Floral patterning is a complex task. Various organs and tissues must be formed to fulfill reproductive functions. Flower development has been studied, mainly looking for master regulators. However, downstream changes such as the cell wall composition are relevant since they allow cells to divide, differentiate, and grow. In this review, we focus on the main components of the primary cell wall—cellulose, hemicellulose, and pectins—to describe how enzymes involved in the biosynthesis, modifications, and degradation of cell wall components are related to the formation of the floral organs. Additionally, internal and external stimuli participate in the genetic regulation that modulates the activity of cell wall remodeling proteins. The process of flower development involves the formation of very complex structures. Flowers generally arise from the inflorescence meristem, where cellular differentiation processes give rise to specialized structures involved in all the aspects of plant reproduction. Once the flower primordium is formed, specific changes can be observed in this area; cell growth, division, and differentiation make up tissues with different identities and functions . A largeThe involvement of cell wall-related genes has been described during plant development. The detection of different cell wall components in different plant organs, including roots, leaves, stems, and flowers has been reported . In addiArabidopsis thaliana L., research has been conducted on genes involved in the formation, composition, and maintenance of the cell wall in the shoot apical meristem (SAM) [CELLULOSE SYNTHASE-LIKE D2, 3 and 5 [CELLULOSE SYNTHASE 3, CELLULOSE SYNTHASE 1, and CELLULOSE SYNTHASE 6 genes lead to the discovery of their involvement in the biosynthesis of cellulose [XYLOSYLTRANSFERASE 1, XYLOSYLTRANSFERASE 2, and α-XYLOSIDASE 1 genes were identified to play a role in the modifications of xyloglucans in the cell wall [In em (SAM) . The cha 3 and 5 , the CELellulose . Furtherell wall ,13. The ell wall . These iIn this review, we summarized recent and classical studies showing the influence of primary cell wall components on flower formation. We focused on the major components: cellulose, hemicellulose, pectins, and cell wall proteins. Most of the information presented here comes from Arabidopsis, though we also included some works on tobacco, rice, tomato, carnation, and other flowering species.One of the main components of the cell wall is cellulose. Cellulose comprises glucose molecules linked by β-(1-4) bonds synthesized by cellulose synthase complexes located in the plasma membrane. The deposition of the cellulose microfibrils influences the direction of the cell wall growth and, for instance, is essential for anisotropic growth. In this process, cellulose synthase complex conformed by cellulose synthases and other accessory proteins may be orientated by cytoskeletal control .Arabidopsis thaliana is the mutation of the RADIAL SWELLING (RSW1) gene, also known as CELLULOSE SYNTHASE 1 (CESA1) gene, which is involved in cellulose synthesis and when not functional causes defects related to reproductive development. These defects are the result of stigmas that protrude beyond the sepals and shortened petals. Although the anthers have normal dehiscence and pollen development, self-pollination is reduced because the stigma is well above the anthers as they have short filaments and show a wrinkled surface [CESA1 and CESA3 genes are pollen defective [CESA3 antisense plants is associated with shorter filament cells [CSLD1 and CSLD4 showed their importance for pollen tube growth. Both CSLD1 and CSLD4 are highly expressed in mature pollen grains and pollen tubes. Mutations in CSLD1 and CSLD4 caused a significant reduction in cellulose deposition in the pollen tube wall and disorganization of the pollen tube wall layers. In csld1 and csld4 single mutants and the csld1 csld4 double mutant, the pollen tubes exhibited abnormal growth both in vitro and in vivo, affecting the pollination capacity [AtCSLA7, a member of a CELLULOSE SYNTHASE-LIKE (CSL) subfamily [It is interesting how a lack of cellulose synthesis can affect the development of floral tissues. An example in  surface . Interesefective . These pnt cells . On the capacity . The impubfamily . We can conclude that the amount of cellulose that is synthesized has a significant effect on flower function. Multiple factors could be indirectly involved in cellulose synthesis since its anabolism depends on the plant nutritional status. For example, plants with enhanced sucrose metabolism showed increased plant biomass and abnormal morphological flower phenotypes, suggesting that the physiological processes related to the nutritional status can modulate cell wall composition .It is worth mentioning that other studies not explicitly related to flower development, showed that the orientation of the cellulose fibers within the cell wall is determinant for proper plant development. In one of these experiments, a compound called cobtorin was used to alter cellulose fiber deposition within the cell wall, which resulted in amorphous and swollen cells in roots and hypocotyls . It woulThe phenotypes mentioned above suggest that in addition to reducing the size of the floral organs, a consequence of decreased cellulose synthesis is abnormal pollen tube elongation, which ultimately affects fertilization and subsequent seed formation. Given the importance of cellulose as the main component in cell wall biosynthesis, it is interesting to analyze genes involved in cell wall biosynthesis that are expressed in floral tissues in Arabidopsis. Most of them have not yet been functionally characterized but many are expressed in floral tissues , as showAnother major component of the plant cell wall are hemicelluloses, which are constituted by a diverse group of sugars such as xylose, mannose, arabinose, galactose, or fucose. This group constitutes roughly one-third of the cell wall biomass and encompasses mannans, xyloglucans, xylans, and mixed-linkage glucans. The structure of these polysaccharides, particularly their substitutions, varies depending on the tissue and the plant species ,25. The Dendrobium crumenatum petals [The structure and complexity of hemicelluloses are crucial factors affecting their activity and function. Synthesis (formation) and hydrolytic (degradation) reactions contribute to the overall distribution of a polymer size. Studies from the early 1990s showed the redistribution in polymer size of hemicelluloses and endoxylanase activity in a partially purified enzyme preparation of m petals , suggestXTH1, XTH30, and XTH33 showed expression in anthers at different developmental stages. The XTH29 gene showed expression in pollen. XTH5 expression was detected in the distal region of the stamen filament, possibly related to elongation. On the other hand, XTH9 showed the highest expression in the youngest flowers and loss of generalized expression as the flower matured, specifically in the terminal portions of the carpel and throughout the stamen filaments. XTH15 and XTH28 also showed complex expression patterns in the developing floral bud, with prominent expression in the carpel. On the other hand, XTH28 was detected only in sepals [XTH genes seems to be tightly regulated at the transcriptional level, resulting in precise expression patterns.Based on the work performed in Arabidopsis by Becnel et al. in 2006 , it is in sepals . The expXTH’s suggest that they are involved in floral organ development; however, future functional work on these genes will be necessary to understand their involvement in floral development.These expression patterns of Solanum lycopersicum), an increase in the presence of arabinans and xyloglucans in the pedicels was observed. In addition to this, a peak in the XTH presence in the abscission zone was observed one day after anthesis, which decreased over time. These results suggested that XTH enzymes may play an essential role in the floral abscission process [Studies from other species suggest the importance of hemicellulose modifications during the organ abscission processes. During floral abscission in tomato ( process .Dianthus caryophyllus L.), the expression of two genes encoding for DcXTH2 and DcXTH3 was detected in large quantities in petals compared with other tissues. The function of XTH in growing petal tissues was verified by an in situ staining of xyloglucan endotransglucosylase (XET) activity, which was detected at all stages of flower opening in all parts of growing petals. However, the activity tended to be higher in the base and boundary regions [MANY SENESCENCE-RELATED 12 (SR12), putatively encoding a β-galactosidase, has been isolated from carnation petals and related as a marker of senescence-associated with ethylene [During flower opening in carnation ( regions . Additioethylene . The xylethylene .Sandersonia aurantiaca), three genes encoding putative β-galactosidases  have been isolated from petal tissue. Expression of all three genes is observed in petals at the onset of flower wilting [α-L-ARABINOFURANOSIDASE 1 (AtASD1) detected in vascular tissues of mature and senescent petals, and α-L-ARABINOFURANOSIDASE 2 (AtASD2) localized throughout the petal in fully mature flowers [XYL1 showing expression in sepals, style, stamens, and the gynophore. The mutant of this gene causes altered growth of sepals [Studies related to the dynamics of the formation of hemicellulose have been linked to petal formation. In Sandersonia  are the most representative group of pectins. They are made up of galacturonic acid monomers linked in α 1-4 bonds . RhamnogOsPMT16 gene showed an alteration in flower formation; the pistils were slightly shorter with delayed growth of the stigma and shorter stamens, which led to significantly reduced fertility [QUARTET1 gene is affected in cell wall degradation, and the microspores remain grouped during microsporogenesis [VANGUARD1 (VGD1) that encodes another pectin methylesterase protein. The vgd1 mutant is affected in the growth of pollen tubes in the style and transmitting tract in the gynoecium. Genetic studies indicated that the vgd1 mutant did not show visible effects in the rest of the flower [PME5 is overexpressed, it can partially recover the phenotype of Arabidopsis plants where ETTIN (ETT/ARF3) is mutated, ettin is a mutant where the valves are affected in their size along the carpels in the gynoecium [It is interesting how the lack of pectin esterification can affect gynoecium development. For instance, a rice mutant of a putative pectin methyltransferase (PMT) encoded by the ertility . These rogenesis . Anothere flower . Interesynoecium .AtPPME1, it was shown that the atppme1 mutant had alterations in pollen tube growth, length, and morphology. However, it had no apparent effect on pollen grain morphology and cell wall patterns [Nicotiana tabacum, silencing of the gene NtPPME1, encoding the main tobacco PME isoform, showed a slight but significant decrease in in vivo pollen tube growth [In another work performed to characterize another pectin methylesterase encoded by patterns . Similare growth . These rAtPPME1 and AtPMEI2 are both expressed in Arabidopsis pollen; furthermore, the proteins physically interact, and that AtPMEI2 inactivates AtPPME1, at least under in vitro conditions. Additionally, in the same work, research performed with transient expression of the AtPMEI2 in tobacco pollen tubes showed increased length of pollen tubes compared to wild type, suggesting its role in cell expansion [Brassica oleracea L. var. italics, the pectin methyl esterase inhibitor encoded by BoPMEI1 showed expression specifically in mature pollen grains and pollen tubes. Ectopic expression of BoPMEI1 in the antisense orientation in Arabidopsis suppressed the orthologous gene At1g10770, resulting in retarded pollen tube growth and sterile buds. These sterile flowers displayed stamens with shorter filaments than wild type with no pollen on the surface of the shrunken anthers [Brassica Rapa ssp. pekinensis (Chinese cabbage), it was argued that the stamens in a male-sterile mutant (ftms) were relatively smaller than the wild-type line. The ftms mutant showed altered anthers without pollen. Interestingly, transcriptome analysis of the ftms mutant elucidated 17 differentially expressed PMEI genes compared to the wild type [The enzymatic activity of PMEs is regulated by the inhibition of Pectin Methyl Esterase Inhibitors (PMEI). These PMEIs act by regulating the demethyl esterification of homogalacturonans affecting cell wall mechanics and consequently plant development. Some PMEIs show differential expression in inflorescences and floral tissues of Arabidopsis, suggesting that they could play specific roles in flower development ,45. Morexpansion . In Bras anthers . Furtherild type .PMEI3; its overexpression in Arabidopsis plants causes reduced growth of the valves and