Phocoena phocoena) on the basis of inner ear analysis. The animal live stranded on the Dutch coast at Domburg in 2016 and died a few hours later. Ultrastructural examination of the inner ear revealed evidence of sensory cell loss, which is compatible with noise exposure. In addition, histopathology also revealed multifocal necrotising protozoal encephalitis. A diagnosis of toxoplasmosis was confirmed by positive staining of tissue with anti-Toxoplasma gondii antibodies; however, T. gondii tachyzoites were not observed histologically in any of the examined tissues. This is the first case of presumptive noise-induced hearing loss and demonstration of T. gondii cysts in the brain of a free-ranging harbour porpoise from the North Sea.Evidence of hearing impairment was identified in a female harbour porpoise (Phocoena phocoena) on the basis of scanning electron microscopy. In addition, based on histopathology and immunohistochemistry, there were signs of unrelated cerebral toxoplasmosis. The six-year old individual live stranded on the Dutch coast at Domburg in 2016 and died a few hours later. The most significant gross lesion was multifocal necrosis and haemorrhage of the cerebrum. Histopathology of the brain revealed extensive necrosis and haemorrhage in the cerebrum with multifocal accumulations of degenerated neutrophils, lymphocytes and macrophages, and perivascular lymphocytic cuffing. The diagnosis of cerebral toxoplasmosis was confirmed by positive staining of protozoa with anti-Toxoplasma gondii antibodies. Tachyzoites were not observed histologically in any of the examined tissues. Ultrastructural evaluation of the inner ear revealed evidence of scattered loss of outer hair cells in a 290 µm long segment of the apical turn of the cochlea, and in a focal region of ~ 1.5 mm from the apex of the cochlea, which was compatible with noise-induced hearing loss. This is the first case of concurrent presumptive noise-induced hearing loss and toxoplasmosis in a free-ranging harbour porpoise from the North Sea.Evidence of hearing impairment was identified in a harbour porpoise ( Phocoena phocoena Linnaeus 1758) is the most abundant cetacean species in the North-East Atlantic Ocean and adjacent North Sea, both in terms of sightings [The harbour porpoise  was recovered on the beach of the town of Domburg on the Dutch North Sea coast in July 2016 and died en route to a regional rehabilitation centre. In the Netherlands, necropsies of marine mammals are conducted at the division of Pathology, Department of Biomolecular Health Sciences of the Faculty of Veterinary Medicine of Utrecht University (UU) following an internationally standardised protocol . The harT. gondii  following a standard avidin–biotin complex protocol [T. gondii surface antigen p30 (SAG1) as described previously [All organs were grossly examined and, samples were systemically collected for histopathology, including skin, skeletal muscle, lung, heart, thymus, thyroid, stomachs, pancreas, spleen, liver, adrenals, kidney, intestine, urinary bladder, reproductive organs, mammary gland, eyes, brain and spinal cord, and various lymph nodes. This suite of tissues and representative samples of gross lesions were preserved in 10% neutral buffered formalin, processed by conventional histologic techniques, embedded in paraffin, sectioned at 4 µm and stained with haematoxylin and eosin (HE). Additional recuts and special stains of the cerebrum tissue, included periodic acid–Schiff (PAS) stain, to assess the presence of fungal organisms, and Ziehl–Neelsen (ZN) stain, to assess the presence of acid-fast mycobacteria. Sections of the cerebral lesions were stained immunohistochemically with polyclonal rabbit antibody against protocol . Controleviously  with theThe inner ears of the harbour porpoise were collected and fixed within 4 h post mortem following earlier published methods . The ear® 488 donkey anti-goat IgG, Alexa Fluor® 568 donkey anti-rabbit IgG, Alexa Fluor® 647 donkey anti-mouse IgG; Molecular Probes, Inc. Eugene, OR, USA A11055, A10042 and A31571, respectively; 1:400). Three small sub-segments were processed as controls: (1) control for the specificity of binding by the primary antibody ; (2) control for non-specific binding of the secondary antibodies  and (3) control for autofluorescence (no primary and no secondary antibodies were used).The right periotic bone was decalcified with 14% EDTA  tetrasodium salt (pH 7.4) for 43 days at room temperature. Then, the bone was removed, and the cochlea dissected using the whole-mount technique. The OHCs of the organ of Corti were labelled with anti-prestin antibody , the IHCs and OHCs with anti-myosin VI antibody , and type I afferent innervation was labelled with anti-neurofilament 200 kD antibody . Nuclei were counterstained with DAPI  and with the secondary antibodies . Brightness and contrast were enhanced, using identical values for treatments and the respective controls.2 and coated with platinum/palladium. The left cochlea was observed using an S-4700 SEM at the UBC Bioimaging Facility.The left periotic bone was decalcified with 14% EDTA (see above) for 37 days. The cochlea was dissected, dehydrated with increasing concentrations of ethanol, critical-point-dried with COThe ovaries were fixed and assessed for corpora scars  withBrucella spp., herpesvirus, morbillivirus, Neospora caninum and T. gondii. For Brucella spp., DNA was extracted from fresh lung, pulmonary lymph node and reproductive tract lymph node tissue, and for T. gondii and N. caninum from frozen as well as paraffin-embedded and fixed lung and cerebrum tissue using the DNeasy Blood and Tissue Kit  and QIAamp DNA FFPE Tissue Kit at Laboklin, according to the manufacturer’s protocol, respectively. An ADIAVET™ TOXO FAST TIME Kit von ADIAGENE Bio-X Diagnostics, according to manufacturer’s instructions, was used for the detection of T. gondii, while an in-house-developed PCR modified after Pereira and colleagues [N. caninum. Isolated DNA was screened by real-time PCR targeting the IS711 sequences of Brucella spp. following Maio and colleagues [Targeted pathogen screening was conducted by PCR for lleagues  was usedlleagues  and by qlleagues . For herlleagues . For morlleagues .Blubber and milk samples were analysed for polychlorinated biphenyls (PCBs), as part of a separate study . Air-expThe brightness and contrast of images were adjusted in Adobe  Photoshop CC 2018.Pseudaliidae) in both the bronchi and in the lumen of multiple pulmonary vessels. There were large numbers of nematodes morphologically consistent with Anisakis simplex present in the lumen of the forestomach, with multifocal mucosal ulceration and numerous invading nematodes  and in the tympanic cavities. A small number of trematodes, morphologically consistent with Campula oblonga, were observed in the hepatobiliary arcade.At necropsy, the adult female measured 146 cm total body length and weighed 46 kg. Based on the absence of visceral fat, muscle atrophy and blubber thicknesses of 6–8 mm she was in poor nutritional condition. Macroscopic lesions included multiple foci of necrosis in the cerebrum a. Additiematodes . A moderHistopathology of the cerebrum revealed extensive necrosis with haemorrhage and degenerated neutrophils, lymphocytes and macrophages b–d. TherToxoplasma gondii cysts in the cerebrum was confirmed by positive staining with anti-T. gondii antibodies .The ∑17PCB in blubber was 5.1 mg/kg lipid weight (lw) and 16.8 mg/kg lw in milk, indicative of active offloading through lactation .Inner ear analysis revealed evidence of OHC death in the apical turn of the cochlea, which in mammals results in permanent hearing loss ,41,42. SPermanent hearing loss can be caused by several factors, including exposure to noise, ototoxic drugs, or PCBs, age, congenital or immunological disorders, and other infections ,47,48,49The type of lesions seen in the cochlea in our study are compatible with noise-induced hearing loss. The location of noise-induced lesions within the cochlea depends on the frequency of the source. Studies on noise-induced hearing loss in terrestrial and marine mammals exposed to high intensity noise levels showed that the frequency of maximum hearing loss was a half octave, up to one octave, above the exposing tone ,65,66,67T. gondii antibodies confirmed protozoal encephalitis due to T. gondii infection while herpesvirus, morbillivirus and N. caninum were negative. This animal live stranded on the Dutch coast in 2016 and the most significant gross findings were multifocal necrosis and haemorrhage of the cerebrum and generalised emaciation. Positive staining of cerebrum tissue with anti-Toxoplasma gondii is generally considered a sporadic infection of aquatic and marine mammals [T. gondii is likely from terrestrial run-off and infection may be predisposed or possibly exacerbated by immunosuppressive, or debilitating factors, such as chemical contamination by PCBs, pregnancy, malnutrition, morbillivirus infection and other processes [T. gondii infection. This animal was born and held in a semi-open outdoor facility [Stenella coeruleoalba), reports of toxoplasmosis are more common [T. gondii life cycle” with more virulent genotypes has been suggested, but not yet confirmed [ mammals ,69. Exporocesses ,72,73,74facility  and faeconfirmed ,76. Thesonfirmed ,77.Severe and multiple-organ metazoan parasitic infections were also detected in this porpoise. The degree of emaciation and parasitic burden in this animal indicates generalised debilitation and impaired health status. However, in harbour porpoises from the southern North Sea, severity of parasite infections increased with total length of the host and all examined adults presented multisystemic parasitism . The lunHarbour porpoises use very high frequency acoustic signals of relative narrow bandwidth, with a frequency peak of ~130 kHz and the main energy between 110 and 150 kHz ,87,88. HThis study presents a case with evidence of hearing loss, which was compatible with noise exposure. In addition, histopathological and immunohistochemical analysis revealed a case of toxoplasmosis in a wild harbour porpoise stranded in the North Sea with severe lesions in the brain.


