actuation on the tenogenic commitment.91Our group has been keen on developing magnetic systems for tendon tissue engineering purposes. The effect of an externally applied magnetic field on tenogenic differentiation of human adipose stem cells (hASCs) was assessed by culturing cells on a magnetic scaffold. An aligned fibrous structure of starch with poly(ε-caprolactone) (SPCL) incorporating iron oxide MNPs was fabricated by 3D printing technology. The results showed that the effect of the aligned magnetic scaffolds combined with magnetic stimulation promotes tenogenic differentiation of hASCs suggesting the potential of the developed system to activate mechanotransduction pathways that are responsible for tenogenic commitment.92 and tropoelastin magnetic sponge-like hydrogels78 were also developed as 3D carriers of magnetic fields to the cells modulating the biochemical, physical and mechanical properties of the surrounding environment. By the application of EMF stimulation, it was possible to control the intrinsic properties of the constructs. Moreover, EMF stimulation of human tendon-derived cells and osteogenically differentiated hASCs was capable of modulating the cellular response of both cellular types.92Magnetically responsive hydrogels of methacrylated chondroitin sulfate (MA-CS) coated with iron-based MNPsIn summary, magnetic materials have the potential to enhance cell behavior promoting the activation of signaling pathways involved in tendon development and homeostasis by delivery of mechanical cues through remote generation of an external magnetic field.3.2.93,94 which have been functionalized with specific receptor targets which can be mechano-activated via remote magnetic fields. Magnetic mechano-activation remotely delivers mechanical stimuli directly to cells which are transmitted through activation of mechanically sensitive receptors available on the cell membrane. This activation initiates signaling pathways enabling cells to respond to mechanical cues in the environment through biochemical signals that dictate downstream cellular responses in many cases leading to differentiation. The use of MNPs, magnetic biomaterials, and magnetic fields is increasingly becoming a hot topic in regenerative medicine to regulate cell fate by manipulating mechanotransduction  for tissue engineering in in vitro and in vivo approaches. To target the mechano-responsive ion channel TREK-1, hMSCs were labeled with TREK-1 functionalized MNPs under magnetic stimulation. The results demonstrated that this approach can directly stimulate cells and selectively activate the mechanosensitive ion channel TREK-1 promoting osteogenic differentiation of hMSCs.57,96–98 Magnetic mechano-activation was also explored to induce tenogenic differentiation of hASCs which were labelled with MNPs functionalized with anti-activin receptor type IIA antibody to remotely activate the TGF-β/Smad2/3 signaling pathway. The results showed phosphorylation of Smad2/3 proteins in MNPs–ActRIIA tagged hASCs potentiating the commitment into the tenogenic lineage via TGF-β/Smad2/3.101 These findings emphasize the role of magnetic actuation in the activation of cell receptors and triggering of signaling cascades, giving rise to a wide range of opportunities to remotely control stem cell commitment. Recent work has translated this approach to large animal models for bone regeneration.96 However, more investigation is needed to fully understand the effect of this approach on tendon regeneration, including studies to explore different target receptors, combining this approach with magnetic biomaterials, and optimizing instrumentation for delivering magnetic stimuli.Previous studies have explored the use of MNPs targeting PDGF,4.in vivo and the ultimate translation of this technology constitutes an important challenge in the field due to the lack of non-invasive techniques which can be tuned at the tissue level.It is becoming increasingly clear that understanding the basis of mechanotransduction plays an important role in developing successful tissue engineering and regenerative medicine therapies. Scaffolding systems can serve as valuable platforms for studying cell mechanotransduction in three-dimensional environments that recap the native cellular niche. Using magnetic biomaterials and magnetic mechano-actuation, we can further explore novel approaches and concepts to target key challenges in the field. Hence, unexplored areas such as triggering mechanotransduction using the most recent nanotechnology tools for tendon tissue engineering present major opportunities for research on advancing regenerative solutions. Magnetic actuation of cells and cellular constructs and promotion of mechanotransduction shed light on the remote activation of intracellular responses and tissue formation in cell therapies. Furthermore, combining magnetic biomaterials and remote magnetic mechano-activation of signaling pathways is a potential regenerative magnetic toolbox as yet hardly explored in tendon tissue engineering, which might harness new biomedical possibilities in the regeneration of tendons. Concomitantly, the delivery of functional stimuli There are no conflicts to declare.


Selection of a single dominant follicle from a cohort of growing follicles is a unique biological process, a key step in female reproductive function in monovular species, and lies at the core of reproductive technologies in cattle. Follicle growth and the number of follicles that ovulate are regulated by precise endocrine, paracrine, and autocrine mechanisms. Most of our current understanding about follicle selection focuses on the role of FSH, LH, and the IGF family in follicle growth and selection of the dominant follicle. However, more recently the role of members of the TGF-ß family has been highlighted, particularly in high fecundity genotypes in sheep. Intercellular signaling between the oocyte and granulosa cells (GC) regulates proliferation and differentiation due to actions of bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) within the follicle. Mutations that either knockout or reduce the activity of BMP15 or GDF9 have been found to increase ovulation rate in heterozygotes and generally cause severe follicle abnormalities in homozygotes. A mutation in the intracellular kinase domain of the BMPR1B receptor (Booroola fecundity gene) increases ovulation rate in heterozygotes with further increases in ovulation in homozygotes. The physiological mechanisms linking these mutations to increased ovulation rates are still not well defined. A recently identified high fecundity bovine genotype, Trio, causes increased expression of SMAD6, an intracellular inhibitor of the BMP15/GDF9 signalling pathways. This bovine model has provided insights into the mechanisms associated with selection of multiple dominant follicles and multiple ovulations in carriers of fecundity alleles. The present review focuses on the mechanisms involved in follicle selection in ruminants with a special emphasis on the contribution made by multiple ovulation models in both cattle and sheep. The evaluation of multiple ovulation models in ruminants has allowed us to construct a new physiological model that relates changes in the BMP15/GDF9 signalling pathways to the physiological changes that result in selection of multiple dominant follicles. This model is characterized by acquisition of dominance at a smaller follicle size but at a similar time in the follicular wave with multiple follicles acquiring dominance in a hierarchal sequence, delaying FSH suppression and, thus allowing additional follicles to continue to grow and acquire dominance. Selection of a single dominant follicle from a cohort of growing follicles is a unique biological process and a key step in female reproductive function. Disturbances in this process can lead to anovulation and infertility or, alternatively, multiple ovulations and multiple births. The improvement in our understanding of follicle development and selection has fueled the development of synchronization protocols for fixed time artificial insemination as well as the development of other reproductive biotechnologies such as superovulation and embryo transfer. As a result, these advances have highlighted the importance of optimal follicle growth and selection as a critical step towards achieving reproductive efficiency in livestock species in order to feed a growing world population.Alterations in follicle selection can lead to the occurrence of codominant follicles and multiple ovulations which are the basis for dizygotic twinning in cattle and sheep. A particularly useful approach, suitable for study of many biological processes, is the examination of abnormal phenotypes. In this regard the occurrence of multiple ovulations in otherwise monovular species provides a physiological model in which the follicle selection mechanism has been altered in such a way to allow multiple follicles to be selected and ovulate. The available multiple ovulation models in sheep . Subsequent growth of the primary, secondary, and early antral follicles involves FSH and a series of paracrine factors, such as C-type natriuretic peptide, that stimulate follicle growth allowing the growing follicles to overcome inhibitory pathways, such as the Hippo pathway, ultimately allowing follicles to