thinner carpels than wild types [Another gene directly affecting gynoecium development is ld types .The biochemical remodeling of pectins seems to be important. In a study performed in potato, different pectin remodeling enzymes were overexpressed, resulting in plants with pectins that contained less arabinan and rhamnogalacturonan residues and these plants showed collapsed pollen grains .PAE1) from populus in Nicotiana tabacum plants, which leads to a decrease of acetyl esters levels of pectin. Deacetylation produced changes in the cellular elongation of floral styles and filaments, the germination of pollen grains, and pollen tube growth [Furthermore, pectins can be modified by apoplastic enzymes; one is the pectinacetylesterases (PAEs) that can remove acetyl-substituents ,51. Theie growth .Among the enzymes that degrade pectin (pectinases), the function of polygalacturonases (PGs) has been studied in several plant species and is related to anthers and pollen formation. When PG activity is absent or reduced in mutants, pollen development, pollen release, and pollen tube growth are altered ,53,54,55Nicotiana benthamiana plants. Significantly higher PG expression was present after incompatible pollination in comparison with the compatible stigma, this suggests a potential function of PGs in regulating stigma incompatibility. Furthermore, the application of exogenous PGs resulted in pollen tube growth inhibition or failure of germination [PGDZAT/ADPG1 (RDPG1), Brassica campestris Male Fertility 16 (BcMF16), PGAZAT/ADPG2, and At1g80170 found that they are consistently expressed in male tissues such as anthers, pollen grains and dehiscence zones in the flower [PGs have been detected in the cytoplasm of mature pollen, pollen tubes, and the pistil of mination . Furthere flower ,58,59.POLYGALACTURONASE INVOLVED IN EXPANSION 1 (PGX1), which is very similar in its DNA sequences to the known PGs, in overexpressing Arabidopsis plants, alterations in the total activity of PGs and the composition of the cell wall were detected. The phenotype of these plants generated flowers with extra petals, which suggests the participation of PGX1 in the early events of flower organ formation [In another study of a putative PG called ormation .Biochemical modifications in pectins appear to be decisive in the development of reproductive structures. As it was reviewed in this section, several works have been done with regard to androecium and specifically, to the development and function of pollen grains, changes in the expression of genes that encode for pectin remodeling enzymes, and its biochemical activity is important, because it can reduce the reproductive capacity of plants. Cell wall proteins play essential roles in all physiological processes and particularly in the dynamics of the components within the cell wall . The WalThese proteins are located in the cell walls, but do not have a direct action on carbohydrates; functional characterization indicates a function on processes affecting floral organ formation.INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), which is a gene that encodes a short protein with an N-terminal secretory signal peptide. It has been detected in the extracellular space, and its signaling is carried out through a protein similar to a leucine-rich repeat-receptor-like kinase (LRR-RLK), encoded by the functionally redundant genes HAESA (HAE) and HAESA-LIKE 2 (HSL2). Overexpressing IDA exhibits early abscission of floral organs [Nicotiana benthamiana showed that the overexpression of NbenIDA1A gene generated shorter corollas, premature senescence of this whorl, and corolla abscission was also accelerated [hae hsl2 generates flowers that do not open after pollination [An interesting example is l organs . This prl organs . Recent elerated . On the lination . WAK4) from Arabidopsis is associated with cell elongation. An antisense line of WAK4 in Arabidopsis exhibited smaller floral buds than wild type [Within the cell wall, the interaction between ligands and receptors is mediated. A gene encoding a cell wall-associated kinase (ild type . BIIDXI (BDX) is the only DUF642 gene detected in the anthers, and when mutated, pollen grain viability is reduced compared to wild type [bdx mutants. Although the mechanism by which BDX modifies the activity of PMEs remains unknown, this phenomenon may be relevant in modifying the cell wall and thus in the formation of reproductive structures [The participation of miscellaneous proteins, which do not have a clear predicted molecular function, i.e., domain of unknown function (DUF) proteins, have been reported related to flower development. In Arabidopsis, four genes encoding for the DUF642 domain have been detected. ild type . Curiousructures .As we have seen, cell wall modifications are a regular feature of flower development. Proteins that act on the main components of the cell wall seem to be relevant, however, there are other proteins with unknown catalytic capacity that appear to alter the architecture of the cell wall through different mechanisms.AtEXPA4 and AtEXPB5 displayed consistent expression patterns in mature pollen grains and pollen tubes and the atexpa4 atexpb5 double mutant was defective in pollen tube growth [PhEXP1 is preferentially expressed in petal limbs during development. Silencing of PgEXP1 in petunia revealed a decrease in petal limb size, reduced epidermal cell area, and alterations in cell wall composition and structure [Expansins are proteins that disrupt non-covalent binding between cellulose microfibrils and other hemicelluloses. Their participation has been described mainly in the expansion of cell walls in response to increased turgor pressure within the cell . Expansie growth . In petutructure . DcEXPA1 and DcEXPA2 transcripts accumulate in petals of opening flowers. Similarly, the expansins EgEXPA1, EgEXPA2, and EgEXPA3 found in Eustoma grandiflorum could be participating in the continuous growth of the petals during the development of the flower [Mirabilis jalapa, a set of expansin transcripts was identified that include α-expansins (MjExp1-MjExp7) and β-expansins (MjExpB1-MjExpB3) with detectable changes in transcript abundance during flower development, particularly in the rapid expansion and senescence of the ephemeral flowers [DcEXPA1 and DcEXPA2) can be associated with petal growth and development during carnation flower opening [The presence of expansin genes was detected in pea petals  and tomae flower . In Mira flowers . The pre opening . In summLeAGP-1 in Lycopersicum esculetum, where overexpressing plants presented a greater number of inflorescences than wild type and most floral buds do not develop completely [AtAGP19 showed relevance in the development of flowers and fertility. In the null mutant, besides less and smaller flowers, stamen and ovule development were affected, resulting in less seed [Brassica campestris, the putative MALE FERTILITY 8 (BcMF8) gene encodes an AGP, which is expressed in pollen and pollen tubes. Antisense plants presented morphological defects in pollen grains and pollen tube growth, causing a lower seed yield [Arabinogalactan proteins (AGPs) are extensively glycosylated hydroxyproline-rich glycol proteins found along plants. They are thought to have important functions in plant growth and development, especially in plant reproduction ,77,78. Smpletely . In Arabess seed . In Brased yield .RICE MALE STERILE2 (RMS2) gene, which encodes a GDSL esterase/lipase, showed shrunken anthers with abnormal pollen, resulting in male sterility in this rms2 mutant. This family of proteins is related to lipid metabolism [Finally, there are studies on other proteins that seem to be good study potentials. In rice, plants with a mutation in the tabolism , howeverBased on the available information, probably many of the cell wall proteins participate in flower development, making them interesting candidates to study in greater depth.A lot of enzyme families are potentially involved in the biosynthesis and modifications of cell wall components see CAZy database . Within YUCCA4 [Most of the genes mentioned in this review encode enzymes, the last acting players in regulatory cascades. An interesting question is how their expression is regulated. Transcription factors (TFs) involved in flower development are good candidates since they often function as master regulators, causing severe developmental defects related to cell wall processes. For instance, the cell wall composition has been studied during gynoecium medial domain development , a zone YUCCA4 . Similarly, little is known about how endogenous regulators such as hormones, including biosynthesis, transport, and perception, may actively participate in this process by controlling the expression of genes that subsequently modify cell wall components in the flower. pin mutant), which can be partially recovered by affecting xyloglucan biosynthesis, suggesting that this xyloglucan synthesis is partially responsible for auxin transport [XTH gene expression [PMEI3 resulted in delocalization of the PIN1 auxin transporter, and these plants had a more rigid cell wall due to lower demethyl esterification of pectins [Auxins are involved in cell wall regulation . Celluloransport . In othepression . The imppression . Overexp pectins .AHP4) affect anther dehiscence by affecting the expression of genes related to cellulose and hemicellulose synthesis [Another hormone necessary for plant development are cytokinins. Cytokinin signaling is clearly present in the carpel margin meristem (CMM) and the valve margins ,102. Somynthesis . In rootynthesis ,105.Although the involvement of these hormones in flower development is widely known, future work is needed to understand the relationship between the response to these and other hormones and cell wall development that contribute to flower development.Modifications in the cell wall are decisive in floral organ development. Alterations in cellulose synthesis, which is the most representative component in the cell wall, show floral organs that do not reach their optimal sizes for processes such as fertilization. Furthermore, loss in biomass is a relatively predictable event since cellulose is the main cell wall component. However, there are many genes involved in the cellulose synthase complex that are not yet properly identified and studied. Hemicelluloses modulate senescence and abscission processes. The biochemical modifications and degradation may have a participation in the development of organs. Specific expression patterns of genes encoding enzymes that synthesize/modify, or degrade hemicelluloses give indications of their relevance in specific regions of the flower and the necessity of tight transcriptional control.Pectins are found in various events, from the formation of pollen grains, the development of fundamental structures in the gynoecium, and participation in the development of the perianth organs. The characterization of regulators that modify and degrade pectins are very representative. Alterations in the methyl esterification of pectins can modify floral structures and hence the fertilization process.Of the proportion of proteins present within the cell wall, those that do not have enzymatic activity also seem to be very relevant for flower development. Expansins have been described in several flowering plant species. However, how they participate during development and what their importance is in collaboration with other interactors within the cell wall still needs more research. The proteins involved in signal transduction are very important in the life cycle of plants. Both receptors and ligands are found in the apoplast zone. Their regulation not only occurs outside the membrane, but it is also believed that genes related to modifying the biochemistry of the cell wall could also be regulated. Another type of protein with unknown function i.e., some domain of unknown function proteins (DUFs), seems to be relevant and quite visible phenotypes in loss-of-function mutants have been observed. However, it is unknown how their mechanism of action could be, it is known that they can influence enzymatic activity at least indirectly of other proteins. The expansion of the cell wall allows the cells to acquire their final size, shape, and identity in the different organs of the plant. Therefore, the synthesis and modification of the main components of the cell wall are important for the formation of the reproductive structures ,9.In It is important to highlight that several genes that could actively participate in flower development have only been detected at the transcriptomic level without being functionally studied. On the other hand, at the proteomic level, the mechanism of action of a large proportion of proteins detected in the cell wall is still unknown, which could be relevant in scaffolding-type processes and signal transduction related to flower development.