Grape seeds are a rich source of flavan-3-ol monomers, oligomers, and polymers. The diverse profile of compounds includes mainly B-type procyanidins  and the key monomers, catechin, and epicatechin that are positively implicated in the ‘French Paradox’. Today grape seed nutraceuticals have become a multi-million-dollar industry. This has created incentives to elucidate the variations in chemistry across cultivars, to identify signs of adulteration, and to understand the intrinsic and extrinsic factors controlling the expression of metabolites in the seeds’ metabolome. This review provides a critical overview of the existing literature on grape seed chemistry. Although the biosynthetic pathways for polymeric procyanidins in seeds have not yet been explained, abiotic factors have been shown to modulate associated genes. Research of extrinsic factors has demonstrated that the control of procyanidin expression is strongly influenced, in order of importance, by genotype  and environment, as claimed anecdotally. Unfortunately, research outcomes on the effects of abiotic factors have low certainty, because effects can be specific to genotype or variety, and there is limited control over physical metrics in the field. Thus, to gain a fuller understanding of the effects of abiotic factors and biosynthetic pathways, and realise potential for optimisation, a more fundamental research approach is needed. Nevertheless, the current synthesis offers insight into the selection of species or varieties according to the profile of polyphenols, as well as for optimisation of horticultural practices, with a view to produce products that contain the compounds that support health claims. Vitis vinifera L.) is one of the oldest and most significant crops in human civilisation [The grapevine  or extrinsic  factors on the chemistry of grape seeds and discusses how these sources of variation can be used to deliberately modulate the chemistry of grape seed extracts to optimise for quality. Finally, the quality of commercial grape seed extracts is variable, and the problems of adulteration mean that greater scrutiny is required as part of the quality control of grape seed products. Therefore, we also suggest detailed guidelines to ensure the quality, authenticity, and efficacy of commercial products.Polyphenolic compounds represent the most abundant and chemically diverse group of metabolites accumulated in grape seeds and the third most abundant constituents in the whole fruit, after carbohydrates and organic acids . Among tIn the present section, the chemical diversity of grape seeds is summarised, giving details from the first reports made in the late 1960s through to early 2020. This information was retrieved from the SciFinder and Google Scholar databases using the key words: “grape seed”, “chemistry”, “flavanol”, and “procyanidins”. Additionally, the main enzymes and intermediates involved in the biosynthesis of procyanidins are discussed and knowledge gaps are highlighted. Grape seed quality is strongly related to biological effects and the specific end-use or intended application, so a discussion of pharmacokinetics and structure–activity relationships is also provided.Most of the quality characteristics of wine, as well as the biological and nutraceutical properties are attributed to the polar components in grape seed extracts, particularly the flavan-3-ols, so the current review focuses on these components. Detailed reviews of the chemical constituents in grape seed oil are provided by ,24,25.trans configuration are known as (+)- or (−)-catechin, and in the cis configuration they are (+)- and (−)-epicatechin  and (−)-epicatechin  [S, 3R) and (+)-epicatechin  (ent-catechin or ent-epicatechin) are significantly less common [Flavan-3-ols represent the most reduced form of the flavonoids. They are characterised by the presence of a 2-phenyl-3,4-dihydro-2H-chromene skeleton, which is hydroxylated at position 3 of ring C . Favan-3catechin . However ,27,28,29  -catechin and (−)-epicatechin produces the monomers (+)-gallocatechin and (−)-epigallocatechin, respectively . These mO-gallate, represent the three most abundant and commonly reported metabolites [O-gallate, (+)-gallocatechin, (−)-epigallocatechin, (+)-gallocatechin-3-O-gallate, (−)-epigallocatechin-3-O-gallate, and (−)-epigallocatechin-3-O-vanillate [O-β-glucoside and (+)-catechin-7-O-β-glucoside [Thus far, the chemical structures of eleven monomeric flavan-3-ols  have beeabolites ,34. The anillate ,31,35,36lucoside  -gallocatechin, (−)-epigallocatechin and their pe seeds ,36, but pe seeds ,30,37. Htandards ,31,36. Sards [−1 ,39,40. TIt is only in the last decade that glycosylated catechins have been assigned in grape seeds . AccordiO-β-glucoside and (+)-catechin-7-O-β-glucoside were obtained through enzymatic hemisynthesis and their structures confirmed by uni- and bi-dimensional NMR spectroscopy [A recent study by Zerbib et al.  confirmetroscopy . Hence, Zerbib et al.  also ideGrape seed flavan-3-ols are found as monomers, dimers, oligomers (3 to 10 units), and polymers  . The conVitis vinifera variety (‘chardonnay’) based on tandem mass spectrometry analyses [Based on the interflavanic linkage of procyanidins, they are often classified as B-type, if a single linkage occurs in C4→C8 or C4→C6 , or A-tyanalyses .Among the 17 dimeric procyanidins reported in grape seeds ,47,48,49O-gallates [O-gallate derivatives of procyanidins B1, B2, B4, B5, and B7 -epicatechin, gallates ,29,47. I, and B7  are commgallates  in grapes et al.  was baseAmong the 16 trimeric procyanidins reported in grape seeds ,45,46,50Backbone types can include different combinations of catechin and epicatechin as the initiation, extension (middle), or terminal subunits. Downey et al.  reportedO-gallate -epicatechin as the extension unit, four have (+)-catechin, and two have (−)-epicatechin-3--gallate . (−)-Epi-gallate , only thO-gallate, with four. This suggests that the initiation units come from different metabolic pools than the extension units, as previously hypothesised [Hence, (+)-catechin is the most abundant terminal unit among procyanidin trimers, with six out of 16, followed by (−)-epicatechin, with five, and (−)-epicatechin-3-thesised ,54. The Aside from the known dimers and trimers, much higher degrees of polymerisation also occur. For instance, procyanidins with a degree of polymerisation from 28–37 units and a degree of galloylation from 8–15 have been observed ,55. FurtIn conclusion, despite recent advances there are still significant gaps in our knowledge about the chemistry of grape seed flavan-3-ols. For example, the absolute structural characterisation of glycosylated flavan-3-ols and oligomeric procyanidins, the presence of galloylated monomers in seeds, and the biosynthetic steps necessary for their formation is still lacking. This information is important because monomeric and polymeric flavan-3-ols are involved in the biological and nutritional properties attributed to grape seed extracts.Even though the mechanism of flavan-3-ol polymerisation is yet to be described, the biosynthesis of the major flavan-3-ol units in grape seeds, (+)-catechin and (−)-epicatechin, is well-known. The main enzymes and intermediates leading to the formation of flavan-3-ol monomers are depicted in The biosynthesis of flavan-3-ol monomers in grapes follows the same initial steps of other flavonoids via the phenylpropanoid pathway, leading to the production of narigenin by chalcone synthase (CHS) and chalcone isomerase (CHI). Narigenin is oxidised by flavanone 3-hydroxylase (F3H) followed by either flavanone 3′-(F3′H) or 3′,5′-hydroxylase (F3′5′H). The latter two enzymes create two possible flavan-3-ol precursors: dihydroquercetin for catechin and epicatechin (by F3′H); or dihydromyricetin for epigallo- and gallocatechins (by F3′5′H). Thereafter, dihydroflavonol 4-reductase (DFR) converts the precursors to leucoanthocyanidins, including leucocyanidins (from dihydroquercetin) and leucodelphinidins (from dihydromyricetin) . In the Considering that prodelphinidins are exclusive to the skin of the grape berry, the enzyme F3′5′H is expected to be active in this site. Studies on the temporal and tissue-specific expression of genes encoding F3′H, F3′5′H in grapes demonstrated that these two enzymes, in addition to having a cytochrome b5 (CytoB5) isotype, are necessary for the hydroxylation of the B ring of flavan-3-ols to produce (epi)gallocatechin . AccordiRegarding the temporal regulation of these genes, Bogs et al.  found thMedicago truncatula (Fabaceae), concluding that MATE1 is an essential membrane transporter for procyanidin biosynthesis in Medicago [O-glucoside results in increased accumulation of procyanidins [O-glucoside rather than free epicatechin is the substrate for procyanidin biosynthesis acting as intermediate in the polymerisation of flavan-3-ols in M. truncatula [O-glucoside in the seeds of different grape varieties further supports this hypothesis [M. truncatula, showed that a loss of function of this enzyme leads to the accumulation of 4β-(S-cysteinyl)-epicatechin, which provides the C4→C8-linked extension units during non-enzymatic procyanidin polymerisation, resulting in increased levels of oligomeric procyanidins.Despite considerable progress in the understanding of the flavan-3-ol biosynthetic pathway, the mechanism of flavan-3-ol polymerisation remains unknown. Furthermore, flavonoid synthesis occurs in the cytoplasm, yet most products are stored in specific compartments, such as cell walls and vacuoles. Hence, an efficient, albeit unknown, transport mechanism exists . Zhao anMedicago . The oveyanidins . Furtheruncatula . A similpothesis ,41. Howepothesis , suggestpothesis . This stIn vitro assays that elucidate important structural features of procyanidins often fail to predict in vivo outcomes. This is because of the unforeseen challenges of natural product pharmacokinetics. For example, it is common to ascribe biological effects to metabolites at concentrations that are many orders of magnitude above the feasible plasma concentrations . FurtherPolyphenols are less likely to be oxidised in Phase 1 reactions