enter the antral stages of follicle development . The final development stages of antral follicle growth occurs in a wave-like pattern in ruminants, a model first proposed by Follicle development is an essential aspect of female reproduction with intricate mechanisms driving all stages of this process. Initial follicle development involves gradual activation of primordial follicles, which occurs throughout the reproductive lifespan of the female. The complex mechanisms involved in primordial follicle activation are slowly becoming clearer with a key role for KIT ligand from the granulosa cells (GC) activating PI3K/Akt/mTOR pathways in the oocyte and eventually blocking the transcriptional machinery that tonically inhibits primordial follicle activation, such as Foxo3a and p27  undergo regression . The folBos taurus and Bos indicus breeds , as shown by the inhibition of follicle growth less than 8 h after FSH suppression and the growth cessation of the largest subordinate follicle within 8 h after the future dominant follicle reaches 8.5 mm in diameter . Evidenc1) deviation occurs in association with FSH concentration reaching its nadir ; 2) initCirculating luteinizing hormone (LH) has also been implicated in follicle selection. Perhaps the most compelling piece of evidence for the role of LH in follicle selection is the lack of continuous growth of the dominant follicle beyond deviation in the absence of LH pulses .Follicle selection and acquisition of dominance not only involves morphological and endocrine changes but also profound changes in follicular fluid, GC and theca cells (TC). Among the intra-follicular components, E2 concentration has been one of the best characterized changes associated with diameter deviation and acquisition of dominance. Intrafollicular E2 has been shown to increase in the future dominant follicle at or immediately after deviation, and this is associated with increased mRNA expression for CYP19A1 (aromatase) in GC . The induction of LHCGR in GC appears to be stimulated, at least in part, by FSH and is mediated by increases in cAMP  in GC has been proposed as one of the initial signatures of the dominant follicle phenotype  using triplet, quadruplet, and quintuplet born ewes and a quintuplet born ram obtained from a commercial operation, Booroola, owned by the Seears Brothers in Cooma, NSW, Australia in 1958 . Since tI, Hannah/FecXH and FecGH led to the discovery of the role of TGF-β signaling in folliculogenesis and ovulation rate. The lines listed above resulted in the identification of two oocyte- secreted factors (OSF), namely bone morphogenetic protein 15 (BMP15), and growth and differentiation factor-9 (GDF9), as key regulators of GC function  the effect of each factor depends on the species of origin for BMP15 and GDF9, as well as the species of origin of the GC; and 3) a cooperative effect between BMP15 and GDF9 has been observed in all species evaluated, albeit differences appear to exist among species regarding which particular GC function is affected by the cooperation. In addition, recent results indicate that BMP15 and GDF9 proteins can occur as dimers, monomers, and multimers and that components of the immature protein such as the pro-region may have important roles in the biological effects of these OSF (The results previously described emphasize that: 1) both GDF9 and BMP15 regulate GC proliferation and differentiation measured through P4 production Multiple ovulation models in cattle can be divided into three main types, namely: the USDA- MARC twinner population . GenomicOne of the most replicated regions is the bovine chromosome 5 segment containing IGF1. QTL mapping work in both the Norwegian dairy cattle population and the US Holstein population has provided strong support for a contribution of this genomic region to variation in twinning rate. In a follow-up study, polymorphisms in the IGF1 gene were identified and tested for association in two samples of US Holstein sires representing different timeframes. IGF1 polymorphisms were identified with repeatable associations across these two data sets, one of which was subsequently further validated in the MARC twinner population . Two other regions with some degree of replication across studies are the region of bovine chromosome 23 containing the steroid 21-hydroxylase (CYP21) gene and chromosome 7 near the anti- Müllerian hormone (AMH) gene. In all three cases, contribution of these positional candidate genes to genetic variation is uncertain, and further work is needed to establish any causal relationship between these and variation in ovulation or twinning rate.-28) of segregation of a single gene, located on chromosome 10, with a large effect on ovulation rate  .The search for major genes affecting ovulation in cattle led to the identification of the cow Treble with an exceptional record of prolificacy (r cycle) . Examinar cycle) . Consider cycle) , suggestMutations in high fecundity ovine genotypes, in general, result in reduced signaling of the GDF9 and/or BMP15 pathways leading to ovulation of multiple smaller follicles as compared to wild type ewes  compared to wild types ewes  or circulating AMH when compared to controls , Inverdale/FecXI  are smaller on an individual basis and have fewer cells than those observed in non-carrier control ewes  of ewes carrying high fecundity alleles  greater plasma and intrafollicular IGF1 (The recent discovery of Trio provides an outstanding opportunity to investigate the physiologic mechanisms associated with selection of a single follicle (non-carriers) or multiple follicles (Trio carriers) during different experimental conditions . The cowet al., 2018; University of Wisconsin- Madison, Madison, WI, USA; unpublished). Mean ovulation rate per cycle was 4.3 ± 0.5 for homozygous Trio allele carrier heifers, indicating that the Trio allele does not result in primary ovarian failure in the homozygous state, unlike many of the high fecundity genotypes found in sheep. However, due to low number of homozygote animals, it is currently unclear if homozygous Trio carriers have similar or increased ovulations compared to heterozygotes.Evaluation of more than 243 estrous cycles in Trio carrier cattle (heterozygote) and half-sib non- carriers, revealed that Trio carriers had a consistent increase in ovulation rate with a mean ovulation rate of 3.5 ± 0.2 while non-carriers had a mean ovulation rate of 1.1 ± 0.1 . In cattle, AMH and AFC have been found to have relatively high correlations ranging from 0.59 to 0.88 .Nevertheless, there is clear evidence of subordinate follicles in Trio carriers, supporting the idea that selection still occurs but at a smaller size and with a greater number of selected follicles in Trio carriers. Thus, diameter deviation between future dominant follicles and subordinate follicles occurred at similar times (~3 days) in Trio carriers and non-carriers albeit at significantly smaller follicle sizes , indicating that combined hormonal output is similar regardless of whether it originates from one large follicle or four smaller follicles.Evaluation of CL volume and circulating P4 indicates that despite individual CL being smaller in Trio carriers, total luteal volume and resulting P4 concentrations are not different than those observed in non-carriers . Overall, average circulating FSH was greater in Trio carriers than non-carriers. However, precise analysis focusing on the time encompassing observed deviation found that FSH was greater only in a narrow window encompassing deviation .The known role of FSH in stimulating follicle growth and the ability to induce multiple ovulations by administration of exogenous FSH  and upon ovulation results in multiple CL of smaller individual size but the same total number of luteal cells with corresponding similar P4 output.An important aspect that stems from the development of smaller follicles in Trio carriers is their reduced growth rate. The reduced follicle growth is likely mediated by a reduction in the rate at which GC proliferate, resulting from attenuation of BMP15 signaling pathways in response elevated SMAD6 (The potential for FSH being the major driver for occurrence of multiple ovulations is extremely appealing. Our findings of elevated FSH concentrations, particularly around the time of expected deviation provides clear support for this mechanism, and this would provide a logical physiologic explanation for increased ovulation rate. Thus, our current model  for Trio