Drosophila, the MYO6 ortholog regulates actin reorganization during spermatid individualization and male KO flies are sterile. In C. elegans, the MYO6 ortholog mediates asymmetric segregation of cytosolic material and spermatid budding through cytokinesis, whereas in mice, this myosin regulates assembly of highly specialized actin-rich structures and formation of membrane compartments to allow the formation of fully differentiated sperm. In this review, we will present an overview and compare the diverse function of MYO6 in the specialized adaptations of spermiogenesis in flies, worms, and mammals.Spermiogenesis is the final stage of spermatogenesis, a differentiation process during which unpolarized spermatids undergo excessive remodeling that results in the formation of sperm. The actin cytoskeleton and associated actin-binding proteins play crucial roles during this process regulating organelle or vesicle delivery/segregation and forming unique testicular structures involved in spermatid remodeling. In addition, several myosin motor proteins including MYO6 generate force and movement during sperm differentiation. MYO6 is highly unusual as it moves towards the minus end of actin filaments in the opposite direction to other myosin motors. This specialized feature of MYO6 may explain the many proposed functions of this myosin in a wide array of cellular processes in animal cells, including endocytosis, secretion, stabilization of the Golgi complex, and regulation of actin dynamics. These diverse roles of MYO6 are mediated by a range of specialized cargo-adaptor proteins that link this myosin to distinct cellular compartments and processes. During sperm development in a number of different organisms, MYO6 carries out pivotal functions. In  The acrosomal vesicle adheres through a cytoskeletal plate called the acroplaxome  to the nuclear envelope. As demonstrated in rodents, the acroplaxome consists of actin filaments and the intermediate filament protein, SAK57 (spermatogenic cell/sperm-associated keratin of molecular mass 57 kDa), which forms the marginal ring terminating the acroplaxome and connecting, together with additional linker proteins, the acrosome to the nuclear lamina ; (Toshimori 6); , which consists of a perinuclear ring, and contains actin filaments and microtubules that extend into the elongating sperm tail  and the tubulobulbar complexes (TBCs); . The apical ES is a specialized actin-rich structure formed between spermatids and Sertoli cells and is composed of parallel actin bundles sandwiched between the spermatid plasma membrane and the Sertoli cell’s endoplasmic reticulum (ER);  mouse contains a spontaneous mutation in the Myo6 gene, which leads to the complete absence of MYO6 in the homozygous mouse . Interestingly, newly formed actin cones are composed exclusively of actin bundles, whereas moving cones develop two domains—a rear region of parallel bundles and a dense actin meshwork at the front  is present throughout the actin cones, whereas later, when the individualization cones move away from the spermatid nuclei, it concentrates at the front of the actin filament meshwork  flies actin filament assembly is disrupted causing loss of the actin meshwork at the front of the cone and as a result the movement of the actin cones stops before individualization is completed  and singed (a fascin ortholog), are localized at the rear end of the cone  and spe-15, the ortholog of human MYO6 (encoded by spe-15) , which may indicate that dimerization/multimerization of MYO6 is required for cytokinesis and spermatid release. Depletion of NMY2 or spe-15 causes defects in the asymmetric segregation of cytosolic components.Morphological rearrangements in nematode spermatogenesis do not lead to the formation of flagellated sperm, but instead give rise to spermatozoa that use amoeboid motility. In ial Fig. ;  male mice  male mice completely lacking an acrosomal granule , which would suggest the premature release of round/elongated spermatids to the lumen of seminiferous tubules (Wen et al. The apical ES not only maintains the close contact between maturing spermatids and Sertoli cells but also enables the migration of spermatids across seminiferous epithelium during spermiogenesis and contributes to the positioning of spermatids with their flagella pointed towards the lumen of seminiferous tubules (Dunleavy et al. Drosophila spermatid individualization, C. elegans spermatid differentiation, and mouse spermiogenesis, MYO6-deficiency in mouse spermatids is less pronounced and the Snell’s waltzer male mice are only sub-fertile, while MYO6-deficient flies and worms are sterile (Kelleher et al. The unique reverse directionality of MYO6 may explain why the activity of this motor is necessary at different stages of spermiogenesis and its absence causes a set of specific phenotypes in mice. During the Golgi phase, MYO6 appears to maintain the spatial organization of the Golgi complex and to facilitate a short-distance transport and fusion of proacrosomal vesicles. During the following cap and acrosome phases, depletion of MYO6 impacts on the correct localization of the acrosomal granule and symmetry of developing acrosome. Finally, during the maturation phase, loss of MYO6 disturbs the spatial integrity of the endocytic compartment at the TBC. In addition, our preliminary observations suggest that MYO6 may regulate actin dynamics linked to the apical ES organization. Overall, our observations suggest that in mouse testes, MYO6 plays a structural role during spermatid development by regulating actin dynamics and anchoring different membranous organelles to the surrounding actin cytoskeleton. Although there are certain parallels in the function of MYO6 and its orthologs in regulating actin filament dynamics during While the role of the actin cytoskeleton in mammalian spermiogenesis is a rapidly developing field, our current understanding and recognition of the involvement of different myosins in this process is still less well documented. As highlighted throughout this review, several questions remain, especially in relation to the actin-based origins of acrosome symmetry, regulation of TBC organization and endocytosis, and function of the apical ES in the adhesion of spermatids to Sertoli cells and spermiation. At present, the suggested functions of MYO6 during mouse spermiogenesis are predominantly based on morphological studies of fixed cells and tissues. Although the functional analysis of mammalian spermiogenesis is very complex, future studies may focus on identifying the wider MYO6 interactome in mammalian testis. This would allow us to understand which binding partners and cargo adaptor proteins regulate its function and recruitment to different testicular compartments during sperm development. Moving forward, the use of in vitro Sertoli cell cultures and a combination of CRISPR/Cas9 KO cells with super-resolution confocal microscopy may provide further data to verify the proposed mechanisms of MYO6 function at different phases of spermiogenesis in mammals.