because the molecule is already highly oxidised. Rather, polyphenols tend to be circulated around the body as xenobiotic conjugates, either as sulphate esters or glucuronides . Cushnieper se, for reducing the perception of astringency. Other key structural characteristics such as the mean degree of polymerisation, the conformation, and hydrophobicity of the procyanidins are also important because they influence their interaction with macromolecules [In vitro assays often demonstrate that the most potent effects are derived from galloyl derivatives  and C4→C6 linked isomers. For example, procyanidin isomers with a C4→C6 linkage showed stronger epidermal lipid peroxidation inhibitory activity than C4→C8 isomers , which molecules .The other phenols in grape seed include phenolic acids, stilbenes, hydrolysable tannins, and flavonols . These aTrans-resveratrol is the most famous stilbene in grape skin and is widely implicated in positive health outcomes. Some studies report traces of both cis- and trans-resveratrol isomers in seeds [trans-resveratrol into its cis analogue has been shown to occur by natural light [cis and trans isomers of piceid, piceatannol, and miyabenol C [While stilbenes are common in the fruit pulp and grape skins , they sein seeds ,82, but in seeds ,83. Sincal light , it is ral light . A recenal light , but conabenol C .V. rotundifolia, or ‘muscadine’ grape [V. vinifera. However, it may be necessary to exclude the possibility of metabolite transfer from other plant parts. For example, studies that utilise grape seeds as by-products from the wine industry, such as the studies by Garcia-Jares et al. [An investigation of hydrolysable tannins was performed by Sandhu and Gu . They ide’ grape ,77,87. Ne’ grape  also ides et al.  and Pasis et al.  may needs et al. ,85, a chO-rhamnoside [O-glucoside [O-glucuronide [Contrary to hydrolysable tannins, which have a restricted distribution across species and cultivars, ellagic acid ,77, querlucoside ,85, quercuronide , rutin [curonide , kaempfecuronide  seem to curonide ,77,78,88In addition to the phenolic compounds, which usually represent between 4 and 7% of the seed weight, grape seeds demonstrate a complex metabolome containing 11% protein, 35% fibre, 16% oil, 3% minerals, and a fraction of other metabolites including free amino acids and simple organic acids ,89. The Several studies have shown that the content of phenolic compounds in grapes and their seeds is influenced by both extrinsic and intrinsic factors. Intrinsically, the variety or species is evidently significant. Extrinsically, the environmental characteristics such as solar radiation, water availability, ambient temperature cycles, location , stage of ripeness, and viticultural practices, are also important ,95,96,97The ideology behind understanding modulating factors is to harness some degree of control to optimise expression of phenolics for their health or aesthetic benefits. One of the first studies comparing the influence of different factors on the accumulation of highly polymerised flavan-3-ols in grape seeds  concludeAlthough the concentration and composition differences in grape seed flavan-3-ols can be influenced by external factors, genetic divergence within the species constitutes a major driving force of chemical differentiation in grapes . FurtherV. vinifera, there are important qualitative and quantitative differences in the phenolic composition of different grape species. Polyphenols in the seeds of 91 grape accessions from 17 species in Vitis demonstrated significant interspecific differences in the concentration of monomeric flavan-3-ols, procyanidins B1 and B2, flavonols, isoflavones, and gallic acid derivatives [While most chemical studies of grape seeds in the published literature are based on ivatives .V. palmata yielded the highest content of total polyphenols, giving 21.02 mg/g , followed by V. vinifera  and V. vulpina (19.49 mg/g FW) [V. champinii (0.95 mg g−1 FW). A similar pattern was observed in the specific analysis of monomeric flavan-3-ols, dimeric and trimeric procyanidins, and flavonols, reiterating that V. palmata, V. vinifera, and V. vulpina provide the highest natural sources of these compounds, except for resveratrol, which was abundant only in V. monticola [From the 17 species, mg/g FW) . These tonticola .V. vinifera is the most cultivated species of grape, other species such as V. labrusca and V. rotundifolia (muscadine grapes), are especially popular in the Eastern and South-eastern United States. By comparison with V. vinifera, these species express less (+)-catechin and (−)-epicatechin in the seeds, but more gallic acid and ellagic acid [V. rotundifolia [V. rotundifolia surpasses other species [V. vinifera, which expresses only trace amounts of tannins [While gic acid ,87,100. ndifolia ,87. Furt species , particu tannins .V. rotundifolia. Studies with different cultivars of V. rotundifolia grown in different places with potentially different climate conditions such as Central Florida, USA [The presence of hydrolysable tannins is a consistent feature of ida, USA , North-wida, USA ,98 and Sida, USA , have coida, USA .V. vinifera to develop high-yielding, biologically active grape seed extracts [In this regard, there is minimal research dedicated to understanding the degree of polymerisation and galloylation of procyanidins across the different species. This has significant implications in the context of health outcomes ,101. Altextracts .Multivariate statistical methods strongly emphasise the importance of genotype in determining the chemical profile of a specific cultivar . PreviouIn any given year, the species’ genotype is the most significant determinant of a seed’s chemical profile. However, if specimens from the same cultivar face differences in extrinsic factors, chemical profiles diverge significantly. Chira et al. , observeIn general, chemical differences between red and white varieties are observed in the proportion of galloylated and non-galloylated procyanidin dimers . One stuGenerally, across varieties, the relative quantity of monomers to oligomers varies, as well as the degree of galloylation ,103 and The impact of the growing environment on grapes has been recognised for centuries; the concept of ‘terroir’ was developed to describe the unique influence of a region’s soil type, temperature, rainfall, and other environmental factors on the characteristics of the grapes it produces. Additionally, agronomic strategies such as alteration of environmental conditions , application of elicitors, stimulating agents, and plant activators have been employed to enhance the biosynthesis of polyphenols in grape ,106,107.Solar radiation has long been known to influence the development of the grape berry and the biosynthesis of phenolic compounds, which ultimately affect wine quality . Howeverper se triggers the expression of key genes, such as phenylalanine ammonia lyase (PAL) and the enzyme chalcone synthase (CHS), which are essential for flavonoid biosynthesis [Although studies have remained inconclusive about the effects of light manipulation on the accumulation of seed flavan-3-ols, it is well known that sunlight ynthesis . A recenynthesis .Conceptually, changes to light will affect plant surfaces first, but it is unclear how this transmits to the internal organs, such as the seeds in which procyanidins accumulate. A possible hypothesis is that solar radiation works indirectly, by stimulating the biosynthesis of flavan-3-ol precursors such as narigenin or dihydroquercetin . Due to In a review of the impact of seasonal temperatures on flavan-3-ol biosynthesis, Gouot et al. , concludOther studies observed that very hot summers were associated with high quantities of procyanidin B1 in seeds . ConversIrrigation of vineyards during the critical developmental stages before ripening is a widespread practice, which has a positive effect on the phenolic content of seeds ,119. ConDue to the complexity of processes leading to the biosynthesis of grape seed procyanidins, the ideal conditions for optimisation of chemical profiles have not yet been realised. It is not a single factor, but many, that control the expression of procyanidins. Unfortunately, experiments fail to control for all possible variables, i.e., shading treatments not only reduce light, but also affect temperature and humidity, causing changes in transpiration and other factors . TemperaGrape seeds develop over three phases. In Phase I, (histodifferentiation) the seed is fertilised, and cells divide rapidly, forming all seed structures paralleling the creation of the immature fruit. In Phase II (expansion/reserve disposition), reserve materials are accumulated, and no further cell division takes place. This phase is associated with veraison, and towards the end of this phase the vascular connections between the seed and the plant close. Finally, in Phase III (maturation/drying), the seeds mature by desiccation ,125.Over the course of the seed and berry’s ripening, the profiles of flavan-3-ols similarly go through phases. In Phases I and II, the concentration of seed flavan-3-ols increase but diminish as the fruit ripens (Phase III) ,126,127.Procyanidins accumulate at a stage different to flavan-3-ol monomers. Procyanidins accumulate very early during seed development ,127, whiProcyanidin biosynthesis, peaking during rapid growth of juvenile berry (Phase I);Flavan-3-ol monomer biosynthesis: as the biosynthesis of procyanidins slows down, monomers increase (Phase II). Kennedy et al. ,127 explProgrammed oxidation: At veraison and after monomer biosynthesis declines. Monomers and procyanidins are oxidised (visible as a browning and hardening of the seed coat);Non-programmed oxidation: the seed is fully desiccated. Little change in extracted polyphenols occurs during this phase.Based on their work, the dynamics of flavan-3ol and procyanidin accumulation in grape seeds were classified into four distinct stages:Interestingly, glycosylated forms of flavan-3-ol monomers do not correlate to concentration changes of free flavan-3-ols and procyanidins. Even after Phase II, their relative concentration continues to