Syntenic cell wall QTLs (SQTLs) can identify genetic determinants of biomass traits in understudied species based on results from model crops. However, their effective use in plant breeding requires SQTLs to display intraspecific allelic variability and to predict causative loci in other populations/species than the ones used for SQTLs identification. In this study, genome assemblies from different accessions of Arabidopsis, rapeseed, tomato, rice, Brachypodium and maize were used to evaluate the intraspecific variability of SQTLs. In parallel, a genome-wide association study (GWAS) on cell wall quality traits was performed in miscanthus to verify the colocalization between GWAS loci and miscanthus SQTLs. Finally, an analogous approach was applied on a set of switchgrass cell wall QTLs retrieved from the literature. These analyses revealed large SQTLs intraspecific genetic variability, ranging from presence–absence gene variation to SNPs/INDELs and changes in coded proteins. Cell wall genes displaying gene dosage regulation, such as PAL and CAD, displayed presence–absence variation in Brachypodium and rapeseed, while protein INDELs were detected for the Brachypodium homologs of the rice brittle culm-like 8 locus, which may likely impact cell wall quality. Furthermore, SQTLs significantly colocalized with the miscanthus and switchgrass QTLs, with relevant cell wall genes being retained in colocalizing regions. Overall, SQTLs are useful tools to screen germplasm for relevant genes and alleles to improve biomass quality and can increase the efficiency of plant breeding in understudied biomass crops. The projection of known quantitative trait loci (QTLs) across species through genome synteny was recently shown to allow for the quick identification of conserved genomic regions underlying traits of interest in large panels of plants, including novel, under-domesticated, crops . On the The concept of SQTLs was demonstrated for plant cell wall compositional traits . These tWhat was just discussed demonstrates that SQTLs represent valuable tools during the pre-breeding steps of crop improvement. Specifically, their availability, combined with the dropping of genome sequencing costs, allows for the potential circumvention (of part of) the pre-breeding studies on trait genetics needed to start breeding programs in under-domesticated crops . Given tArabidopsis thaliana, Solanum lycopersicum, Brassica napus, Zea mays, Oryza sativa, and Brachypodium distachyon—for which multiple genomes representing diverse plant accessions were available. On the other hand, the SQTL value for predicting relevant loci in novel populations/species was tested by assessing the colocalization between cell wall SQTLs from Miscanthus sinensis and Panicum virgatum and genomic regions associated with cell wall variability identified through association mapping in miscanthus and switchgrass, respectively. Miscanthus and switchgrass are C4 biomass crops with high potential to fulfil diverse industrial applications, but at the same time still largely under-domesticated [The two latter aspects are the focus of this study. On the one hand, the intraspecific allelic genomic variability of cell wall SQTLs was assessed in six angiosperm species—sticated ,13. The sticated ,14. The To investigate the intraspecific genetic variability of SQTLs, the nucleotide sequences of 1184 cell wall SQTLs previously identified in six angiosperm species  [Brachypodium distachyon involved in lignin synthesis , and two CAD genes operating the in muro monolignol polymerization from Brassica napus  . The majority of these genes is absent in only one target assembly , while 475 genes across all the species tested are missing in >50% of the target assemblies assessed for a particular species . Finallyssembly) . To concSQTL alignments were also used to quantify intraspecific nucleotide variation at SQTL regions consisting of single nucleotide polymorphisms (SNPs) and insertions/deletions  between reference and target assemblies . The resp = 0.000; p < 0.001; p < 0.001; t-test’s p < 0.001 for both; A more detailed analysis revealed that the number of SNPs/kbp and INDELs/kbp varies substantially between species  displayed polymorphisms leading to one or more protein sequence changes between reference and one or more target assemblies . Of thesCOBRA (COB) and COBRA-like (COBL) gene families responsible of the Brittle-culm cell wall rice mutants [FERULATE 5-HYDROXYLASE (F5H), CINNAMOYL-CoA O-METHYLESTERASE (CCOAOMT), and PHENYLALANINE AMMONIA-LYASE (PAL) [CELLULOSE SYNTHASE (CESA), CELLULOSE SYNTHASE-LIKE (CSL), and IRREGULAR XYLEM (IRX) gene families [WRKY12, NAC SECONDARY WALL-THICKENING FACTOR1 (NST1), and C3H14 [To assess the potential effect of the intraspecific protein sequence variability on cell wall quality traits, how such patterns impact functionally relevant and highly syntenically conserved candidate SQTL genes identified in our previous SQTL study was studied . These g mutants ; importaSE (PAL) ; key hemfamilies ,18; impond C3H14 ,20. For BRITTLE CULM-LIKE loci OsBCL1, OsBCL8, and OsBCL9, and of their related syntenic homologs conserved through SQTLs [OsBCL9 displayed three amino acid changes within the COBRA domain of the protein, of which one involving a change in polarity  and leading to the removal of a β-sheet domain in the 2D protein configuration , right next to the GPI-anchoring-related ω-site  displayed massive protein rearrangements in three of the 45 Brachypodium genome assemblies analyzed . These changes involve large deletions of the BdBCL8 protein sequence, including a consistent part of the COBRA domain in the line “ABR3”, and the complete COBRA domain in the lines “Bd2” and “Adi12”  displayed two regions with relatively large INDELs and different amino acid substitutions within the GT43 functional domain (CESA7 gene (XP_003574029.1), also retained within SQTLs, displayed large deletions within the cellulose synthase catalytic domain in the accession “TR9” compared to the reference Brachypodium genome , across different Brachypodium accessions for the BdWRKY12 gene , and between the rice reference genome and three rice cultivars  for the OsNAC43 gene   .Miscanthus sinensis and Panicum virgatum [Miscanthus sinensis collection and QTLs mapped by other researchers in an F1 progeny of a biparental cross of two switchgrass lines diverging for cell wall quality traits [In addition to evaluating the level and relevance of the intraspecific genetic variability of SQTLs in diverse species, another aim of this study was to assess the validity of SQTLs for predicting genomic loci associated with biomass quality traits in novel plant populations and species. This aspect was studied by assessing the degree of colocalization between the SQTLs previously detected in virgatum  and celly traits . These pThe GWAS conducted on the miscanthus population identified a total of 91 QTLs associated to eight traits related to cell wall content and composition . These 9An analogous procedure to the one just described for miscanthus was performed in switchgrass to test the colocalization between the 56 cell wall-related QTLs identified by Ali, Serba, Walker, Jenkins, Schmutz, Bhamidimarri, and Saha  Supplem, and theMsBLH6 (GQ_01G471400 and GQ_02G130600), the MsWRKY12 (GQ_12G168300), the miscanthus homolog of arabidopsis VND4 (GQ_12G158800), and the MsMYB103 (GQ_07G174800) (PAL and CCOAOMT copy (GQ_12G150500 and GQ_07G169800), or the miscanthus homolog of the maize BRITTLE-STALK 2 locus   . In addiG165800) . FinallyG165800) A,B. HoweFRAGILE FIBRE 1 locus  and a homolog of the arabidopsis KOR gene (IV_1NG408000). Moreover, an IRX gene involved in xylan synthesis (IV_5NG144100), and two MYB42/MYB85 genes important for lignin biosynthesis (IV_2NG449200 and IV_6NG352900). All these genes are retained in QTLs from Ali, Serba, Walker, Jenkins, Schmutz, Bhamidimarri, and Saha [For switchgrass, the most relevant cell wall genes found in colocalizing regions between SQTLs and QTLs from Ali, Serba, Walker, Jenkins, Schmutz, Bhamidimarri, and Saha  are summand Saha  that areMiscanthus sinensis was therefore used to study if SQTLs can, at least partly, overcome this limitation. First, the analysis of population structure computed to perform the “standard” GWAS described in In the final step of this research, it was evaluated whether SQTLs, given the incorporation of information from multiple genomic loci previously shown to determine variability in a trait of interest , can be used to overcome limitations of genetic mapping approaches. Specifically, a common issue encountered in association mapping—and especially GWAS—is that if patterns of population structure overlap with patterns of phenotypic variability across accessions for the trait(s) evaluated, population structure correction can hide (part of) the relevant loci that govern the trait(s) . The GWAKOR gene (GQ_05G142200) that was found within a QTL associated to cellulose dry matter content. Moreover, an MsCSL gene (GQ_19G142500), a homolog of the Arabidopsis and maize CslD5, was also found within four colocalizing “extra QTLs” associated with total cell wall dry matter content, ADF dry matter content, cellulose dry matter content, and cellulose NDF percentage. Finally, two peroxidases , and a XYLOGLUCAN ENDO-TRANS-GLYCOSYLASE/HYDROLASE .In total, 17 “extra QTLs” were detected . Their aPAL and CAD genes, which displayed PAV among some of the Brachypodium distachyon and Brassica napus accessions assessed. PAL and CAD are multigene families, and gene dosage is thought to be important for their functionality [Brassica napus, Brassica rapa, intraspecific copy number and PAV of PRX genes, which together with CAD determine the efficiency of lignin production at the final steps of the lignin pathway [In a previous publication, it was shown that SQTLs are useful breeding tools to project known genetic information on the architecture of traits of interest—specifically cell wall quality traits—from model species to understudied crops . This reionality ,42,43. M pathway , was sug pathway . TherefoBCL8 locus and its Brachypodium syntenic homolog identified through SQTLs are