Mitochondria are dynamic organelles, the morphology of which is tightly linked to their functions. The interplay between the coordinated events of fusion and fission that are collectively described as mitochondrial dynamics regulates mitochondrial morphology and adjusts mitochondrial function. Over the last few years, accruing evidence established a connection between dysregulated mitochondrial dynamics and disease development and progression. Defects in key components of the machinery mediating mitochondrial fusion and fission have been linked to a wide range of pathological conditions, such as insulin resistance and obesity, neurodegenerative diseases and cancer. Here, we provide an update on the molecular mechanisms promoting mitochondrial fusion and fission in mammals and discuss the emerging association of disturbed mitochondrial dynamics with human disease. In thAs a result of their endosymbiotic origin, mitochondria carry their own genome, which is denoted as mtDNA and encodes 13 components of the OXPHOS system that are synthesized by ribosomes present within mitochondria. The remaining subunits of the system, as well as proteins required for mtDNA replication, transcription and translation, are encoded by nuclear genes . StructuAs highly dynamic structures, mitochondria undergo morphological changes and spatial rearrangements in order to adapt to cellular demands and to maintain energy homeostasis. These changes are modulated through the coordinated cycles of mitochondrial fusion and fission, commonly referred to as mitochondrial dynamics, which control mitochondrial number, size, shape and distribution within the cell ,9. The dIn mammals, mitochondrial dynamics are mainly progressed by large GTPases that belong to the dynamin superfamily. In particular, mitochondrial fusion requires three key proteins, mitofusin 1 and 2 (MFN1 and MFN2) and optic atrophy 1 (OPA1) while mitochondrial fission is promoted by the dynamin-related-like protein 1 (DRP1) ,14,15,16In the present review, we discuss the dynamic nature of mitochondria and summarize the current knowledge with regards to the molecular mechanisms of mitochondrial fusion and fission in mammals. In addition, perturbations in the dynamic transitions of the mitochondrial network caused by aberrant fusion and fission will be highlighted in the context of representative human diseases.As previously mentioned, under physiological conditions, mitochondrial dynamics are regulated by the balanced interplay between the opposing processes of fusion and fission. The identification of several key molecules involved in mitochondrial fusion provided important insights into the molecular mechanisms underlying this process . The terDrosophila fuzzy onions (Fzo) was the first mitochondrial-fusion-related gene to be identified [Drosophila’s Fzo [The entified . In mammla’s Fzo ,15. As pla’s Fzo . Both MFla’s Fzo . Mitofusla’s Fzo . Furtherla’s Fzo .OPA1 gene encodes the mammalian homologue of the mitochondrial genome maintenance 1 (MGM1) protein that was originally described in yeast [alpha-helix-rich N-terminal region that lacks specific domains. The functional domains of the C-terminus include a GTPase domain, a middle domain, a pleckstrin-homology region and a GTPase effector domain [The in yeast . Like miin yeast  or in thin yeast , ensuingin yeast ,35,36. Sin yeast ,37,38. Er domain .The fusion of biological membranes is a generic cellular process that occurs during several vesicular trafficking processes. Endosome–lysosome fusion, synaptic vesicle fusion and ER to Golgi tethering are a few examples of biological membrane merging, all sharing a common set of steps promoting fusion. At first, a close apposition of membranes known as membrane “docking” is facilitated through the interaction of proteins on opposing membranes and the assembly of protein complexes. During the second step of contact, the bridging of the two membrane compartments takes place, driven by the assembly of helical bundles that brings membranes into closer proximity, thereby promoting their fusion . It has Despite the fact that the fusion of the outer and inner mitochondrial membranes are separate events that are mechanistically distinct, the fusion machineries must be coordinated to complete fusion . To dateDRP1 result in hyperfused mitochondrial tubules secondary to the inhibition of mitochondrial fission [Mitochondrial fission describes the division of one mitochondrion into separate organelles through a multistep process in response to cellular cues. Fragmentation of the mitochondrial network can occur upon stimulation of fission activity and/or inhibition of mitochondrial fusion. As in the case of mitochondrial fusion, the core components of the fission machinery were originally identified in yeast ,51, whil fission ,54. DRP1 fission . It is c fission . MFF in HeLa cells resulted in reduced DRP1 aggregation on mitochondrial membranes, which was associated with fused and elongated mitochondria [Although the precise scission mechanism remains elusive, it has been shown that the initial step of mitochondrial fission begins with the translocation of soluble DRP1 to the OMM, where it binds to membrane-anchored receptors, oligomerizes and constricts the membrane at the fission site by forming a ring-like structure  Figure . The recchondria . Converschondria . Furtherchondria ,66. The chondria . An addichondria . On the chondria . The mitchondria . Accordichondria ,71,72,73Mitochondrial fission enables the separation of defective and depolarized mitochondria. It is thus of no surprise that mitochondrial fragmentation is often associated with conditions of cellular stress and mitochondrial dysfunction. Furthermore, a reciprocal relationship exists between mitochondrial fission and the process of mitophagy. In particular, mitochondrial fragmentation was found to be necessary for mitophagy since the inhibition of fission decreases mitophagy . ConversThe observation that the diameter of DRP1 polymeric rings (~150 nm) is much smaller than the average diameter of mitochondrial tubules (~300 nm) led to the postulation of the existence of a “pre-DRP1 constriction” step that reduces mitochondrial tubule diameter . As reveFurther insights into the mechanism of mitochondrial constriction at ER–mitochondria contact sites and mitochondrial fission were provided by a study that identified the ER-associated form of inverted formin 2 (INF2) as an important player in this process. INF2 was found to act upstream of DRP1 and promote actin polymerization at ERMCS, providing the force to facilitate the initial mitochondrial constriction followed by DRP1 recruitment and assembly to complete the division process . It has In addition to their role in determining the sites of mitochondrial fission, ERMCS have been identified as important glucose-sensing hubs in tissues such as the liver and muscle . The avaUnequivocally, the steady-state and dynamic equilibrium of the mitochondrial network is critical for preserving optimal function at the organismal level. Disruption of the delicate equilibrium between the two opposing processes of fusion and fission results in a fragmented or elongated mitochondrial network that has been associated with several pathological conditions. Besides the mutations in nuclear or mitochondrially encoded genes that are associated with monogenic diseases of mitochondrial dysfunction , aberratSystematic studies have shown that a plethora of human diseases are associated with excessive mitochondrial fragmentation as a result of defective fusion. MFN2 gene represent the most common molecular cause of Charcot–Marie–Tooth disease Type 2A (CMT2A), a dominantly inherited form of peripheral neuropathy. To date, more than 100 different mutations affecting MFN2 have been reported in patients with CMT2A (available from: uantwerpen.vib.be). The typical clinical symptoms of CMT2A involve the progressive atrophy of distal limb muscles that usually leads to wheelchair dependency in patients and a decrease of deep-tendon reflexes linked to foot deformities and distal sensory loss [MFN2 are mapped to four distinct functional zones and are mostly associated with severe CMT2A phenotypes, whereas mutations beyond the functional domains lead to milder symptoms. At the molecular level, MFN2 mutants encoding truncated or dimerization-incompetent proteins are more likely to cause disease due to decreased levels of normal MFN2, whereas mutants capable of dimerization and fusion tend to be pathogenic by hijacking normal MFN1 and MFN2 activity [MFN2 mutations have been also linked to another type of CMT disease, designated as hereditary motor and sensory neuropathy (HMSN) Type IV, which is characterized by subacute optic neuropathy, bilateral visual impairment and color-vision defects. Interestingly, mutations in the IMM fusion mediator OPA1 underlie the most prevalent form of autosomal dominant optic neuropathy, suggesting a functional overlap of OPA1 with MFN2. The above further implies that balanced mitochondrial dynamics are of particular importance for the proper function of the optic nerve [Neurons are cells of extremely high energy demand and are thus particularly vulnerable to mitochondrial dysfunction. Alterations in the mitochondrial network and consequent mitochondrial dysfunction have been suggested to occur as prominent early features in neurodegenerative diseases . Nowadayory loss . Structuactivity . In a reactivity . Moreoveactivity . MFN2 muic nerve ,95,96. Given their key regulatory role in energy metabolism, mitochondria respond to the availability of nutrients and energy demands by adjusting mitochondrial dynamics to maintain homeostasis. As mentioned earlier, under conditions of nutrient shortage and increased energy demand, the mitochondrial network appears elongated, whereas ample nutrient supply and decreased energy demand are associated with mitochondrial fragmentation ,98. It iAs mentioned earlier, OPA1 mutants are responsible for the autosomal dominant optic atrophy-1 (ADOA), a neuro-ophthalmic condition primarily characterized by impairment of visual acuity and generalized color-vision deficits ,104. BesIn addition to aberrations in the function of mitochondrial fusion mediators, perturbations of the mitochondrial fission machinery have received special attention with regards to their relevance in the development and progression of human pathologies.DRP1-based large-scale analysis of cancer genomes across various cancer types revealed a robust association of DRP1 with cell-cycle genes, postulating a role for DRP1 and mitochondrial fission in the regulation of cell proliferation [Disturbances in mitochondrial fission and in particular in the function of the DRP1 protein have been proposed to play a principal role in the initiation and progression of cancer, though different cancers are known to have distinct oncogenic backgrounds and etiologies. It has been shown that lung-cancer cell lines and lung adenocarcinoma cells from patients exhibit excessive mitochondrial network fragmentation as a result of increased DRP1 expression and reduced MFN2 levels . Moreoveferation . It is nOver the last few years, mitochondrial fission has been increasingly implicated in a number of neurodegenerative disorders, including Alzheimer’s disease (AD), the leading cause of senile and presenile dementia. Pathologically, AD is characterized by generalized cortical atrophy and aggregations of neurofibrillary tangles and amyloid plaques, the density of which appears to correlate with the clinical presentation . ExpressBesides AD, mitochondrial dysfunction in neuronal cells is also regarded as one of the main mechanisms for the development of Parkinson’s disease (PD), a degenerative disorder of the central nervous system affecting the motor activity of millions of people worldwide. The histopathological hallmark of PD is the intraneuronal build-up of misfolded proteins such as a-synuclein in the substantia nigra leading to the loss of dopaminergic neurons and concomitant dopamine deficiency . DespiteMitochondrial fusion and fission fine-tune cardinal processes of cellular life, and cumulative evidence has attracted remarkable interest into the role of mitochondrial remodeling in biological function. Undoubtedly, the scientific conception of mitochondrial dynamics and a comprehensive understanding of the mechanisms controlling mitochondrial architecture have progressed considerably in the last years. Of course, there are still many unresolved questions and additional proteins implicated in mitochondrial dynamics that remain to be identified. Nonetheless, the association between imbalances in mitochondrial fusion and fission with a steadily increasing number of pathological conditions and the provided proof-of-concept that restoration of mitochondrial network structure ameliorates disease renders mitochondrial dynamics as a major culprit for disease pathogenesis and an attractive target for therapy.