increase and peak at Phase III . There aAs previously mentioned, it is problematic to discuss a single abiotic factor because such factors are usually linked to other effects. For example, linking an aesthetic improvement of wines to the physical location and elevation of the vineyard is not reliable because there may also be rainfall differences, among others. Even local microclimates can create chemical variation, i.e., a study exploring the chemical composition of the ‘albariño’ variety of white grape, cultivated in different vineyards restricted to the region of Galicia (Spain), found that flavan-3-ol profiles varied widely. Seeds from grapes grown in the Ribeira Sacra region contained higher concentrations (up to double) of flavan-3-ols than those grown in other regions, such as O Rosal .While difficult to validate findings related to elevation, patterns related to altitude have been observed. A Turkish cultivar (Ekşikara), grown at two different altitudes of 1000 m and 1500 m , demonst50) is higher than 4 g/kg in rats [Catechins and procyanidin-rich grape seed extracts started appearing in the market as nutraceuticals in the early 1990s . Over th in rats . In huma in rats .The quality and efficacy of commercial grape seed products depends upon the chemical composition of monomeric catechins and oligomeric procyanidins. While grape seed procyanidins are non-specifically listed as the active ingredient in dietary supplements, the most important are the oligomeric procyanidins  because The degree of polymerisation is not the only factor influencing absorption or bioactivity. As previously mentioned, the degree of gallate esterifications on procyanidin subunits, their conformation, and hydrophobicity are also important ,72,101. O→C7 or C2→O→C5 bond  were adulterated with peanut skin extracts and six samples (28%) were determined to be devoid of grape seed extract . Furtherm/z 577.1352, its A-type analogue (procyanidin A2) gives a deprotonated molecule at m/z 575.1195. The fragmentation pattern of these molecules also shows significant differences that can be used to discriminate between both isomers (m/z 423.072 (negative ion mode) on the MS2 spectrum, resulting from a Retro-Diels-Alder fragmentation, indicates the presence of A-type procyanidins, while B-type procyanidins show a fragment ion at m/z 425.088 via the same mechanism  are the main bioactive constituents. Ideally, these metabolites are quantified to give an actual oral dose, particularly of the ingredients that are absorbed . AccordiThe problems of adulteration in grape seed extracts are recognised as a serious concern by the American Botanical Council in their Botanical Adulteration Program . The mosIn addition to the issues of adulteration and safety, the sale of poor quality extracts for use in nutraceutical supplements or in cosmetic products undermines the potential benefits of quality plant-derived products .Despite the popularity of grape seed-based nutraceuticals, there is more to learn on the chemistry. Since the first reports in the late 1960s to the current date, 45 flavan-3-ols have been unambiguously characterised in the seeds of different grape species and varieties. However, the real number of grape seed flavan-3-ols is likely to be double or triple of this figure. There is a high diversity of glycosylated catechins and polymeric procyanidins that has not been elucidated. Furthermore, many of the chemical studies in the published literature do not comprehensively characterise components, with assignments stopping at the level of class or subclass. Unfortunately, greater specificity is required to make predictions of quality, absorption, and health benefits. Furthermore, when metabolites are identified in the seed extract that originate from other fruit parts, such as the skin , this is reflective of the quality of processing before extraction. Unfortunately, these observations require proof in the form of biosynthetic knowledge, but almost nothing is known about the biosynthesis of oligomeric and polymeric procyanidins in grape seed. Hence, it is necessary to direct studies toward these end goals to take extract optimisation a step forward.The recognition of the intra- and inter-specific variability of bioactive metabolites has generally been overlooked by both researchers and industry. While the genotypic factors influencing chemical variability are accepted, studies that seek to identify abiotic factors are not conclusive, due to limited control over overlapping variables. Researchers should not be discouraged by this, but greater monitoring of the effects of changing variables should be included in studies, i.e., higher altitude environments may also have different rain regimes. Lastly, given their widespread popularity and high monetary value, the quality, safety, and efficacy of commercial grape seed products represent a fundamental concern. The adulteration of grapeseed dietary supplements is a common problem, particularly with other procyanidin-rich extracts from peanut skin and pine bark. The methods for detecting adulteration prescribed by pharmacopoeias are inadequate to combat this problem. Hence, additional measures are necessary to confirm authenticity and to standardise the chemical profile, particularly in clinical trials when it is necessary to link bioactive ingredients to specific health outcomes.


The longitudinal associations between hearing impairment and higher-level functional measures and the potential confounding role of vestibular function have not been assessed. We investigated these associations in 831 participants of the Baltimore Longitudinal Study of Aging (2012–2019). Hearing was measured using pure-tone audiometry and categorized using WHO standards. Physical function was assessed with the Health Aging and Body Composition Physical Performance Battery  and walking endurance with time to walk 400 meters. Multivariable regression models tested the hypotheses that participants with hearing impairment have poorer physical outomes. In a subset, we further adjusted for vestibular function. Hearing impairment was associated with decrements in higher-level physical performance and walking endurance, and faster decline over time, regardless of vestibular function. Among participants with any hearing impairment, hearing aid users were faster in the 400-m walk. Early screening for higher-level functional loss among older adults with hearing loss is warranted.


PJVK gene cause the DFNB59 type of autosomal recessive non-syndromic hearing impairment (AR-NSHI). Phenotypes are not homogeneous, as a few subjects show auditory neuropathy spectrum disorder (ANSD), while others show cochlear hearing loss. The numbers of reported cases and pathogenic variants are still small to establish accurate genotype-phenotype correlations. We investigated a cohort of 77 Spanish familial cases of AR-NSHI, in whom DFNB1 had been excluded, and a cohort of 84 simplex cases with isolated ANSD in whom OTOF variants had been excluded. All seven exons and exon-intron boundaries of the PJVK gene were sequenced. We report three novel DFNB59 cases, one from the AR-NSHI cohort and two from the ANSD cohort, with stable, severe to profound NSHI. Two of the subjects received unilateral cochlear implantation, with apparent good outcomes. Our study expands the spectrum of PJVK mutations, as we report four novel pathogenic variants: p.Leu224Arg, p.His294Ilefs*43, p.His294Asp and p.Phe317Serfs*20. We review the reported cases of DFNB59, summarize the clinical features of this rare subtype of AR-NSHI and discuss the involvement of PJVK in ANSD.Pathogenic variants in the  Inherited hearing impairment is clinically and genetically very heterogeneous. Hearing loss can be an isolated condition  or it can be part of the clinical signs that are characteristic of specific genetic syndromes . Over 12PJVK gene (MIM #610219) [The DFNB59 type of autosomal recessive (AR) NSHI MIM #610220) is caused by pathogenic variants in the #610219) , which i10220 is #610219) . In othe#610219) ,9.PJVK have been reported as causative of AR-NSHI in families from diverse geographic origins [sirtaki mouse, which was obtained by ENU mutagenesis and carries a nonsense variant in Pjvk [PJVK variant spectrum and the resulting phenotypes, through the investigation of large cohorts of hearing-impaired subjects, with or without ANSD.Up to 19 different variants in  origins ,24,25,26 in Pjvk . ClarifiPJVK pathogenic variants, four of them novel, were found in three unrelated cases whose clinical characterization further illustrates the phenotypic variability of the PJVK type of hearing impairment.In this study, we have screened a cohort of 77 familial cases of non-DFNB1 AR-NSHI, and a cohort of 84 subjects with isolated ANSD. We report the first European cases of DFNB59 NSHI. Five GJB2 gene and by testing for the common del(GJB6-D13S1830) and del(GJB6-D13S1854) deletions, which revealed causative variants in 63 families. The remaining 77 families were investigated for variants in the PJVK gene. The second cohort consisted of 84 simplex cases  with isolated AN in whom pathogenic variants in the OTOF gene, encoding otoferlin, had been excluded previously. After approval by the Ethical Committee of Hospital Universitario Ramón y Cajal (in accordance with the 1964 Declaration of Helsinki), written informed consent was obtained from all participating subjects.Two cohorts of subjects were enrolled in this study. The first cohort consisted of 140 Spanish familial cases of autosomal recessive NSHI (AR-NSHI), with at least two affected siblings and unaffected parents. Prior to this work, they were investigated by Sanger sequencing of the coding region and splice sites of the Hearing was evaluated by pure-tone audiometry, testing for air conduction (frequencies 250–8000 Hz) and bone conduction (frequencies 250–4000 Hz). The degree of hearing impairment was defined by the pure tone average (PTA) threshold levels at 0.5, 1, 2 and 4 kHz, and was classified as mild (21–40 dB HL), moderate (41–70 dB HL), severe (71–95 dB HL) and profound (>95 dB HL). ANSD was diagnosed on the basis of absent or grossly abnormal auditory-evoked