particularly relevant. In fact, the protein modifications reported for these genes sometimes break the integrity of the COBRA domain of the protein or affect the membrane-anchoring domains at the C-terminus. These mutations are highly similar to the ones found in the well characterized OsBC1 and OsBCL1 mutants, which are close homologs of the OsBCL8 gene [COBL gene mutations [BCL8 protein—displays differences in the relative content of cell wall components, including cellulose, compared to the reference of this study, cv. “Nipponbare” [In addition to PAV, nucleotide polymorphisms were also found in high numbers in the intraspecific analyses of SQTL regions. Specifically, multiple genes that were previously defined as relevant SQTLs candidates  display CL8 gene ,47. Bothutations ,46,47. Tponbare” . NeverthTo conclude, irrespective of the functional relevance of the specific patterns of genomic variability observed for the SQTL regions in the intraspecific comparisons performed, it is still highly relevant that SQTL genes were proven to show such patterns. This demonstrates that the high conservation of gene presence and order across diverse genomes does not preclude the existence of intraspecific allelic variability for those highly conserved regions. Therefore, synteny and SQTLs can be effectively used to mine interesting alleles by genomically analyzing SQTL regions in target species and searching for specific mutations previously identified as particularly relevant in model crops. If this step would be performed during the screening of useful material for breeding programs, it would lead to faster and more effective breeding activities in novel (under-domesticated) biomass species.Miscanthus sinensis and Panicum virgatum, respectively, colocalized significantly with SQTL regions of these two species as compared to random genomic loci. This result demonstrates that, in a breeding context involving novel species, SQTLs can be valuable tools to pinpoint relevant target loci that are likely responsible of variability in traits of interest. Moreover, by using the annotations of SQTL genes from the multiple species that are represented within SQTLs—as performed here for cell wall genes—it is possible to filter candidate genes based on functional and literature information. Finally, syntenic conservation of target loci through SQTLs can quickly display the functional conservation of interesting target genes through multiple (model) species for which studies might be available to better evaluate the functional relevance of cell wall genes within QTLs and SQTLs.In the second part of this study, it was tested if SQTLs represent effective tools to predict relevant loci associated to biomass quality in novel mapping panels or crop species. The results showed that two sets of QTLs from BLH6 is known to deeply affect the properties of plant cell walls, not only within different species [BLH6, genes such as WRKY12, VND4, and MYB103 are all central cell wall genes, demonstrating the modulation of cell wall regulation and composition across a range of species [By applying the approach just described, in addition to the colocalization between SQTLs and miscanthus and switchgrass QTLs, several genes involved in these colocalizations were identified that represent valuable candidates for determining variability in biomass composition in these species. Specifically, some of the genes found from the miscanthus GWAS and conserved through SQTLs appear of particular interest. For example, the transcription factor  species ,49, but  species . Therefo species ,51,52,53In the last part of this study, whether SQTLs can be used to make the population structure correction more efficient in GWA studies was also tested. While it needs to be clearly stated that the accounting of population structure in GWAS is pivotal to enabling accurate mapping, the results obtained here indicate that the inspection of colocalization between SQTLs and significant SNPs normally discarded as false-positives after population structure correction could represent a useful complement of a “standard” GWAS pipeline. Specifically, as they are defined, SQTLs represent genomic regions that are syntenically conserved between species and in multiple model species were shown to be associated to QTLs for a trait of interest . TherefoArabidopsis thaliana, Brassica napus, Solanum lycopersicon, Brachypodium distachyon, Oryza sativa, and Zea mays were collected from online databases after the literature search . Finally, all the statistical analyses reported in this manuscript were performed using R or SPSS v27.0 .Custom R scripts were developed to process the NUCmer outputs and extract different information. First, the aligning regions and coordinates of SQTLs in every target accession; second, SQTL coverage for each alignment; third, the SQTL  genes included in each alignment; finally, the SNPs and INDELs found along alignments. To extract these data, the custom R scripts made also use of a previously developed list of cell wall genes from 169 angiosperm genomes . MoreoveThe assessment of the effects of SQTLs genomic variability on protein sequences was performed by using a custom R script were the coordinates of the exons of every SQTL gene included in alignments and concurring to code the main gene transcripts were used to retrieve gene CDS and translated proteins in the target genome assemblies. On the retrieved proteins, sequence changes between reference and target assemblies were assessed with ClustalW . MoreoveMiscanthus sinensis was performed by using a miscanthus collection established in 2013 in Wageningen (The Netherlands) and composed of 94 accessions originated from various international gene banks and breeding programs. Genotypes were planted in square-like plots with 16 clonal replicas, and the four central plants of each plot were harvested every spring for five years, starting in 2017.The GWAS on Phenotyping was performed for eight cell wall quality traits, including NDF cell wall as percentage of dry matter, ADF cell wall as percentage of dry matter, ADL lignin as percentage of dry matter, lignin as percentage of NDF, cellulose as percentage of dry matter, cellulose as percentage of NDF, hemicellulose as percentage of dry matter, and hemicellulose as percentage of NDF . PhenotyMiscanthus sinensis reference genome [The genomic DNA of all the accessions was isolated from random young leaves from the four central plants of all the plots in the collection, following a CTAB-based protocol . Extracte genome , resultie genome   incorporating SNP data and the kinship matrix, as implemented in the statgenGWAS R package . FDR corColocalization between SQTLs and the 91 QTLs found by the miscanthus GWAS was performed by developing 100 sets of 91 random QTL regions from the miscanthus genome mirroring the size distribution of the QTLs from GWAS results (custom R script). The proportion of QTLs colocalizing for >50% of their bp length with SQTLs was then calculated for every set, and binomial tests were performed to assess if random QTLs co-localized with SQTLs significantly less than the QTLs from the GWAS (custom R script).In addition to calculating the statistical significance of the colocalization between miscanthus SQTLs and QTLs, the cell wall genes in colocalizing regions were identified by using the set of angiosperm cell wall genes developed in our previous SQTL study . MoreoveA total of 56 QTLs related to cell wall traits in switchgrass were retrieved from the results of Ali, Serba, Walker, Jenkins, Schmutz, Bhamidimarri, and Saha  Supplem. ColocalAs performed in miscanthus, the cell wall genes in colocalizing regions were identified by using the set of angiosperm cell wall genes developed in our previous SQTL study . MoreoveThis study aimed at assessing the validity of SQTLs to assist breeding of (novel) biomass crops, by (i) analyzing the intraspecific level of SQTLs allelic genomic variability in panels of available good-quality plant genomes, (ii) assessing the SQTLs predictive value of relevant biomass-quality-related loci in miscanthus and switchgrass, and (iii) suggesting possible side-uses of SQTLs to complement standard approaches of genetic mapping. The results showed that SQTLs are valuable tools to improve breeding activities in novel crops, where they can be applied in different ways to either screen plant material for genes or alleles of interest, or to pinpoint relevant loci based on the information from model species in novel accession panels or crops. Moreover, the research performed to achieve the goals above allowed the finding of relevant alleles at candidate SQTL loci, as well as of important candidate cell wall genes for miscanthus biomass improvement. Future research could investigate the phenotypic relevance hypothesized for the intraspecific patterns of SQTLs variability revealed in this study, by phenotyping cell wall composition in the plant lines showing promising variability. Moreover, the miscanthus loci highlighted in this study could be targeted by genetic modification to study their specific relevance for miscanthus biomass quality. Finally, SQTLs might start to be included in breeding programs of under-domesticated biomass crops as proposed in this research, to test their usefulness in novel, important, breeding settings.