Mitochondria are crucial bioenergetics powerhouses and biosynthetic hubs within cells, which can generate and sequester toxic reactive oxygen species (ROS) in response to oxidative stress. Oxidative stress-stimulated ROS production results in ATP depletion and the opening of mitochondrial permeability transition pores, leading to mitochondria dysfunction and cellular apoptosis. Mitochondrial loss of function is also a key driver in the acquisition of a senescence-associated secretory phenotype that drives senescent cells into a pro-inflammatory state. Maintaining mitochondrial homeostasis is crucial for retaining the contractile phenotype of the vascular smooth muscle cells (VSMCs), the most prominent cells of the vasculature. Loss of this contractile phenotype is associated with the loss of mitochondrial function and a metabolic shift to glycolysis. Emerging evidence suggests that mitochondrial dysfunction may play a direct role in vascular calcification and the underlying pathologies including (1) impairment of mitochondrial function by mineral dysregulation i.e., calcium and phosphate overload in patients with end-stage renal disease and (2) presence of increased ROS in patients with calcific aortic valve disease, atherosclerosis, type-II diabetes and chronic kidney disease. In this review, we discuss the cause and consequence of mitochondrial dysfunction in vascular calcification and underlying pathologies; the role of autophagy and mitophagy pathways in preventing mitochondrial dysfunction during vascular calcification and finally we discuss mitochondrial ROS, DRP1, and HIF-1 as potential novel markers and therapeutic targets for maintaining mitochondrial homeostasis in vascular calcification. The process of calcification takes place due to the precipitation of insoluble salts of calcium carbonate and calcium phosphate. In soft tissues, three different types of calcifications are present, dystrophic (occurs in damaged or necrotic tissue), metastatic  and calcinosis   mice , medial arterial (associated with ESRD and diabetes), and aortic valve  6OH2] deposition within tissues 2] observed in a mouse model of uremic arterial calcification  overload and mitochondria are the key regulators of intracellular Ca and Pi levels  reduces calcification both in VSMCs VSMCs are the most abundant cells of the tunica media in the vessel wall. Their pivotal role is to maintain vessel structure and function. VSMCs in the vessel wall are often exposed to blood pressure variability, particularly in disease settings e.g., increased blood pressure variability during hypertension Parati, . RecentlAdditionally, VSMCs maintain a contractile phenotype under physiological conditions, which facilitates blood flow through the arteries. However, under biological stress or injury, the cells differentiate into an osteogenic phenotype with an acquired ability to proliferate, migrate and synthesize extracellular matrix vesicles. These differentiated VSMCs migrate into the intima layer of the vessel wall and can induce arterial stiffening  has a crucial role in maintaining mitochondrial homeostasis in VSMCs. COMP is present both in the extracellular matrix and in the cytoplasm where it binds to mitochondria. Mitochondrial transplantation experiments in VSMCs suggest that mitochondrial COMP regulates the contractile phenotype of VSMCs  and oxidative stress caused by ROS, which are by-products of aerobic metabolism  DNA haplogroup analysis in a cohort of aortic stenosis patients has revealed an increased presence of haplogroup H, suggesting haplogroup H as a risk factor in developing aortic stenosis  is reported in human stenotic aortic valves  induces calcification with a concomitant increase in osteogenic markers including TNAP, collagen type I alpha1 (Col Iα1), Runx2 and osteocalcin  induced VSMC calcification, suggesting a pivotal role for Runx2 in regulating oxidative stress-mediated vascular calcification   generation, thus reducing ROS  and TNAP in the medial layer of the vessels , a crucial component of the mitochondrial electron transport chain  is a rare premature aging disorder. A recent study investigated the mechanisms underpinning excessive vascular calcification in HGPS patients  and ROS. Generation of ROS leads to a vicious cycle of further ROS production, dysfunctional mitochondria and more AGEs  are post-translational modifications caused by the non-enzymatic binding of free reducing sugars and reactive carbonyls to proteins or lipids. AGEs are glycotoxins with pathological significance in diabetes, aging and several other chronic conditions / pyruvate dehydrogenase kinase 4, (PDK4) activation , hexokinase 1 (Hex1), and Pdk4  fed on a Pi-enriched diet show reduced mitochondrial respiration and increased oxidative stress, with aggregates of fragmented and swollen mitochondria observed in the cytoplasm, suggesting that damaged mitochondria are not cleared in this mouse model  is the process of removal of unwanted cellular cytoplasmic contents via double membraned autophagosomes. This unwanted cytoplasmic content is passed over to the lysosome for degradation and further recycling. Recent studies have demonstrated the role of autophagy in protection against vascular calcification   results in impaired osteogenesis  and interleukin 1β (IL-1β) are known inducers of VSMC phenotype switching and calcification  is a crucial regulator of vascular calcification. Increase in ROS levels stabilize and activate HIF-1α, which triggers VSMC transdifferentiation and calcification  and, ENPP1, (to reduce Pi overload) could provide an upstream approach to restoring mitochondrial homeostasis in vascular calcification.This is a renaissance period for mitochondrial research in vascular calcification. Mitochondrial dysfunction seen in vascular calcification pathologies is strongly associated with mineral dysregulation, ROS generation, phenotypic switching of VSMCs and metabolic shift from oxidative phosphorylation to glycolysis. It would be beneficial to investigate the sequence of mitochondrial dysfunction in a spatiotemporal manner in vascular calcification models.Runx2 (Lin et al., Enpp1 (Li et al., ApoE ablation or CKD induction will allow the investigation of mitochondrial architecture, biogenesis and turnover via mitophagy during the progression of calcification.Whilst Ca and Pi overload during vascular calcification is a likely cause of mitochondrial dysfunction, the mechanisms of Ca and Pi transport to the mitochondrial matrix from the extracellular matrix remain elusive. Moreover, investigating mitochondrial dysfunction during the progression of vascular calcification in mouse models lacking key calcification regulators e.g., In conclusion, maintaining mitochondrial homeostasis is crucial to protect cells against vascular calcification. Future therapeutic strategies targeted toward molecular regulation of mitochondrial ROS, DRP-1 and HIF-1 could be successful tools in the prevention of vascular calcification pathology and warrant further investigation.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sorghum bicolor (L.) Moench] production systems, threatening grain quality, safety, and nutritional value as both human food and livestock feed. The crop’s nutritional value, environmental resilience, and economic promise poise sorghum for increased acreage, especially in light of the growing pressures of climate change on global food systems. In order to fully take advantage of this potential, sorghum improvement efforts and production systems must be proactive in managing the sorghum grain mold disease complex, which not only jeopardizes agricultural productivity and profitability, but is also the culprit of harmful mycotoxins that warrant substantial public health concern. The robust scholarly literature from the 1980s to the early 2000s yielded valuable insights and key comprehensive reviews of the grain mold disease complex. Nevertheless, there remains a substantial gap in understanding the complex multi-organismal dynamics that underpin the plant-pathogen interactions involved – a gap that must be filled in order to deliver improved germplasm that is not only capable of withstanding the pressures of climate change, but also wields robust resistance to disease and mycotoxin accumulation. The present review seeks to provide an updated perspective of the sorghum grain mold disease complex, bolstered by recent advances in the understanding of the genetic and the biochemical interactions among the fungal pathogens, their corresponding mycotoxins, and the sorghum host. Critical components of the sorghum grain mold disease complex are summarized in narrative format to consolidate a collection of important concepts: (1) the current state of sorghum grain mold in research and production systems; (2) overview of the individual pathogens that contribute to the grain mold complex; (3) the mycotoxin-producing potential of these pathogens on sorghum and other substrates; and (4) a systems biology approach to the understanding of host responses.Grain mold is a major concern in sorghum [ Sorghum bicolor (L.) Moench] is grown internationally for its robust ability to withstand harsh climates, underlined by great water use efficiency and a diverse biochemical profile boasting high antioxidant potential. Sorghum exhibits robust environmental resilience and yield stability while being capable of reaching potential yield levels at lower input costs than other major cereals. These desirable traits have the crop posed for increased acreage. However, in many areas of agronomic sorghum growth such as regions in the tropical and subtropical climates, high heat and extended periods of humid conditions (relative humidity > 70%) are common. Regions such as these demonstrate inherently warm air capable of holding increased levels of water vaper, and will become more common and expansive as climate change occurs  , fungal constituents of the grain mold complex can infect and colonize spikelet tissue prior to grain development , resultiea mays) . In addiea mays) . In liveea mays) .Worldwide, mycotoxin exposure through food is widespread  and the The USDA estimates that 300–400 mycotoxins have been identified to date , with a The pathogenic relationships between SGM-associated fungi and their sorghum host have been difficult to characterize due to the dynamic spatiotemporal development and multi-organismal nature of the disease complex. SGM has generally been regarded as a single entity, yet the complex does not have a single causal agent. Rather, SGM is a syndrome attributable to a diverse assemblage of fungal taxa , the comThe diverse fungal species associated with SGM each take on unique physical and biochemical relationships with their host, involving both pathogenic and saprophytic lifestyles. Given the breadth and complexity of these relationships, host molecular defense mechanisms and the underlying genetics have been difficult to characterize. The need to breakdown the multi-species complex and scrutinize its various components has been expressed in previous literature , and numFusarium, which contains numerous mycotoxigenic species and is among the most common genera in the disease complex (F. verticillioides (synonymous with F. moniliforme in the literature for the purposes of this review), F. thapsinum, and F. proliferatum, although many others have also been isolated from sorghum grain  biosynthetic potential. F. moniliforme isolates from sub-Saharan Africa, 6 produced detectable FB1 in vitro with a range of 95–2448 μg/g when incubated at 25°C for 31 days  on sorghum medium in vitro when incubated at 28°C for 21 days  was collected in South Africa, vast ranges in MON production by F. verticilliodies was observed across the isolates of the various geographical regions (Moniliformin (MON), another mycotoxin produced by  21 days . Leslie  regions .Fusarium genus is a prolific producer of trichothecene mycotoxins such as deoxynivalenol (DON) and zearalenone (ZEN), and others, which have been documented in sorghum. DON biosynthetic potential or sorghum-derived Fusarium isolates has also been studied to some depth in a range of environments. In India, isolates cultured in glucose-yeast extract-peptone broth at 28°C for 14 days exhibited relatively low levels (0.01–0.04 μg/g) of DON biosynthesis .rescence . There hdiseases . While tmonisins . Abbas eF. verticillioides were shown to colonize stalks at an equal rate and caused similar levels of ear rot. Additionally, both fumonisin producing and non-producing strains were able to create similar levels of maize