brainstem responses (ABR) and preserved otoacoustic emissions (OAE) . Speech PJVK gene are shown in DNA was extracted from peripheral blood samples by using the Chemagic MSM I automated system . Microsatellite markers D2S148, D2S2173, D2S324 and D2S2310 were amplified using fluorescently-labeled primers and PCR conditions as previously reported . AmplifiPathogenicity of DNA variants was assessed according to the guidelines from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) , as implPJVK. In 23 families in which haplotype analysis could not exclude genetic linkage, we sequenced all exons and exon/intron boundaries of PJVK from one affected sibling. We found likely causative variants in Spanish family S269. The two affected brothers were compound heterozygous for the novel variants c.671T>G (p.Leu224Arg) and c.880del (p.His294Ilefs*43), whereas the father carried c.671T>G, and the mother carried c.880del  and c.950del (p.Phe317Serfs*20). His father carried c.950del, and his mother c.880C>G. Subject DAN7-1, from the cohort recruited in Denmark but of Tamil ethnic origin, was homozygous for the previously reported c.1028G>C (p.Cys343Ser) variant. His parents were heterozygous carriers for this variant. Cys-343 has been shown to play a crucial role in the interaction between pejvakin and LC3B, an autophagosomal marker in the pexophagy pathway [We also screened a cohort of 84 simplex cases  with isolated ANSD in whom pathogenic variants in the >G p.His24Asp and Two of the novel variants are single-base deletions that result in frame shifts, leading to truncated polypeptides or mRNA degradation by nonsense-mediated decay. The two other novel variants are missense, which affect evolutionarily conserved residues in the pejvakin polypeptide C. They wIn Spanish family S269, the two affected brothers had not been subjected to newborn hearing screening. Subject II:1 was diagnosed with non-syndromic hearing impairment by age four years. Because of this familial history, his brother (II:2) received a similar diagnosis earlier, at age two years. Both presented with a severe hearing loss, which seems to be stable, as shown by serial pure-tone audiograms along 10 years of evolution a–d. TheiIn Italian case E1471, subject II:1 had normal growth and motor development, and he showed no risk factors for hearing loss. He had not been subjected to newborn hearing screening and was referred for assessment because of parental concern regarding his hearing at the age of two years. ABR recordings showed no response at the maximum stimulation intensity (90 dB nHL) while OAE were detected in both ears and disappeared thereafter. Behavioral reinforced audiometry, performed at the age of four years, indicated profound hearing loss. Pure tone average (PTA) threshold at 0.5, 1, 2 and 4 kHz measured in the free field was higher than 110 dB HL. The child was first fitted with power hearing aids, which resulted in a considerable improvement in pure tone sensitivity e; howeveAt age five years, the child received a cochlear implant  in the right ear. Electrically-evoked auditory nerve responses  were recorded through the cochlear implant. The aided thresholds, measured in the free field with the child wearing the cochlear implant f, fell wAt age nine years, the child was using the cochlear implant in the right ear and a hearing aid in the other ear. Scores of the speech perception tests were as follows: recognition of disyllabic words, in bimodal configuration and quiet environment: 85%; recognition of sentences, in bimodal configuration and quiet environment: 90%; recognition of disyllabic words, with cochlear implant only, in quiet environment: 70%. Overall, speech perception is satisfactory. In contrast, language is poorly developed for the chronological age. Indeed, scores on lexical comprehension and production would be adequate only for the age of five years .Case DAN07 was recruited in Denmark, but the family is of Tamil origin. Subject II:1 was diagnosed with hearing impairment at age six months. Both CT and MRI scans were normal. ABR recordings and pure-tone audiometry revealed a profound hearing loss g. ElectrPJVK. Both the Spanish and Italian cases had only Spanish and Italian ancestors, respectively, beyond at least three generations, which supports their European origins. Taking into account the four novel variants, 23 pathogenic variants have hitherto been reported in this gene  and in previous works, which did not find any DFNB59 case in a series of cohorts from different European countries .Although clinical data in the literature are far from being complete , it is pPJVK variants as a cause of AR-NSHI [ANSD was postulated to be a clinical feature of DFNB59 on the basis of the study of four Iranian families, in the first report of  AR-NSHI . In thre AR-NSHI , this woPJVK shows ANSD [sirtaki mouse, which carries a nonsense variant in Pjvk [Pjvk-null mice carrying a deletion of whole exon 2 [Pjvk in spiral ganglion neurons did not result in hearing impairment [Murine models do not shed light on this issue, as they reproduce the situation that has been observed in humans. A knock-in mouse model for the p.Arg183Trp variant in ows ANSD . However in Pjvk , and in e exon 2 ,13. Howepairment . As regapairment . Pejvakipairment . Accordipairment . ConsequOn the basis of the expression of pejvakin in the auditory pathway, it was hypothesized that the outcomes of cochlear implants in DFNB59 patients may not be good. Here we report case E1471, with an early diagnosis of ANSD, who received a cochlear implant in the right ear at the age of five years. Four years later, speech perception tests show good results, but language development is delayed. Although this delay could be related to the relatively late age of implantation, careful follow-up is needed for a correct evaluation of this case. Data on the outcome of cochlear implants in many other DFNB59 patients should be collected before any recommendation may be issued to orientate the choice of therapy in subjects with this subtype of AR-NSHI.


The coronavirus disease 2019 (COVID-19) outbreak has caused significant destruction, claiming over three million lives worldwide. Post SARS-COV-2 invasion, immunosuppression with hyperglycemia and elevated ferritin levels along with steroidal treatment creates a perfect storm for opportunistic infections. There is increasing evidence of mucormycosis co-infection in COVID-19 patients, during or post-treatment. A worse prognosis, a late diagnosis, and limited guidelines of screening and management of COVID-19 associated mucormycosis have made healthcare professionals fear an epidemic alongside a pandemic. This review geographically reports cases of COVID-19 associated mucormycosis (CAM), evaluates characteristics, clinical manifestations, and outcomes of mucormycosis in COVID-19 active or recovered patients. It further describes preventive strategies and recommendations for optimal management therapy that can be adopted worldwide to curtail an impending threat to the healthcare system. The novel severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) first reported in Wuhan, China on 21 December 2019 . The mosAspergillus flavus, one case of Candida glabrata, and three cases of C. albicans [A. fumigatus [According to a study conducted in China, Chen et al. found 5% cases of fungal co-infections out of 99 cultures isolated from COVID-19 positive cases, including one case of albicans . A Germaalbicans . In Nethumigatus . Amongstumigatus .Rhizopus spp., Mucor spp., Rhizomucor spp., Syncephalastrum spp., Cunninghamella bertholletia, Apophysomyces spp., and Lichtheimia (formerly Absidia) spp. [Rhizopus oryzae is the most common type and responsible for nearly 60% of mucormycosis cases in humans and accounts for 90% of the Rhino-orbital-cerebral (ROCM) form [Mucormycosis  is a serious fungal infection caused by a group of molds called mucoromycetes . The typia) spp. . The RhiCM) form .These fungi live particularly in soil and in decaying organic matter, such as leaves, compost piles, or rotten wood. It is transmitted by coming in contact with the fungal spores in the environment. Mucormycosis may be associated to different clinical manifestations depending on the organs affected. There has been a rise in the number of cases of mucormycosis in patients with COVID-19 worldwide, particularly in India. The reason behind this is the favorable environment in the affected patient that allows the spores to grow. These include hypoxia, high glucose levels due to diabetes or steroid-induced hyperglycemia, acidic medium created by diabetic ketoacidosis or metabolic acidosis, high ferritin levels due to inflammation, and a decreased activity and count of white blood cells along with several underlying conditions that promote the germination of spores and lead to the catastrophic picture of rhino cerebral mucormycosis co-infection with COVID-19 . In thisThis systematic review was carried out along the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines statement . An exteInclusion criteria of the study includes published cases of COVID-19 and mucormycosis co-infection. All articles in a language other than the English, studies that did not confirm diagnosis of mucormycosis, websites which reported unpublished cases were excluded from our review.Rhizopus, Mucor spp, followed by Lichtheimia spp., Rhizomucor spp., Cunninghamella spp., Apophysomyces spp., and Saksenaea spp. [Previously known as zygomycosis, mucormycosis belongs to the Mucorales group of fungi . The mosaea spp. . This opaea spp. . Immunocaea spp. .Rhizopus has shown an increased affinity for serum iron at pH < 7.3, allowing it to multiply rapidly in the body [R. oryzae to bind more frequently to endothelial cells in the lungs, brain, and sinuses, and thus, cause damage [The immune system has macrophages and neutrophils as the primary defense system against spores. An insufficient immune response allows the spores to germinate into hyphae, and establish infection ,23. In dthe body ,23,24. Te damage . Virulene damage . Usage oe damage ,28.Additionally, the incidence of mucormycosis has exasperated as the