Maximal safe surgical resection plays a key role in the care of patients with gliomas. A range of technologies have been developed to aid surgeons in distinguishing tumor from normal tissue, with the goal of increasing tumor resection and limiting postoperative neurological deficits. Technologies that are currently being investigated to aid in improving tumor control include intraoperative imaging modalities, fluorescent tumor makers, intraoperative cell and molecular profiling of tumors, improved microscopic imaging, intraoperative mapping, augmented and virtual reality, intraoperative drug and radiation delivery, and ablative technologies. In this review, we summarize the aforementioned advancements in neurosurgical oncology and implications for improving patient outcomes. In this article, we discuss novel intraoperative technologies that are under development, which aid in distinguishing tumor from normal brain with the goal of greater tumor resections.Maximal safe surgical resection is a cornerstone of treatment for patients with low- and high-grade gliomas.3 iMRI can provide updated information on tumor location as a case progresses thereby accounting for potential increasing brain shift.Magnetic resonance imaging (MRI) has long been used in the preoperative evaluation of patients with brain tumors, providing valuable insight into tumor characteristics and proximity to eloquent structures. Similarly, it has been used in the postoperative setting to evaluate tumor extent of resection (EOR). Intraoperative MRI (iMRI) for the evaluation of tumor EOR in the operating room was originally developed in the 1990s.4 One RCT included in their review was an interim analysis and included only 14 patients, finding no benefit to iMRI use.5 The second RCT by Senft et al. included 58 patients with known or suspected glioma with contrast enhancement on MRI. They found that patients who received iMRI were significantly more likely to undergo complete resection of their tumor, with no difference in the rate of new postoperative neurological deficits.6 A subsequent meta-analysis by Golub et al. used seven studies, with 236 iMRI cases and 233 conventional navigation cases, to compare iMRI to conventional navigation; iMRI was associated with increased incidence of gross total resection .7 Finally, a recent retrospective analysis by Shah et al. of 640 patients with supratentorial glioblastoma, 332 of whom were treated with iMRI, found iMRI to be independently associated with GTR.8 Overall, these studies indicate that iMRI may meaningfully contribute to increased EOR of gliomas, although some practical limitations include the availability of the technology and optimizing its use in the operating room. Interestingly, the combination of iMRI with 5-aminolevulinic acid (5-ALA) for intraoperative tumor visualization may also be beneficial, although available data in the literature is mixed and of meager quality, making this an area in need of additional investigation.9–12There are few randomized controlled trials (RCTs) evaluating the use of iMRI in glioma surgery. A systematic review of the literature in 2021 by Fountain et al. identified two RCTs assessing the efficacy of iMRI.13 Similarly, an RCT of ioUS demonstrated a higher incidence of GTR in patients with presumed GBM where ioUS was used.14 Novel improved ultrasound modalities are further increasing the utility of intraoperative ultrasound for surgeons. Advanced ultrasound imaging techniques, such as navigated ioUS,15 contrast-enhanced ultrasound,17 and intraoperative strain elastosonography18 have shown promise in glioma surgery and are interesting avenues of further development. Finally, the use of contrast-enhanced ultrasound in combination with 5-aminolevulinic acid (5-ALA) has been shown to increase EOR in a retrospective study of 230 GBM patients by Della Pepa et al., highlighting the synergy of these two technologies.19 Overall, these various ultrasound-based technologies are limited in part by the availability of the technologies, but warrant additional investigation given their potential ability to improve EOR.In contrast to the relatively high costs associated with iMRI, intraoperative ultrasound (ioUS) is a less expensive, more accessible technology to aid in intraoperative tumor identification and resection. However, limitations of traditional ioUS include its relatively low spatial resolution and poor image quality. A meta-analysis assessing the efficacy of ioUS by Mahboob et al. found an estimated GTR rate of 77% in cases where ioUS was used.20 Briefly, limitations of 5-ALA include its limited utility in low-grade gliomas as well as its sensitivity and specificity for infiltrating tumor cells.21–23 Additional potential alternatives for the intraoperative visualization of tumor cells include indocyanine green24 and fluorescein , neither of which are molecularly tumor-specific. Tumor-specific fluorescent tags include labeled antibodies against EGFR,28 labeled chlorotoxin (which binds to a number of solid tumors),29 and labeled proteoglycan glypican-1 (GPC-1) antibodies.30 Preclinical experiments using triple-modality magnetic resonance imaging–photoacoustic imaging–Raman imaging nanoparticles have also shown promise, with highly specific accumulation in tumor relative to normal brain, highlighting the efficacy of this technology.31 Fluorescence lifetime imaging (FLIM), which uses time-gated intensified cameras, can be used to view nicotinamide adenine dinucleotide  and/or 5‐ALA induced protoporphyrin IX (PPIX).32 Studies have demonstrated the use of FLIM to highlight areas of weak 5-ALA fluorescence34 and to increase the ability to differentiate tumor from normal brain when used to detect NADH in addition to PPIX.355-ALA is the primary drug used for fluorescence-guided surgery in GBM. Stummer et al. demonstrated the efficacy of 5-ALA in increasing GTR and 6-month progression-free survival (PFS) in a phase III trial of high-grade glioma patients, supporting its continued use.37 Indeed, a meta-analysis by De Witt Hamer et al. of 90 studies, including 8,091 patients, demonstrated reduced neurological deficits and an increased incidence of GTRs in patients who underwent intraoperative stimulation mapping when compared to those who did not.38 Intraoperative mapping has been combined with iMRI in a number of small studies and has been shown to be safe, albeit with little robust evidence around efficacy or impact on EOR.39–42 Similarly, 5-ALA has been used in combination with intraoperative mapping, with some evidence suggesting a potential benefit of using the two technologies together, but with conclusions limited by the lack of larger-sized studies.44Intraoperative mapping is frequently used for gliomas near functional brain areas and plays a significant role in maximizing safe resection.36 Similarly, a manuscript by Bander et al. highlighted the utility of high-frequency bipolar train-of-five (TOF) stimulation that combines the reliability and low incidence of intraoperative seizures seen with monopolar TOF stimulation with the focus of traditional low-frequency bipolar stimulation. Intraoperative mapping will undoubtably continue to become safer and more accurate as mapping techniques and technologies further develop.Intraoperative mapping techniques also continue to develop and improve. A recent report by Gogos et al. described favorable outcomes associated with asleep triple motor mapping in which bipolar and monopolar stimulation, as well as transcranial or transcortical motor evoked potentials (MEPs), are utilized to preserve cortical and subcortical motor systems during tumor resection.45 Alexiou et al. published a similar protocol with the ability to differentiate cell cycle of tumor cells, providing insight into tumor grade as well as information regarding surgical margins.47 A more recent study by Saito et al. also hinted at the potential utility of MI from intraoperative flow cytometry as a prognostic tool for patients with glioblastoma.48Various intraoperative cell and molecular profiling techniques have been investigated to aid in identifying and characterizing tumor cells. Flow cytometry utilizes lasers to measure various characteristics of cells and fluorescent antibody tags; it is commonly utilized in hematologic malignancies and can be performed relatively quickly. Two recent studies