ear rot. F. verticillioides, F. verticillioides growth and alluded to a more constitutive form of defense in resistant genotypes  of fumonisin production to F. verticillioides infection  and ochratoxin. The Aspergillus species common in the SGM disease complex include soil-borne saprophytes that can opportunistically infect the developing caryopsis following events such as insect damage  and red-pericarp (r2 = 0.600) sorghums. The authors added that the lower rate of chitinase activity in red to white-seeded sorghums is likely due to red-seeded sorghums reliance on a constitutively higher presence of polyphenols. Chitinase and glucanase production in an effort to fragment a pathogen cell wall is a common defense mechanism in host plants  is another important contributor to SGM symptoms in some production systems and has also been implicated in the production of tenuazonic acid. A recent study from Brazil documented substantial tenuazonic acid production by sorghum-derived isolates between 0.0986 and 148 μg/g incubated on rice substrate at 25°C for 21 days (Sorghum contamination with  28 days . Epicocc 21 days .r = 0.47) and grain pigmentation to decrease severity (r = −0.45). It should be noted that the relationship between grain pigmentation and SGM is difficult to determine using visual methods due to potential bias, as it is inherently easier to observe mold incidence on white pericarp grain than pigmented grain.Previous publications dating from the 1980s to the early 2000s have described important sources of host resistance to sorghum grain mold, recognized and selected upon through visual features such as panicle compactness , grain pOpen panicle structures common in the guinea race of sorghum allow increased resistance to mold colonization by maximizing airflow and minimizing moisture within the panicle. Additionally, guinea sorghum exhibits large glumes with high grain coverage, a trait shown to limit grain mold infestation . HoweverWhile panicle architecture is a compilation of types of inflorescence branching, grain pigmentation is a culmination of a diverse and abundant set of biochemical compounds housed within the pericarp, testa, and endosperm . PresencPhenolic compounds underlying pigmentation in sorghum demonstrate a range of antioxidant potential that provides an effective source of phytochemical resistance to diseases such as SGM through scavenging of reactive oxygen species generated during fungal infection by both the host and pathogen . While cThe presence of a pigmented testa contributes a large amount of condensed tannins and a considerable portion of phenolic content to the biochemical profile of sorghum grain . HoweverTriticum aestivum) and maize (that encounter similar head mold diseases), has guided the characterization of cereal grains response to grain mold-specific pathogens to limit host stress and mitigate mycotoxins production. Increased characterization of the head mold pathosystems has provided a framework to further understand host phytochemical resistances of sorghum that are derived from metabolic pathways. The following sections seek to highlight specific host chemical mechanisms that are active throughout SGM disease development to provide a general understanding of these interactions to assist in the further research and development of sorghum cultivars with effective phytochemical grain mold resistances while maintaining and even improving important agronomic traits.Improved knowledge of the transcriptional responses and metabolic architecture of not only sorghum, but crops such as wheat  , which tp-coumaric acid have been shown to have inhibitory effects for Fusarium growth and mycotoxin production , flavanone-3-hydroxylase (F3H) as well as dihydroflavonol 4-reductase (DFR) and the DFR-like enzyme flavanone 4-reductase (FNR) . The ultSorghum demonstrates a high level of host sensitivity to fungal ingression, as spatial accuracy in the induction of phytoalexins critical to SGM resistance occur through site-specific synthesis at place of infection. Naringenin is a precursor to many of these phytoalexins and marks an important junction in the flavonoid pathway. A resistant response to fungal ingression is dependent on the availability of naringenin to enzymes that synthesize SGM resistance-related compounds. As described in detail in later sections, synthesis of 3-DA phytoalexins is an inducible resistance-related host response to SGM, and a lack of activity by F3H can be a consequence of inducible synthesis of 3-DAs resulting from increased channeling of DFR3/FNR on naringenin . As willCondensed tannins (proanthocyanidins) are well understood for their roles in increased antioxidant capabilities and significant correlations with SGM resistance . The preSbF3H1 of sorghum codes the flavanone-3-hydroxylase (SbF3H) enzyme that is capable of converting naringenin to condensed tannins ( tannins . As anot tannins . SbF3H c tannins .Tannin-1 (TAN1) transcription factor   were more susceptible to grain mold on average compared to accessions with the functional allele. Interestingly, the Genome-Wide Association Studies (GWAS) of the SAP did not associate TAN1 with grain mold resistance, illustrating the complexity of the SGM resistance phenotype that supports the author’s conclusion that resistance is a culmination of many additional factors in addition to the concentration of tannins (el (SAP) , an inte tannins .TAN1 coding region. The authors suggested TAN1’s role in the flavonoid pathway affecting biosynthesis of 3-DAs and tannins could contribute to TAN1’s role in grain mold resistance. While widespread presence of TAN1 across mapping populations is a factor of both aforementioned studies, a key difference is the differing levels of resistance between the two populations, with the former (SAP) demonstrating increased susceptibility overall than the latter (Ethiopian). This notable difference in SGM resistance between mapping populations may be a consequence of varying levels of TAN1’s role in transcriptional regulation of the enzyme-coding genes in the flavonoid pathway, and could be factoring into why TAN1 was not associated with SGM resistance in the SAP, but consistently identified in the Ethiopian panel.Another recent SGM association study employing 635 Ethiopian genotypes by Additional GWAS such as those by Flavan-4-ols have been subject to many early studies regarding grain mold resistance, and repeatedly shown to be a critical component of sorghum defense mechanisms active against SGM . StudiesH. maydis and C. graminicola, accumulates at slower rates following infection  (YELLOW SEED2 (Y2) as previously described by Y1 (YELLOW SEED3 (Y3). In addition to mapping efforts by the previously illustrated study, Two overlying concepts in the understandings of 3-DA activity in the SGM disease complex outline two shortcomings in current understandings: (1) Antifungal properties and pathogen-related induction of 3-DAs suggests a considerable role in grain mold resistance, but evidence of significant contributions to phenotype are inconsistent; and (2) the analysis of inducible 3-DA activity in SGM specific-interactions and host tissues  compared to other fungal pathogens of sorghum has been lacking. Recently however, with the addition of association and expression studies, ED1 (Y1) , that Boed by Y1 . The secSbDFR3 underwent significantly increased expression in resistant genotypes following inoculation of Fusarium spp. and Alternaria spp. Alongside of maintaining constitutive levels of 3-DAs, induction of genes such as SbDFR3 suggest a significant degree of responsiveness is present within the grain and glume. Grain and glume SbDFR3 expression shows transcriptional response to infection, however SbDFR3 is responsible for the conversion of flavanones to flavan-4-ols, and the enzyme responsible for the conversion of flavan-4-ols to 3-DAs, while likely a form of ANS, remains unknown  and demonstrated it to decrease fumonisin concentration in the seed. The authors also speculate as to the ability of phlobaphenes to inactivate fungal proteins by the formation of irreversible complexes as part of host detoxification mechanisms. Additionally, the enzymatic machinery which synthesizes phlobaphenes from flavan-4-ols has not yet been elucidated. While showing promise in resistance to Fusarium spp. of ear rot in maize, phlobaphenes are mainly regarded as a contributor to grain pigmentation in sorghum, and have not been scrutinized for their contribution to other phenotypes such as biotic resistance.The alternate fate of flavan-4-ols are phlobaphenes - insoluble phenolic compounds typically stored in the pericarp that contribute to visible red pigmentation of maize and sorghum . While mPericarp Color 1 (P1) of maize, an ortholog to sorghum Y1, has been shown to be functionally similar in the maize flavonoid biosynthesis pathway, with a confirmed role in the accumulation of phlobaphenes  enzymes are responsible for the synthesis of the flavones apigenin and luteolin , and likDarker pericarp color and purple plant sorghum lines have been generally shown to be more resistant to disease . KawahigThe findings described above from ZmFNSI-1 is commonly coexpressed with maize R2R3-MYB transcription regulator P1  infection point toward induction of flavone synthase as a defense response  plays a potentially important role in the SGM response of sorghum as it is responsible for the conversion of naringenin to the flavanone intermediate eriodyctiol . ThroughResearch has shown grain mold resistance of sorghum to be a vastly complex and highly quantitative trait . Recent As fungal assemblages change throughout regions, the plasticity of grain mold resistance in current germplasm justifies increased attention. Many studies have been performed over the years screening germplasm in a variety of regions under the stress of different fungal assemblages of the SGM complex . As warmP1 of maize demonstrate both similar and dissimilar functions, as divergence of orthologous genes can be significant even in species such as maize and sorghum with a close evolutionary relationship (Many of the underlying transcription factors related to SGM resistance like Y1 and TAN1 have shown widespread transcriptional regulation of a multitude of biochemical compounds. Orthologous genes such as tionship . Consequin planta, knowledge of the contribution of heterosis to host plant resistance, changes in host response through various physiological growth stages, relationships between the individual pathogens in the fungal makeup of SGM, and the effects of fungal endophytes on host resistance. Fungal endophytes have demonstrated the ability to reprogram the host transcriptome and metabolome to favor secondary metabolism in ryegrass (Fusarium thapsinum, Epicoccum sorghinum, Alternaria alternata, and Curvularia lunata. This study by Many additional topics related to the understanding of the SGM disease complex suffer from a lack of general knowledge in sorghum. Included within this group is mycotoxin detoxification ryegrass . While sryegrass . Rajini SGM is poised to increase in relevance as regions boasting warm temperatures and humidity increases due to climate change. For this reason, increased efforts are required to understand the plasticity of the grain mold complex between environments, how this affects the fungal hierarchy, and ultimately how these changes affect SGM pathogenicity on sorghum as a host. While the use of individual pathogens to isolate host response is critical to research, understanding the relationships within the disease complex is critical to understanding SGM and finding sources of resistance adapted to SGM complexes native to specific regions in international germplasm. Maintaining grain quality as a food and feed and increasing SGM resistance are both one in the same. Achieving this objective is achievable largely in part to sorghums abundant phenolic profile. However, due to the intricate and changing makeup of the SGM disease complex, continued research ventures into SGM show both notable obstacles and great potential for sorghum improvement in increased resilience, quality, and yield.AA and AW drafted the manuscript. RB is the professor advisor who oversaw the creation of the manuscript and provided final edits. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chips are obtained by subtractive processes such as machining workpieces and until recently considered as waste. However, in recent years they are shown to have great potential as sustainable raw materials for powder technologies. Powder production from metal chips, through the application of solid-state processes, seems to be an alternative to conventional atomization from liquid cooled with different fluids. However, chip material and processing have an essential role in the characteristics of powder particles, such as particle size, shape, size distribution and structure (4S’s), which are essential parameters that must be considered having in mind the powder process and the metallurgy applications. Moreover, different approaches refereed in the application of this new “powder process” are highlighted. The goal is to show how the actual research has been transforming subtractive processes from a contributor of wastes to clean technologies. The evolution of technology and increase in population have directed human beings to consumerism, facing exponential industrial growth with environmental impacts, such as consumption of minerals, generation of energy and production of wastes, highly seen in the steel industry. Thus, circular economy is highlighted regarding recycling or adding value to chips through the definition of new applications . Hence, Other application for metal chips can involve powder production by milling. This approach seems noticeable since the use of these materials appears sustainable ,8. MoreoThe metal chips used for recycling can involve industrial residues  or as maMilling techniques can include: (1) attritor ball milling that can be categorized as dry grinding or wet grinding, using regular speed (to 400 rpm) or high speed 400–1800 rpm) attritors ,16; (2) 00 rpm atRegarding mechanical milling processes, such as the planetary technique, it is important to consider the processing parameters and conditions. Milling energy is influenced by milling time and revolutions per minute (rpm), even ball to powder ratio (BPR). Other conditions, such as the milling atmosphere, the use of process control agent (PCA), and the temperature of milling, are significant too. These conditions affect the evolution of the 4S’s of the milled product . MoreoveThough the application of cryogenic ambient during milling seems to be effective for powder fragmentation, influencing ductile to brittle fracture temperature transition, some authors reported that disc milling was more productive than ball milling or cryogenic milling/grinding system, taking into account the time of milling for transferring all chips into powders .The jet milling technique was also applied for the powder production from bronze chips . This teFragmentation is greatly influenced by the type of milling process applied on chips, a higher efficiency of powder production was highlighted for the mill shaker technique than the planetary milling, achieving a faster size refinement was attributed to the milling energy induced by the shaking process . Moreove50 of 100 µm to 325 µm; however, the finest particles (d50 of ~140 µm) were produced by introducing 0.5 wt.% SA to the chips before milling [In addition, the application of PCA is important; it is usually used to avoid adhesion between particles to balls or to prevent cold welding between particles in order to dominant fracturing; PCA can define the distribution size and morphology of the milled particles. Methanol and stearic acid (SA) are common PCAs. One study presented the production of a d milling .Regarding the milling atmosphere, the most common is Argon gas, which is very useful to avoid oxidation of metal particles, as contaminations, during milling . HoweverRegarding the effect of BPR, the application of more balls increases collisions with particles increasing fragmentation. Therefore, the application of a higher BPR can increase the efficiency of milling . Thus, iParticle size analysis determines the progress of fragmentation during mechanical milling chips for powder production. This analysis can be performed by several techniques: sieving that is reported in wt.%, image analysis acquired by transmitting optical microscope (OM) or stereoscopic macroscope (SM) or scanning electron microscope (SEM). Other techniques include static light scattering (SLS), laser particle size analyzer (LPSA) or laser diffraction (LD) particle size analyzer (PSA).The evolution of particle size is governed by the fragmentation of metal chips and is dependent on the materials characteristics and processing conditions as well. Regarding the influence of material, some authors showed the production of particles in dissimilar sizes from similar milling of different chips; that difference was attributed to the primary conditions of the chips used for milling . Table 12-Ar mixture atmosphere, on decreasing particle size, the production of fine particles was attributed to the formation of brittle TiH2 compound during the pre-heat treatment step [Some authors revealed the success of applying a pre-heat treatment on a Ti alloy, heating in an Hent step . Another6Al4V chips mixed with 10 wt.% of alumina particles, showing a sharp reduction of particle size [Regarding the achievement of smaller particle size, some studies performed showing that the fragmentation can increase in the presence of a harder phase mixed with the matrix during milling; some authors proved this concept by milling Ticle size .6Al4V particles [Milling time also influences fragmentation and particle size formation, e.g., an increase from 3 h to 5 h milling increased yield fragmentation . Fragmenarticles . Figure 50 of almost 100 µm, by the addition of SA in the beginning of the milling process [The particle size distribution (PSD) can be affected by the presence of additives, such as PCA and even reinforcements , vanadium carbide (VC), silicon carbide (SiC) or Titanium carbide (TiC)), milling time or by the technique chosen for milling. Regarding the use of SA as a PCA, the procedure of addition is very important.  process . As rega process .A comparative study between jet milling and ball milling revealed a larger peak broadening for the PSD of particles obtained by ball milling attributed to the hardening of particles that occurred during the ball milling process .Moreover, the powder shape is influenced by the milling technique, the production of angular and agglomerate-free particles was remarked by the performance of shaker milling that was independent on milling time . The diaIn addition, other authors showed that the use of PCA, such as SA, influenced the particle shape, depending on the milling time in the presence of SA .Regarding particle shape applied in powder metallurgy, ISO 3252:2019 provides complete information, defining acicular particles to spheroidal type, defined by microscopic observations. However, microscopic images can be evaluated quantitatively, defining aspect ratio (proportion of the longest diameter to the shortest aspect of one particle). Nonetheless, a recent study applied the plasma spheroidization technique on milled particles in order to accomplish the aspect ratio of one so that spherical shaped particles could be achieved; however, the shape factor was dependent on the powder feeding rate during the spheroidization process .The efficiency of shake or jet millings can exceed ball milling due to a higher energy inherent to the technique. Moreover, disc milling shows a similar trend. Regarding milling conditions, an increase in milling time leads rise yield fragmentation. However, agglomeration of fine particles and formation of clusters can happen, caused by a cold welding effect. Similar behavior can be achieved by the application of the highest ball number. In what concerns temperature, a cryogenic ambient during milling also means high powder fragmentation for materials that promote a ductile to brittle fracture. The use of controlling agents such as stearic acid or toluene also influences the fragmentation. The use of a controlled atmosphere such as argon can avoid oxidation and contamination of milled particles. Moreover, the presence of additives or milling time affect powder characteristics, in particular particle size distribution. Regarding particle shape, the diameter of the balls appears to have the strongest effect on reaching near spherical powder particles. Moreover, an increment in milling time leads to a modification of shape factor to high values. Nevertheless, shaker milling lets angular shapes and non-agglomerate structures, independently of time. Concerning particle structure, shaker milling reveals a fast rate to achieve nanocrystallinity over planetary milling. However, crystallite size can decrease by increasing the milling speed and not milling time. The presence of a hard phase mixed with the matrix as reinforcement increases the chips fragmentation, promoting particle size reduction. Moreover, the addition of hard reinforcement encourages the reduction of powder crystallite size. The phase transformation from austenite to martensite phase can occur, particularly in austenitic stainless steel, due to intensive plastic deformation.The recycled powder particles obtained from metal chips recovery are noteworthy for powder metallurgy applications, conventional and advanced processes, such as pressing and sintering, hot isostatic pressing (HIP) or additive manufacturing (AM), respectively, including direct energy deposition (DED) or selective laser melting (SLM) 6Al4V powder, but the densification of commercial powders was still higher than powders produced by milling [Some researchers studied compacted disks of Al powder using recycled chips, chemically cleaned prior to milling to reduce oxide on surfaces, attaining a green density of 80%; these authors attributed the lack of strength of sintered powders to the weak densification . Other s milling . With re milling .6Al4V powder and 4 µm in Ti6Al4V-10 wt.% nanoalumina [6Al4V powders showed significant grain coarsening [Metal chips were applied also for producing metal matrix powder composites. Taking into account the milling time, increase in particle size by extending milling time caused by cold welding, some authors overcame that shortcoming in the production of a duplex stainless steel composite powder mixed with vanadium carbide (VC) particles . This stoalumina . The samarsening . Other rarsening . Some rearsening .90 of 40–140 µm for direct laser deposition (DLD) or d90 of 20–50 μm for selective laser melting (SLM) and d90 of 10 μm for fused filament fabrication (FFF) [Additive manufacturing, despite the high costs involved, has been acquiring significant importance due to its sustainability and the reduction of waste , obtaineon (FFF) ,40. Someon (FFF) . Howeveron (FFF) .The ball milling can induce plastic deformation as well as accumulation of internal strain, leading to the grain refinement of powder structures. This effect can induce strengthening, e.g., some authors reported the increase in hardness 850 HV) for stainless steel recycled powder (100 h of milling), the initial chips a hardness of 374 HV. This influence is attributed to the formation of a nanosized martensite phase 50 HV for,23,24,32Regarding the mechanical properties of materials after densification, The reduction in mechanical properties, such as strength and hardness, of recycled powders is attributed to efficiency of densification ,10,23,32From machining work parts is feasible for a significant number of metallic material chips  to produce powder through milling techniques. In general, these new “raw materials” take the advantage of severe plastic deformation to proceed with the fragmentation of chips. Moreover, the addition of other materials  could also promote the fragmentation of ductile metallic alloys.Depending on the pristine material, powder milling of chips have a main role in the final powder characteristics (4S’s). The new powder particles resulting from the milling of metallic chips can be applied mainly to powder metallurgy technology approaches, from conventional processes (subtractive and replicative) to additive manufacturing. This review highlights the role of metallic chips in manufacturing, and it enhances that the production of wastes is not a prerogative of subtractive process, it could be essential to additive manufacturing to guarantee its sustainability.Regarding future perspectives, it can involve the transformation of chips in outstanding powder raw material with characteristics not available in atomized powder particles. This target will be attained by a detailed study of microstructures function of waste material and processing, milling conditions and the nanoreinforced additions. Particular attention will be dedicated to the technologies selected for 3D object production.