deadly SARS-CoV-2 alters the immune response. SARS-CoV-2 spreads mainly through droplets and is characterized by the invasion of angiotensin-converting enzyme 2 (ACE2) receptors in humans. ACE2 is present in the lungs, heart, liver, and kidneys. Downregulation of ACE2 by the coronavirus via its spike protein negatively influences the inflammatory-protective renin-angiotensin system ,30. The Classification of mucormycosis is based on the site of manifestation. It is enlisted as rhino-cerebral-orbital mucormycosis (RCOM), pulmonary, gastrointestinal, cutaneous, and disseminated mucormycosis, as illustrated in Rhyzomucor spp. together infect the lungs, the common site of invasion, and induce dyspnea, cough, and airway bleeding [Neutropenia, the use of corticosteroid, and induction chemotherapy increase the risk of pulmonary mucormycosis in individuals ,34. Neutbleeding ,37. Moldbleeding . Additiobleeding . Other tbleeding ,82.Cutaneous mucormycosis results from trauma or burns to the skin, and mostly occurs on the arms and the legs. Surrounding edema and black discoloration at the site of infection leads to gangrene in susceptible hosts. Progressing gradually, nodular lesions appear on the skin ,42. WhilSong et al. identified that critically ill patients in ICUs, and on mechanical ventilation were prone to various fungal infections, as in SARS. Other fungal infections were reported, however, until May 2020, there were no confirmed reports of mucormycosis .A post-mortem study conducted between March 2020 and April 2020 from the UK revealed pathological findings in a patient, which upon biopsy, PCR, and DNA extraction confirmed the presence of disseminated mucormycosis . Since tThis systematic review reveals a total of 201 published cases of CAM, and 70 deaths across 13 countries, as shown in Mucorales are saprophytes found in soil and decomposing organic waste. They thrive at temperatures ranging from 25 to 55 °C on most common bacterial , and fungal culture media . Mucorales in clinical specimens grow at 37 °C , formingA CT scan is considered better than MRI for fungal infections, especially for visualizing bony destruction. It is also an economically cheaper alternative. It helps visualize any opacification, periosteal thickening, or bony disruption of the sinuses. CT scan has made it possible to detect pulmonary or sinus abnormalities earlier than with traditional sinus and chest radiography. A characteristic reverse halo sign  is observed on CT scans in most patients with mucormycosis. Additional findings like vascular cut-off signs (abrupt termination of a pulmonary artery branch), cavitation, multifocal pneumonia pattern, and peripherally distributed lesions are also seen. The size and extent of lesions point towards the seeding of the infection, most likely to the pleura, and an impending complication like hemorrhage or infarct due to vascular invasion. The development of pulmonary infiltrates in neutropenic patients already indicates tissue damage, angioinvasion, thrombosis, necrosis, bleeding, and edema. Even before localizing symptoms, early CT findings in immunocompromised individuals at high risk for invasive pulmonary mucormycosis can identify pulmonary or Sino nasal lesions in the absence of radiological abnormalities on conventional radiographs. However, the cost and cumulative radiation exposure associated with serial or screening CT scans are some of the commonly disregarded limitations .MRI, on the other hand, is useful for soft tissue imaging and assessing the extent of the disease. A hypotense rim is observed near the lesion edges, which can be attributed to blood products or metals like iron and magnesium accumulated by the fungus although it is common to observe only subtle sinus mucosal thickening or thickening of extraocular muscles and detect no abnormalities in the bones of sinuses even in the presence of the disease clinically in some cases .Orbital invasion, in the case of rhino-cerebral mucormycosis, is followed by rapid involvement of the brain stem and hypothalamus, as seen on MRI. This predisposes the individual to a cavernous sinus infection or internal carotid thrombosis, thus, contributing to higher mortality. Although larger studies are required on this finding, a “black hole” sign has also been observed, which refers to the complete lack of contrast uptake by the lesions .The internal transcribed spacer (ITS) region of fungus DNA is the most extensively sequenced, and it is considered as a first-line technique for Mucorales species identification. When used on tissues, molecular-based techniques have acquired acceptability for confirming infection. Methods for detecting Mucorales DNA in blood have yielded encouraging results in terms of detecting the disease sooner and more quickly .Optimal management of mucormycosis can be achieved by coordinated and interdisciplinary efforts by various sectors. Owing to its high mortality, even the slightest clinical suspicion should warrant initiation of antifungal therapy. The management primarily involves a combination of surgical debridement and antifungal therapy. In a multivariate study of 929 reported instances of mucormycosis, Roden et al. found that antifungal medication plus surgery were strongly associated with better survival rates (69%), while death was practically certain (97%) for patients who got no treatment at all .Amphotericin B is the first-line management of the condition and can contribute significantly to patient outcomes. This was established in a retrospective analysis of 70 patients with mucormycosis who had delayed amphotericin B treatment (initiation of treatment 6 days after diagnosis), which culminated in a nearly twofold increase in death 12 weeks post-diagnosis (83% vs. 49%) .Treatment with amphotericin B is recommended till evident clinical improvement is observed, which usually takes a few weeks. A lipid formulation of IV amphotericin B is mostly used rather than amphotericin B deoxycholate, which is a cheaper and more toxic alternative . MetabolDue to its nephrotoxic nature, individuals with impaired renal function should be started on triazoles, namely posaconazole and isavuconazole, which act by inhibiting ergosterol synthesis in the fungal cell membrane . These aRhizopus spp. and combination therapy with caspofungin plus Amphotericin B Lipid Complex (ABLC) as compared to monotherapy or placebo. Liposomal Amphotericin B plus either micafungin or anidulafungin has resulted in better outcomes in disseminated mucormycosis as well. If LFAB-echinocandin treatment for mucormycosis is considered in the current scenario, it should be administered at doses approved by the US Food and Drug Administration (FDA) [Isavuconazole is a recently approved drug in the United States and Europe for mucormycosis and has the potential of becoming the mainstay treatment for invasive fungal infections . Like oton (FDA) .Angioinvasion and blood vessel thrombosis can result in poor drug bioavailability to the infection site. Patients should be prepared and prioritized for surgery, even with the slightest suspicion of mucormycosis. Surgically debridement of the affected site has been shown to contribute significantly to decreasing mortality . An MRI/In patients with hematologic abnormalities, any effort to reverse neutropenia should be made, whether using hematopoietic growth agents or, in certain circumstances, white cell infusions. If feasible, individuals with corticosteroid-induced immunosuppression, such as those with autoimmune disorders, should be weaned or switched to non-steroidal treatment. Antiretroviral medication should be initiated for HIV/AIDS patients to help with their immunity. For individuals with uncontrolled diabetes and/or ketoacidosis, rigorous glycemic management is critical. Although they warrant more studies, iron chelator therapies also remain to be a plausible therapy for patients with DKA. By chelating with unbound iron in patients with diabetes, especially ketoacidosis, they could potentially benefit in conditions with iron overload. Physicians must also prioritize the management of any other existing comorbidities .Hyperbaric oxygen (HBO) therapy has also been seen to be an adjunctive therapy along with the others. The higher oxygen pressure that is provided is known to improve neutrophil functioning and increase AMB activity by correcting acidosis. Finally, increased oxygen pressure reduces fungus development by inhibiting spore germination and speeds wound healing. As a result, HBO treatment for mucormycosis has been recommended as an addition to surgical and antifungal therapy . In suchTo ensure the best outcomes, healthcare providers must also keep an eye out for linked co-morbidities and create individual-specific treatment regimens and drug dosages. There needs to be more awareness among the public regarding mucormycosis and common individual precautions against it. This is a highly important issue with long-term consequences, and the government must prioritize and devise measures to tackle the endemic by providing enough resources before it becomes an epidemic.The current study had few limitations. Since it is a systematic review, there are no studies available that establish a causal effect relationship between COVID-19 and mucormycosis. It appears to be a number of factors that may contribute to superimposed mucormycosis in individuals co-infected with COVID-19. However, since majority of literature is based on findings of case reports/series which are subjected to publication bias and considerable heterogeneity. So, they may be an underrepresentation of the burden of disease since establishing a microbiological or histopathological diagnosis is difficult. While the lack of a reliable laboratory marker may also hinder the true estimation of mucormycosis co-infection incidence in COVID-19, it also compounded the effect of treatment in these patients.Mucormycosis is an emerging problem in individuals with COVID-19 as well as the healed cases and denotes a poor prognosis. The multisystem involvement and rapid progression associated with the disease warrants additional medical intervention and must be given priority.