have evaluated the use of intraoperative flow cytometry in tumors. Shioyama et al. describe a 10-minute protocol with the ability to calculate the malignant index (MI) of analyzed cells. They demonstrated a significant difference in MI between normal tissue and tumor, highlighting the ability to use their 10-minute protocol to quickly determine surgical margins.49–51 DESI MS has also been utilized to identify tumoral onco-metabolites. Santagata et al. demonstrated the ability for DESI MS to identify 2-hydroxyglutarate (2-HG), a downstream byproduct of mutated isocitrate dehydrogenase 1 and 2 (IDH 1 and 2), providing rapid intraoperative information on the IDH mutational status of a tumor, as well as further contributing to the determination of tumor margins.52 Intraoperative determination of IDH status may play an important role in decision making around EOR, as previous studies have implicated certain IDH1 mutant tumors as more amenable to maximal surgical resection.53 Indeed, given the potential utility of this information, PCR based platforms have also been developed for the intraoperative determination of IDH and telomerase reverse transcriptase promoter (TERTp) mutations.54–56Mass spectrometry (MS) is an additional technology with potential utility in the intraoperative setting. MS allows for the detailed analysis of the various lipids and proteins in a tissue sample. Desorption electrospray ionization (DESI) MS primarily provides information regarding the lipid composition of a tissue; it has been used for the intraoperative molecular diagnosis of brain tumors and to determine tumor margins, correlating well with traditional histopathological analysis.58 As technology continues to improve, it is possible that rapid flow cytometry or DESI MS will be performed directly from CUSA specimens, educating a surgeon not only on tumor margins, but, in the case of flow cytometry, potentially leveraging the range of antibodies available to yield additional information on the cellular composition of the tumor microenvironment. As we continue to move towards increased precision medicine approaches for GBM, insights from intraoperative flow cytometry or DESI MS could also inform surgical decision making and the intraoperative delivery of targeted therapies.Interestingly, there is evidence that tumor specimens obtained by a cavitron ultrasonic surgical aspirator (CUSA) can be used successfully for flow cytometry and other cellular analyses.59 Primary advantages of exoscopes relative to traditional operating microscopes are: higher magnification of the surgical field, increased tissue clarity, a larger field of view, improved ergonomics, and the potential ability to view the field in 3D or using augmented reality.59–61 Both exoscopes and microscopes can allow for results from preoperative Diffusion Tensor Imaging (DTI) and fiber tractography to be overlaid onto real-time images of the brain intraoperatively, potentially improving the safety of resections.61 Despite these theoretical benefits, few studies have assessed the efficacy of exoscopes in glioma surgery. A recent study by Baron et al. including 26 GBM patients who underwent surgical resection with the use of a 2D exoscope, demonstrated a median EOR of 94.8%.61 Additional larger studies directly comparing exoscopes to traditional operating microscopes are needed to further assess the impact of exoscopes on EOR, postoperative deficits, survival, and surgeon comfort in glioma surgery. Research efforts are also being directed at the combination of exoscopes with 5-ALA, which may potentially enhance the efficacy of both technologies.62–64 Practical limitations of exoscopes include the learning curve associated with their use and concerns surrounding depth perception with 2D exoscopes.65Several different technologies have attempted to utilize robotics to improve and enhance glioma surgery. While many of these are in their infancy, one of the most well-described is the use of exoscope platforms. Exoscopes are digital microscopes that are fixed above the surgical field and allow a surgeon to view the surgical site on a screen, rather than looking through a lens, as in a traditional microscope.66–68 The two most well-described Raman-based methods in glioma surgery to this point include Stimulated Raman scattering (SRS) microscopy and Raman spectroscopy.Raman-based intraoperative imaging methods have been increasingly studied within brain tumor surgery and seek to bring detailed information regarding tumor infiltration and pathology to the operating room.69 Ji et al. subsequently demonstrated the potential promise of this technology in preclinical brain tumor models, highlighting the ability of SRS microscopy to accurately identify tumor margins.70 These results were followed by a study in human samples, which similarly showed that SRS microscopy was highly sensitive and specific for tumor infiltration.71 To take SRS microscopy directly to the operating room, Orringer et al. then developed a portable SRS microscopy system as well as stimulated Raman histology (SRH) which generated highly accurate virtual hematoxylin and eosin-stained slides.72 Most recently, SRH has been used in combination with deep convolutional neural networks to provide a brain tumor diagnosis in under 150 seconds; this was shown to be noninferior to pathologist interpretation of histologic images in a multicenter clinical trial including 278 patients.73 SRH is undoubtably an exciting tool with the ability to provide surgeons with rapid and accurate information on tumor margins and classifications.SRS microscopy was originally described in 2008 and allows for the rapid label and processing free analysis of biological tissues.74 Jermyn et al. then subsequently combined multiple label-free optical tools, including optical coherence tomography, Raman spectroscopy, intrinsic fluorescence spectroscopy, and diffuse reflectance spectroscopy to create an intraoperative system with the ability to detect brain, lung, colon, and skin cancers with high accuracy, sensitivity, and specificity.75In contrast to SRS microscopy, which requires samples to be viewed ex vivo, Raman spectroscopy has been adapted for use in the setting of handheld systems albeit with significantly increased artifact and reduced resolution. Jermyn et al. described the utilization of a commercially available handheld contact fiber optic probe for intraoperative Raman spectroscopy; they initially tested the system in 17 glioma patients, identifying cancer cells with an accuracy of 92%.76 In addition, preclinical studies have demonstrated an antagonistic relationship between systemic chemotherapy and the efficacy of immunotherapies.78 As a result, the local delivery of therapeutics directly to a tumor is an attractive treatment modality. Carmustine wafers, a form of local chemotherapy, is perhaps the most well-known and validated locally delivered treatment for glioblastoma.79 However, in practice, local delivery of a novel drug or biologic can be difficult and inaccurate, potentially contributing to the variability in results of various clinical trials investigating locally delivered treatments.Local drug delivery to a glioma has the potential of avoiding the blood-brain barrier and allowing for higher therapeutic dosing while reducing the risk of systemic toxicities. Indeed, systemic temozolomide treatment has been tied to increased immunosuppression in GBM patients.80–82 CED techniques continue to improve through improvements in catheter design, methods for chronic treatment delivery, and imaging and modeling of treatment delivery.81 As CED technology continues to develop it may play an important part in the delivery of treatments to aid with unresectable or microscopic areas of tumor.Convection enhanced delivery (CED) involves the stereotactic placement of catheters directly into a region of interest within the brain and allows for the targeted local delivery of a treatment into a tumor. CED has been used to deliver a number of anti-tumor therapies, including chemotherapies, immunotherapies, and virotherapies.83 It can theoretically damage or destroy residual microscopic tumor cells on the resection cavity edge that are unable to be identified or resected. Additional potential advantages include the ability to direct the radiation to the resection cavity intraoperatively, potentially