Amyotrophic Lateral Sclerosis (ALS) is a mid-life onset neurodegenerative disease that manifests its symptomatology with motor impairments and cognitive deficits overlapping with Frontotemporal Lobar Degeneration (FTLD). The etiology of ALS remains elusive, with various mechanisms and cellular targets implicated, and no treatment can reverse or stop the progression of the pathology. Therapeutic interventions based on passive immunization are gaining attention for neurodegenerative diseases, and FDA recently approved the first antibody-based approach for Alzheimer's disease. The present systematic review of the literature aims to highlight the efforts made over the past years at developing antibody-based strategies to cure ALS. Thirty-one original research papers have been selected where the therapeutic efficacy of antibodies were investigated and described in patients and animal models of ALS. Antibody-based interventions analyzed, target both extracellular molecules implicated in the pathology and intracellular pathogenic proteins known to drive the disease, such as SOD1, TDP-43 or C9ORF72 repeats expansions. The potentials and limitations of these therapeutic interventions have been described and discussed in the present review. Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that primarily affects motor neurons (MN) of the motor cortex and spinal tract. Ninety percent of ALS cases have sporadic origin , whereas the remaining 10% develop the disease due to inherited gene mutations . The clinical manifestations of these two groups of ALS are indistinguishable, suggesting a convergence of varying pathways onto one unambiguous outcome, the degeneration of motor neurons and loss of muscles functions. Although several efforts have been made to decipher the pathogenic mechanisms of ALS, its etiology remains elusive, with various mechanisms and cellular targets suggested.To date, there is no effective treatment for ALS. Patients are currently treated with Riluzole and Edaravone, two drugs approved by the Food and Drug Administration (FDA), that increase the survival up to 14 months  that have been developed and tested in patients and/or models of ALS. Here, we summarize the main publications, based on original and experimental data, that proposed therapeutic interventions with a focus on target, antibody used, outcomes and limitations.*,” that allowed the inclusion of both “antibody” and “antibodies” wordings.The present review was developed following the PRISMA protocol . Titles and abstracts of manuscripts were fully screened to assess their relevance for our review and excluded if they were not describing a therapeutic intervention based on the development or evaluation of an antibody-based approach. A full-text screening was then performed on the four following criteria of eligibility: nature, finality, method, and availability  for further screening. We first eliminated duplicates, and then performed an “exclusion on format” analysis, aimed at removing all studies that were not original articles based on empirical data, thus only research articles, clinical trials and case reports were further considered. We proceeded to an “exclusion on language” and kept all the studies that were in English, French or Italian as authors could further select and analyze the papers in these languages. Using Endnote we performed an “exclusion on keywords.” Titles, abstracts and keywords were screened for the presence of population keywords: “amyotrophic lateral sclerosis” and “ALS,” keeping only papers focusing on ALS, and the presence of intervention keywords: “therapSelected papers were compiled in one table  have been unequivocally implicated in ALS. SOD1 mutation accounts for 2–5% of ALS cases   specific to misfolded SOD1 (DSE2-3H1), binding to a.a. 125-142 of extended electrostatic loop of full-length SOD1 protein, demonstrated to confer prion-like properties to the protein , specific to human misfolded SOD1  every day for 3 weeks in SOD1G93A mice before the onset of symptoms  domain of TDP-43  injection of AAV2/9:VH7Vk9 mitigated cognitive impairment, neuroinflammation and TDP-43 pathology as early as 2 months after injection. Moreover, in symptomatic TDP-43A315T mice, injected IT with AAV2/9:VH7Vk9, the VH7Vk9 scFv antibody improved motor performance and neuromuscular junction innervation, together with reduced neuroinflammation and TDP-43 cytoplasmic accumulation n repeat expansion in the C9orf72 gene was found to be implicated in frontotemporal lobar degeneration and amyotrophic lateral sclerosis as the most common known genetic cause  and investigated the potential of an antibody therapy to inhibit this phenomenon , a humanized monoclonal antibody (HuMAb) targeting the N- terminus of Nogo A was generated by Schnell and Schwab by immunization of mice in 1990  or 15 mg/kg of Ozanezumab (n = 152) delivered intravenously every 2 weeks for 46 weeks. Assessments were performed at 48 and 60 weeks after treatment. The study was a combined analysis of the ALS Functional Rating Scale-Revised (ALSFRS-R) and overall survival. Even though Ozanezumab was generally well tolerated, and that reported AEs were similar between treatment and placebo groups, no difference was observed in scores nor survival rates at 48 weeks. No efficacy was therefore observed for Ozanezumab, suggesting a poor reliability of anti-Nogo-A treatments for ALS patients . These studies were published between 2006 and 2013. Both used a recombinant humanized monoclonal antibody named Tocilizumab.in vivo effects of Tocilizumab infusion therapy in sporadic ALS patients  and in the CSF of ALS patients through inhibition of IL-6R by treatment with Tocilizumab, a humanized antibody (HuAb) against IL-6R , a destabilizing factor for axonal innervation of neuromuscular junction, triggers distal axonopathy and muscle denervation through its receptor, Neuropilin-1 (NRP-1). Both proteins were upregulated in pre-symptomatic ALS mouse models and ALS patients  of NRP-1 (α-NRP-1A), provided by Genentech Inc , previously generated from hybridoma cells obtained from mice immunized with recombinant myostatin  is a tumor necrosis factor receptor which, after oligomerization, activates different pathways that can lead to neuronal death  until 140 days of age promoted motor neuron survival, decreased astrocytes activation, and induced oligodendrocyte proliferation in the spinal cord and protected NMJ in gastrocnemius muscle from denervation. Moreover, 5D10-treated mice showed decreased pNfH levels in serum and 79% increase in hindlimb grip strength, suggesting improved motor function . In treated mice, authors observed a significant increase of surviving motor neurons but no changes in mice survival. The beneficial effect was observed only for the period during which the antibody was present in the CNS  is a matricellular protein, found up-regulated in spinal cord tissues of ALS patients  is an ubiquitously expressed nuclear protein but also an extracellular mediator of RAGE and TLR4 activation, two proteins implicated in the neuroinflammatory pathways observed in ALS  form of HMGB1  or physiological (TDP-43) forms. Targeting a toxic intracellular protein as SOD1, TDP-43 or C9ORF72 repeats in ALS is an essential requirement, as these proteins are presently the main triggers of the pathology. Full-length antibodies possess two main advantages as therapeutic intracellular strategies. First, they can be specifically up-taken by neurons and microglial cells through the clathrin-dependent Fcγ receptor endocytosis . The use of single chain antibodies could overcome this issue since they are smaller in size and possess higher cellular penetration capacity. Moreover, scFv antibodies have the potential to enhance the target protein degradation, and the fact that they can be easily engineered increases their future therapeutic potential. On the other hand, it is worth mentioning that the delivery of a single chain antibody through viral vectors might present further limitations in controlling and modulating the treatment in case of adverse effects. Despite the limitations posed by BBB, most of the antibody-based interventions identified through the present systematic literature review used a systemic delivery of the immunoglobulin with efficient target engagement and therapeutic effect. The use of a systemic rather than localized delivery represents a huge advantage for patients since therapeutic interventions localized in the CNS might be riskier and more dangerous for them. That being said, systemic delivery might increase the risk of side effects as some of the targeted proteins are expressed also outside the CNS. This subject has been poorly investigated in the reviewed papers but appear necessary to characterize therapeutic interventions. Future studies should consider evaluating the presence and effect of antibodies delivered systemically also outside the CNS. Moreover, novel strategies should be considered to improve the bioavailability of a systemically delivered Ab into the CNS. It is worth mentioning that no intervention analyzed has used local muscular delivery. The neuromuscular junction is a crucial structure in the context of ALS as it represents the contact surface between degenerating motor neurons and atrophic muscles. This synaptic structure appears to be underestimated as a delivery hub and future therapeutic interventions should also consider this administration strategy to target both motor neurons and muscle cells or molecules involved in NMJ degeneration.Another relevant issue to point out following the analysis of the selected literature is the use of ALS models. Although not all targeting SOD1, most of the analyzed studies used the mutant SOD1 model. This model is so far the most utilized as it perfectly recapitulates the symptomatology and pathological events occurring in ALS but, unfortunately, it also presents some limitations. First, it is highly heterogeneous. The different number of transgene copies, the various levels of mutant protein expressed, or the genetic background of the animals .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.