Camponotus japonicus) by removing a pair of antennal cleaning apparatuses from the forelegs and investigated their behavioral change in response to exposure to the alarm pheromone or to encounters with nestmates or non-nestmates. Comparisons between self-grooming-nonlimited and self-grooming-limited ants showed that the self-grooming-limited ants gradually exhibited decreased locomotion activity in their fight-or-flight response to the alarm pheromone and experienced increased failure in nestmate and non-nestmate discrimination. Thus, the results of the present study suggest that antennal sensory system maintenance supports social communication, which is indispensable not only to the individual workers but also to the survival of their society.Grooming is a common behavior in animals. It serves the function of removing foreign materials and excessive amounts of self-secreted materials from the body’s surface. Social insects, such as honeybees or ants, use various types of pheromones, some of which propagate information about the environment to conspecific individuals, for chemical communication. The individuals that receive such information can respond with suitable behaviors to protect themselves and their society. Hence, grooming is important for the maintenance of the correct performance of their sensory organs on antennae for pheromone perception. Here, we experimentally limited self-grooming of the antennae in workers of the Japanese carpenter ant (C. japonicus) to limit the self-grooming of the antennae, the worker ants demonstrated the self-grooming gesture as usual, but the antennal surface could not be sufficiently cleaned. By using scanning electron microscopy with NanoSuit, we observed the ants’ antennae for up to 48 h and found that the antennal surfaces gradually became covered with self-secreted surface material. Concurrently, the self-grooming-limited workers gradually lost their behavioral responsiveness to undecane—the alarm pheromone. Indeed, their locomotive response to the alarm pheromone diminished for up to 24 h after the antenna cleaner removal operation. In addition, the self-grooming-limited workers exhibited less frequent aggressive behavior toward non-nestmate workers, and 36 h after the operation, approximately half of the encountered non-nestmate workers were accepted as nestmates. These results suggest that the antennal sensing system is affected by excess surface material; hence, their proper function is prevented until they are cleaned.Self-grooming of the antennae is frequently observed in ants. This antennal maintenance behavior is presumed to be essential for effective chemical communication but, to our knowledge, this has not yet been well studied. When we removed the antenna-cleaning apparatuses of the Japanese carpenter ant ( Grooming is a common behavior in many terrestrial animals. Its purpose is to remove foreign materials from the body surface ,3,4,5,6 Self-grooming of animal antennae is important for the olfactory function and olfactory sense-involved behaviors. When cockroaches are unable to groom their antennae, they become significantly less responsive to periplanone-B, a sex pheromone component ; thus, aC. japonicus, workers use undecane as an alarm pheromone to warn nestmates of an urgent situation. The nestmates that receive the undecane odor exhibit a fight-or-flight response, sometimes rushing to the odor source and at other times escaping the threat. Their locomotion velocity highly fluctuates. The central neural routes in response to alarming information have been investigated in Camponotus obscuripes stimulated with undecane [C. japonicus wears cuticular hydrocarbon (CHC) mixtures of 18 common components, and the mixing ratio is used as a colony-specific chemical sign. Hence, they can discriminate between nestmates and non-nestmates by detecting differences in these mixing ratios with a specific antennal olfactory organ called the basiconic sensillum [Camponotus species [C. japonicus in the present study and revealed the effects of self-grooming on the above two types of social chemical communications by comparing video recordings of self-grooming-limited and -nonlimited worker behavior.Ants are social insects that rely heavily on their olfactory sense; hence, they frequently exhibit self-grooming of the antennae with a pair of special cleaners on their front legs to keep their olfactory sensors functional ,29,30. Iundecane ,32. C. jensillum ,34. Cons species ,37,38,39Technically, we could easily limit self-grooming of antennae in worker ants by manually removing the antenna-cleaner apparatus pairs. As we adopted the NanoSuit method for antennal observation under a scanning electron microscope (SEM), we obtained clear antennal surfaces images together with surface materials without drift by charging.C. japonicus, were collected from two different colonies found on the campus of Kobe University, Japan, and were kept in separate plastic cases (40 × 32 × 12 cm) at 20–24 ℃ under a 12L12D cycle in the laboratory. The ants were provided with water and food  at an acceleration voltage of 1.0 or 5.0 kV. The observation chamber was maintained at a vacuum of 10.3–10.6 Pa. Secondary electrons were accumulated by a lower detector within the instrument. Other experimental parameters were as follows: working distance = 8 mm; aperture size = 100 µm; scan speed = 10–15 fps.The NanoSuit method  was usedThus, the antennae of different individual NanoSuit-treated ants were observed under the SEM before and 24 and 48 h after the operation. Using this method, we took pictures of the antennal surface with the secession material on it without any inconvenient pretreatment processes for sample preparation and drift by charging. During SEM observation, a specimen ant attempted to move antennae; hence, the fine SEM images could be successfully acquired only when the ants were motionless ,43. NeveC. japonicus, undecane in n-hexane, or plain n-hexane as a control was gently added to the glass wool using a micro-syringe. For recordings, a CCD camera  with a micro lens  was used. Using the “Windows Live Movie Maker” software , the ants’ images at 720 × 480 pixels were recorded for 3 min from the start of undecane or n-hexane exposure at 29.97 frames per second, and the video data for the first 10 s were used for locomotion analyses. The recording was repeated 10 times at 2, 12, 24, 36, and 48 h after the antenna cleaner removal operation, and at 2, 12, 36, and 48 h after the sham operation.We randomly selected eight workers obtained from the same colony and introduced them to a round arena of a glass dish (12 cm diameter). At the center of the arena, a small piece of glass wool was set 2.5 cm from the base of the dish by hanging it from a plastic strip bridge, which was used as a releaser. Five min after the ants were introduced, we commenced recording their behavior when 10 μL of 1:1000 diluted alarm pheromone of For the locomotion analyses, the two-dimensional locational data of each of the eight individuals were obtained frame-by-frame from the recordings using “K-Track” which was originally developed as the multiple bees’ tracking program by Kimura et al. (2014)  and the 2-anesthetized ant was placed into a round plastic arena . After the test ant awoke, a conspecific encounter ant of the same or a different colony from the test ant or a heterospecific encounter ant was gently introduced using a toothpick. Their behaviors were observed, and the test ant’s aggressive or nonaggressive behaviors were recorded until five-round interactions had occurred with the encounter ant. This behavior test with different pairs of the test and encounter ants was repeated six times immediately and every 4 h until 48 h after operation. The number of rounds in which the test ants exhibited aggressive  or nonaggressive (light antennation or ignored the presence of the encounter ant) behavior was counted.The sham-operated and antenna cleaner removal-operated ants were individually placed into a round glass container  for up to 48 h and were provided with water. Then, a five-round aggression behavior assay was conducted as follows: a single COThe antennal surfaces observed before the antenna cleaner removal operation or at different times after the operation are shown in n-hexane. The velocity changed over time after the antenna cleaner operation. As shown in p = 0.039, n = 8) or 12 h  rather than at 24, 36, or 48 h after the operation . This was not the case in the presence of n-hexane .Based on the ant location images in the recordings, we calculated their locomotion velocities in the presence of undecane or n-hexane a, right.n-hexane c. Those n-hexane at 2  and 12 h , but not at 24, 36, or 48 h after the operation    see a arrow, , n = 8) b. In mos, n = 8) c.We conducted another behavioral assay at various times after the sham or antenna cleaner removal operation. Each test ant encountered six conspecific nestmates, non-nestmates, or heterospecific workers, respectively, with 5 min intervals, and we counted how many times during the five trials the test ant exhibited aggressive or nonaggressive behavior. The results of the sham-operated, i.e., grooming-nonlimited ants, are shown in n-hexane 2 and 12 h after the antenna cleaner removal operation. In fact, the sham-operated ants put in an arena mainly exhibited smooth locomotion along the edge, while they increased irregular locomotion with rushing, turning, or stopping in the presence of certain odor stimulus. However, in our experiments of n-hexane without undecane  and toward heterospecific workers only at 40 and 48 h after the antenna cleaner removal operation . An appropriate balance of secretion and sweeping or spreading of antennal surface material including self-CHCs is presumed to be essential for precise nestmate–non-nestmate discrimination.The effects of antennae self-grooming on aggressiveness are shown in In cockroaches, self-grooming physically removes excessive native cuticular lipids as well as extrinsic chemicals from olfactory sensilla, thus maintaining the insect’s olfactory acuity to all odorants . This maC. japonicus). Our results indicated the importance of antennal cleaning maintenance for the highest performance of the olfactory sensors. Indeed, when the self-grooming of the antennae was limited, the correct performance of two types of chemical communication-related social behaviors, i.e., velocity fluctuation as a fight-or-flight response to the alarm pheromone and nestmate and non-nestmate discrimination by colony-specific CHCs, gradually ceased. Although ants possess finely designed sensory machinery, this cannot be fully utilized without self-grooming of the antennae. Thus, antennal maintenance is indispensable for the survival of ants, not only as individuals but also as societies.Insects have evolved sophisticated systems to interact with the environment via multimodal information, developing various designs of sensors, processors, and actuators. Here, we focused on the ant olfactory sensory organ, the antennae, and the olfactory sensilla in the ant (