increasing accuracy, and the ability to deliver radiation immediately after resection, reducing the time between surgery and the start of radiation for patients. Historically, IORT results have not demonstrated a convincing survival benefit in GBM patient.84 However, more recently an international pooled analysis of 51 GBM patients treated with IORT in addition to standard of care by Sarria et al. suggested a 25% increased overall survival in treated patients at 3 years. Similarly, a subsequently published dose-escalation phase I/II trial of IORT by Giordano et al. involving 15 patients with GBM demonstrated the safety of IORT and integration into the neurosurgical operating room, with a treatment time of approximately 30 minutes, prompting a phase III study.85 Results of future studies assessing the efficacy of IORT are necessary to determine the utility of this technology in patients.Intraoperative radiotherapy (IORT) involves the delivery of radiation therapy, frequently a single high dose, to a resection cavity immediately following resection.86 Complications associated with LITT include hemorrhage, significant tumor edema, and neurological deficit.87 The clinical efficacy of LITT for gliomas is still unclear, but may be associated with ablative coverage of the lesion.86–89 Additional prospective studies are needed to fully elucidate the clinical role of LITT, including its potential ability to open the peritumoral blood-brain barrier and synergize with chemo- or immunotherapies.90The most common ablative technology other than radiation therapy studied for gliomas is laser interstitial thermal therapy (LITT). LITT involves the interstitial transmission of laser light through fiberoptic wires into a target tissue. MRI-guidance and thermography are then utilized to track the resulting thermal damage that is induced. LITT has been primarily used for deep-seated, focal, smaller tumors or in patients who are otherwise poor surgical candidates. There is a paucity of prospective studies evaluating LITT. Current evidence suggests that LITT is relatively safe with a recent study of 58 LITT treatments in recurrent and newly diagnosed GBM suggesting a 30-day morbidity of 16%.91 A subsequent case report examining the use of magnetic resonance-guided focused ultrasound (MRgFUS) to ablate a GBM demonstrated successful ablation, but only in a small percentage of the tumor.92 As a result, due to technical restraints, the direct ablation of most tumors by focused ultrasound may not be feasible. However, focused ultrasound may be used in the settings of sonodynamic ablation in which focused ultrasound is used to activate a sonosensitizer, leading to tumor cell death.93 This has been demonstrated in preclinical rat glioma models using 5-ALA as a sonosensitizer.94 Further investigation is needed to understand the efficacy and safety of sonodynamic ablation in humans.Focused ultrasound has also been investigated for the ablation of GBM in small studies with limited efficacy. A study of three patients with GBM who underwent focused ultrasound thermoablation demonstrated an inability to achieve thermal coagulation of the tumors.95 Sanai et al. demonstrated the initial feasibility of intraoperative confocal microscopy for a variety of brain tumor types.96 Subsequent studies have demonstrated the potential promise of confocal microscopy for in vivo histopathological imaging of brain tumors and in combination with 5-ALA for the identification of low-grade gliomas.98 Pavlov et al. also demonstrated the identification and diagnosis of low- and high-grade gliomas in a small cohort of patients using probe-based confocal laser endomicroscopy in combination with fluorescein.99 Additional large studies are needed to determine the efficacy of this technology in improving EOR and providing an intraoperative tumor diagnosis.Confocal microscopy allows for the intraoperative histological imaging of cells and has been utilized in other surgical specialties to aid in oncologic diagnoses.100 These technologies, however, have since continued to be used more frequently in preoperative planning and intraoperatively.101 While still in the early stages of evaluation, the use of intraoperative AR and VR has been suggested to aid in glioma resection.101 In a study of 134 glioma patients, Sun et al. compared the outcomes between patients whose surgeries had been carried out using AR and VR based on functional neuronavigation and iMRI to a control group using anatomic neuronavigation; they found that the use of intraoperative AR and VR improved EOR and functional postoperative outcomes, highlighting the promise of these technologies.102Both augmented reality (AR), the overlaying of digital elements over real-life structures, and virtual reality (VR), the complete immersion into a virtual environment, have been investigated in brain tumor surgery. Interestingly, these technologies have been used by both patients and surgeons. Until more recently, surgeons primarily used AR and VR for educational purposes.103 This is highlighted by Mazerand et al. who reported the accuracy of VR in detecting visual field defects and the case of a patient with a parietotemporal GBM in which VR was utilized in combination with subcortical mapping to identify and preserve the optic radiations.104 Similar studies have used VR to map social cognition105 as well as language function.106 Overall, VR and AR technologies offer exciting avenues of future investigation in brain tumor surgery with the ability to improve EOR as well as enhance postoperative functional outcomes. Practical limitations of of AR and VR technologies include lack of familiarity their use and potentially high operating costs.AR and VR technologies have also been utilized by patients during awake mapping to expand the repertoire of brain functions that can be mapped accurately.Multiple novel technologies are in development to aid with the intraoperative identification and removal of tumor cells, while reducing patient morbidity . The hig


The severe acute respiratory syndrome coronavirus 2 pandemic has continued to impact global health. However, while immunity acquired by vaccines has been developed, 40% of the world's population has still not been vaccinated. Economic problems associated with acquiring novel therapies, misinformation, and differences in treatment protocols have generated catastrophic results, especially in low-resource countries. Understanding the pathophysiological aspects of coronavirus disease and the therapeutic strategies that have been validated to date is essential for successful medical care. In this review, I summarize the historical aspects of the virus, molecules involved in infecting the host, and consequences of viral interactions with and in tissues. •1.- The viral interaction with multiple structural proteins explains to a great extent the diversity of the different routes of viral access to the organism.•2.- The involvement of the various receptors seems to broadly explain the constellation of clinical manifestations seen in affected patients.•3.- The “silent hypoxemia,” which has captured much of the attention in the scientific community, is primarily explained by mechanisms that include intrapulmonary anastomoses, shunt, and perfusion alterations as a whole and not individually.•4.- Efforts aimed to understand the pathophysiological bases of SARS-CoV2 disease could improve the understanding of clinical manifestations of the disease, contribute to effective treatment, and develop new technologies that allow viral non-interaction with the organism. ACE2angiotensin-converting enzyme 2ARDSacute respiratory distress syndromeCOVID-19coronavirus diseaseFiO2fraction of inspired oxygenHPVhypoxic pulmonary vasoconstrictionIBAintrapulmonary bronchopulmonary anastomosesIFNsinterferonsMERSMiddle East respiratory syndromeNOnitric oxideNRP1neuropilin 1PaO2partial pressure of oxygenPEEPpositive end-expiratory pressureRNAribonucleic acidSARS-CoV-2severe acute respiratory syndrome coronavirus 2TMPRSS2transmembrane serine protease 2VSELsvery small embryonic stem cell1The 2019 coronavirus disease pandemic, which began in China in 2019, continues, with a cumulative death toll exceeding five million worldwide. The heterogeneous descriptions described in clinical symptoms and paraclinical and different phenotypes of the disease suggest a variety of pathophysiological aspects involved in this entity, which differ significantly from those presented in other forms of respiratory distress syndrome.Understanding the origin of coronaviruses, their characteristics, and the scientifically proven evidence during this pandemic shed light on the pathophysiological aspects of the disease, which should not be left aside for the establishment of therapeutic strategies.The approach carried out in this review covers the historical perspective of the disease and the analysis and interpretation of the pathological aspects of the disease, which distinguish covid-19 infection from other similar diseases.The published information is extensive, so we try to take the outstanding evidence available today and summarize it simply for the reader, which allows for a better understanding of the disease process and timely medical care of infected patients.22.1Coronaviruses are microorganisms that have been identified for almost a century . They be33.1Coronavirus disease 2019 (COVID-19) spreads via respiratory droplets and aerosols during person-to-person contact . This agA mutation in N501T has also been identified in the SARS-CoV-2 spike protein, which could significantly improve the affinity for ACE2 binding; this has not been previously identified in other types (SARS-CoV) .Once membrane fusion is performed, viral entry into alveolar epithelial cells is initiated. The viral content is released, the replication process begins, and ribonucleic acid (RNA) is subsequently formed by RNA polymerases ,21. NegaThe ACE2 receptor in cells is the main factor for host entry and is highly expressed in the epithelial cells of the adult respiratory tract ,24. The The CD209L receptor, which represents the pathway of SARS-CoV infection, is a type C lecithin transmembrane glycoprotein expressed in type II pneumocytes and pulmonary endothelial cells . ImmunofOnce in alveolar epithelial cells, the virus initiates replication and increases the number of viral nucleocapsids ,29. AlveThe vascular endothelium plays a fundamental role, represents a very efficient barrier between the circulation and adjacent tissues, and has critical functions in maintaining vascular tone, hemostasis, inflammatory response, coagulation, and a component of the extracellular matrix . TherefoEndotheliitis represents a vascular inflammation that separates the basement membrane from the subendothelial lymphocytes, and is the most important hallmark of viral infection. Additionally, histopathological findings showed diffuse endothelial inflammation and apoptosis . FurtherTherefore, endothelial dysfunction and subsequent endotheliitis result from the direct endothelial invasion of SARS-CoV, leading to endothelial inflammation and leukocyte recruitment. These trigger inattentive immune responses and are responsible for microthrombotic complications. More evidence of endothelial involvement was shown in a study that found extensive endothelial and subendothelial complement deposits (C4d and C5b-9) in the affected vessels and peak and viral envelope glycoproteins  Fig. 4)Fig. 4).FOther effects of endothelial dysfunction associated with viral infection include biosynthesis of nitric oxide (NO), which is responsible for vasodilation, angiogenesis, and proliferation of endothelial cells. NO availability depends on the balance between reactive oxygen species production and endothelial nitric oxide synthase activity . IncreasThe diversity in the mechanisms of viral entry could explain the significant differences in the manifestations between symptomatic and asymptomatic carriers and the severity between different groups of vulnerable patients.44.1Silent hypoxemia is undoubtedly one of the distinctive features of COVID-19. However, the underlying mechanisms are not fully understood, especially in patients with preserved lung elastance . PreservSeveral studies have reported the presence of patent foramen ovale and transpulmonary bubbles during contrast echocardiography, with a prevalence of 10–20% among infected patients. Although it was first considered the basis for the explanation of silent hypoxemia, it cannot be entirely attributed to the existence of short circuits . The pre2) [Additional investigations have reported the presence of prominent intrapulmonary bronchopulmonary anastomoses (IBAs) between the pulmonary and bronchial arteries as a potential source of a right-left shunt, which is not exclusive to COVID-19 infection and has been reported in idiopathic pulmonary hypertension and chronic thromboembolic pulmonary hypertension . These I2) . Trials 2) .The effects of previously addressed endotheliitis and the presence of large shunts with secondary vascular dysregulation are also convincing explanations for the significant hypoxemia that appears early in the hypoxemic phase of the disease.Dysregulation of the pulmonary vasculature is related to perfusion abnormalities and includes dilated peripheral vessels with a “tree-in-bud” configuration. The prevalence of these dilated vessels significantly increases the duration of hospitalization and ventilation . The forThis vascular dysregulation abolishes the hypoxic pulmonary vasoconstriction mechanism, causing an increase in venous mixing, excessive pulmonary vasoconstriction, and micro-and macrothrombosis, with a consequent increase in dead space ,58.2-induced ventilation mechanism, and aggravate hypoxemia. Finally, this interaction between the pulmonary shunt that generates hypoxia and the hypercapnic ventilatory inhibition clarifies hypoxemia without increased respiratory effort or even without dyspnea, referred to in the literature as “silent hypoxemia,” in a better way [It is challenging to identify shunts to visualize their typical presentation as pulmonary involvement progresses . Currenttter way .55.1The distinctive features of classic ARDS are similar to those of SARS-CoV-2, including diffuse alveolar damage, local edema with protein leakage, hyaline membranes, and hyperplasia of type II alveolar cells. However, severe endothelial injury and an increase in the prevalence of thrombotic phenomena may explain the accentuated effect on the pulmonary circulation. These alterations in local perfusion can also explain hypoxemia . An incrIt has been previously reported that in ARDS induced by COVID-19, the presence of short circuits that force redistribution and overflow of normally aerated units, in addition to interstitial edema that increases pulmonary vascular pressure with embolized units, leads to hypoxemia with differences in V/Q ,68. CapiA low V/Q ratio (areas of high perfusion with poor ventilation) and intrapulmonary shunting are caused by alterations in respiratory mechanics, vascular obliteration, and alveolar infiltration, as well as impaired perfusion, which worsens if the coexistence of more of these persists in the same clinical entity, distinctive of COVID-19 .Intrapulmonary shunts caused by pulmonary capillary distention or the presence of intrapulmonary arteriovenous anastomoses are closely related to the hyperdynamic state of patients with ARDS . This haThese V/Q regions can change suddenly from high to low, and vice versa, if increased cardiac output is not followed by proportional alveolar ventilation . IncreasIt is relevant to note that there are significant differences in perfusion, shunt, and ventilation-perfusion in the models studied, secondary to the heterogeneity of alveolar units. The severity of hypoxemia in infected patients cannot be explained without extensive perfusion defects, mismatch V/Q, or overperfusion of non-oxygenated regions . A smallDiffusion is also limited when inflammation and edema impair the exchange barrier (blood gas), worsening hypoxemia  Fig. 5)Fig. 5).F2)/FiO2 <300 mmHg (62). The models analyzed have estimated that a limited fraction of the injured lung between 0 and 30% with an absent exchange mechanism and vasodilation in the injured regions are determining factors for PaO2/FiO2 <300 mmHg, and have demonstrated that the HPV mechanism by itself does not sufficiently explain the observed levels of hypoxemia [The normofunctional HPV mechanism generates lower fractions of shunts and limited hypoxemia, which prevents a partial pressure of oxygen : NoneDr. Francisco Javier González Ruiz.The author declares that there is no conflict of interest.