Background: The immunoglobulin G synthesis rate (IgG SR) and immunoglobulin G (IgG) index are indicators of abnormal intrathecal humoural immune responses, and the albumin quotient (QALB) is an indicator used to evaluate the completeness of the blood-cerebrospinal fluid barrier (BCB). No systematic reports regarding differences in Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) are available. We assessed differences in the IgG SR, IgG index and QALB between GBS and CIDP patients in a Chinese cohort.Methods: A total of 234 patients were retrospectively enrolled in this study, and 167 clinically confirmed GBS and CIDP patients were selected. Meanwhile, 67 non-GBS and non-CIDP patients requiring cerebrospinal fluid (CSF) examination were enrolled as the control group. The IgG SR, IgG index and QALB were calculated using formulas. The relevant clinical data were subjected to statistical analysis.Results: Among the GBS and CIDP study groups and the control group, the QALB had the highest positive rate (80.00%) in the CIDP group (P < 0.01). The QALB stratification analysis showed that the ranges of 10 < QALB ≤ 30 were dominant in the GBS and CIDP groups, and the positive rate of CIDP was higher than that of GBS. Furthermore, a QALB ≤ 7 was dominant in the control group, and a QALB > 30 was dominant in the CIDP group. In receiver operating characteristic (ROC) curve analysis with the CIDP group as the trial group and the GBS group as the control group, the differences in the QALB were statistically significant (P < 0.01). To achieve a high specificity of 98~99%, the diagnostic cut-off value for the QALB was above 57.37 (sensitivity: 9.33%) or below 0.60 (sensitivity: 4.35%). Multivariate logistic regression analysis showed that the CIDP patients had a QALB higher than 57.37, and compared with that in the GBS patients, the difference in the QALB was statistically significant (P < 0.01).Conclusion: QALB elevation was associated with CIDP, while QALB values above 57.37 or below 0.60 had high specificity in differentiating between GBS and CIDP. In CIDP, the BCB is generally moderately to severely damaged; in GBS, the BCB is generally moderately damaged. Guillain-Barré syndrome (GBS) is an immune-mediated demyelinating disease of the peripheral nervous system (PNS) and spinal root and is characterized by the rapid progression of symmetric weakness, disappearance of tendon reflexes, electrophysiological characteristics of demyelination, dysfunction of the blood-cerebrospinal fluid barrier (BCB) and normal cell counts. Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a chronic autoimmune disease of the PNS that shares many common immunopathological changes with GBS .Proteins produced only outside the central nervous system (CNS), such as albumin (ALB), cannot easily cross an intact BCB. However, when the BCB is damaged by CNS diseases, such proteins can cross the blood-brain barrier (BBB) and enter the cerebrospinal fluid (CSF). The CSF/serum albumin quotient (QALB) is currently the indicator most frequently used to evaluate BCB permeability .The sources of immunoglobulin (Ig) in CSF are as follows: (1) blood diffusion through the BCB and (2) local synthesis in the CNS. The determination of the amount of locally synthesized Ig in the CNS, i.e., Ig with components that are not from blood, is essential . IntrathThis study compared the differences in the IgG SR, IgG index and QALB between GBS and CIDP with the aim to determine whether the combined detection of IgG SR, IgG index and QALB could contribute to the diagnosis, treatment and prognosis of peripheral nerve demyelinating disease.Routine cerebrospinal fluid, cerebrospinal fluid biochemistry and IgG SR for GBS and CIDP patients in Beijing, China, from October 2014 to April 2020: A retrospective study. The inclusion criteria included (1) a diagnosis of GBS or CIDP at Beijing Tiantan Hospital, Capital Medical University according to the Guillain–Barré and Miller-Fisher syndromes—new diagnostic classification  and the The exclusion criteria included (1) transient ischaemic attack, stroke, subarachnoid hemorrhage, or encephalitis as the discharge diagnosis; (2) a history of malignant tumors, severe heart, liver and kidney diseases, blood system diseases, and serious infectious diseases; (3) the presence of disabilities and sequelae caused by the patient's neurological diseases; (4) the definite presence of rheumatic diseases; and (5) ventilator-assisted ventilation or death due to GBS or CIDP.Non-GBS and non-CIDP patients hospitalized between October 2014 and April 2020 who required CSF examination were enrolled as the control group. The patients suffered from various conditions, such as headaches, multiple sclerosis, leukodystrophy and minor strokes.In total, 234 patients were retrospectively enrolled. The GBS group (92 cases) included 59 cases of classical acute inflammatory demyelinating polyneuropathy, 16 cases of acute motor axonal neuropathy (AMAN), four cases of acute motor-sensory axonal neuropathy, 10 cases of Miller-Fisher syndrome and three cases of acute sensory neuropathy. The CIDP group (75 cases) included 63 cases of typical CIDP, one case of pure motor CIDP and 11 cases of multifocal acquired demyelinating sensory and motor function (MADSAM). Two acute-onset CIDP (A-CIDP) patients were identified through dynamic patient follow-up and review. The control group contained 67 patients, all of whom underwent cerebrospinal fluid examinations.A venous blood sample was drawn from the cubital vein in the morning within 48 h of admission. The blood samples were stored for 30 min at room temperature and then centrifuged at 3,000 rpm for 10 min in a Hettich centrifuge device. The CSF examination was performed within 2–5 days after admission, and the data from first examination were used if multiple CSF examinations were conducted. The IgG and albumin contents in the serum and CSF samples were determined by rate-scattering turbidimetry using a Siemens BNII automatic protein analyser.(1) QALB: We calculated QALB to determine BBB integrity as follows: [CSF albumin (mg/ml): serum albumin (mg/ml)] × 1000 . The calP < 0.05 was considered indicative of a statistically significant difference.SPSS statistical software 24.0 was used for the data analysis. The measurement data exhibiting a normal distribution are described as the mean ± standard deviation. Data that did not conform to a normal distribution were compared using the Kruskal-Wallis H test and are expressed as the median, 25th percentile and 75th percentile. The count data are expressed as a percentage (%). The differences between two groups were compared using a chi-squared test. A receiver operating characteristic (ROC) curve analysis was performed using MedCalc 19.1 to evaluate the potential use of the IgG SR, IgG index, QALB and QIgG to predict the GBS and CIDP groups. Indicators with predictive ability were extracted from ROC curve analysis. The cut-off point corresponding to the specificity of the predictive index (98–99%) was used as the standard. Patients with values beyond the cut-off value and those in the control group were analyzed by a binary logistic regression to determine the baseline features of the 1–2% of patients with levels above the cut-off who were considered to have the most severe disease presentation. A linear regression analysis was used to analyse the correlation between the IgG SR and QALB. P < 0.01), while age and the HFGS score did not significantly differ between the GBS and CIDP groups (P > 0.05). Regarding sex, significantly more females were included in the GBS and control groups than in the CIDP group (P < 0.05) . The CIDP group consisted of 75 patients . The control group consisted of 67 patients . The GBS and CIDP patients were older than the control patients ( < 0.05) .P < 0.05), S-ALB and the IgG index (P < 0.01) in the GBS and CIDP groups were lower than those in the control group. The S-IgG in the GBS group was higher than that in the control group (P < 0.01).Cerebrospinal fluid-Protein (C-Pro), Cerebrospinal fluid-Albumin (C-ALB), C-IgG, QALB and QIgG were higher in the GBS and CIDP groups than in the control group, while Cerebrospinal fluid-white blood cell (C-WBC) (P < 0.05), C-ALB, QALB and QIgG than the GBS group (P < 0.01). The Cerebrospinal fluid-total cell (C-TC) levels (P < 0.05), S-IgG (P < 0.01) in the CIDP group were lower than those in the GBS group. The differences in the C-WBC, IgG SR, S-ALB, C-IgG and IgG index between the two groups did not reach statistical significance (P > 0.05) (The CIDP group had higher levels of C-Pro ( > 0.05) .P > 0.05). QALB: The positive case numbers and positivity rates were 58 (63.04%) in the GBS group, 60 (80.00%) in the CIDP group and 14 (20.90%) in the control group. A statistically significant difference was identified among the three groups (P < 0.001). According to the results, the positive QALB rates in the GBS and CIDP groups were significantly higher than that in the control group. QALB stratification analysis showed that the ranges of 10 < QALB ≤ 30 were dominant in the GBS and CIDP groups, and the positive rate of CIDP was higher than that of GBS. Furthermore, a QALB ≤ 7 and an IgG index > 0.7 were dominant in the control group, and a QALB > 30 was dominant in the CIDP group, which may indicate that in CIDP, damage to the BCB is mainly moderate to severe. The GBS group showed mainly moderate injuries to the BCB. IgG index: The positive case numbers and positivity rates were 19 (20.65%) in the GBS group, 14 (18.67%) in the CIDP group and 31 (46.27%) in the control group. A statistically significant difference was observed among the three groups (P < 0.001), and the positive IgG index rates in the GBS and CIDP groups were significantly lower than that in the control group  in the GBS group, 39 52.00%) in the CIDP group and 25 (37.31%) in the control group. No statistically significant difference was found among the three groups  < 0.05. Among the patients with moderate-to-severe increases in the QALB (QALB >10), the typical CIDP subtype was the most common (74.60%). No statistical differences in the IgG SR and IgG index were found between the GBS and CIDP subtypes (P > 0.05)  .P < 0.01). The IgG index and IgG SR did not have a statistically significant effect (P > 0.05). The cut-off value was determined by the maximum Youden index. The best cut-off value for QALB was 11.39, which achieved a specificity of 66.30% and a sensitivity of 61.33% : 0.568–0.718). When the cut-off was > 57.37, QALB had a sensitivity of 9.33% and a specificity of 98.91%; when the cut-off was lower than 0.60, QALB had a sensitivity of 4.35% and a specificity of 98.67% . When CIDP patients with QIgG > 36.74 were compared with GBS patients, the difference in QIgG was not statistically significant (P > 0.05)  .2 = 0.127, P < 0.01, A linear regression analysis was used to analyse the correlation between the IgG SR and QALB in the GBS and CIDP groups. The results showed a linear correlation between the IgG SR and QALB . Relevant animal experiments have shown that in active demyelinating lesions, BCB permeability is significantly increased, while in inactive demyelinating lesions, there is very limited or almost no damage to the BCB . This stAnalysis of the GBS and CIDP subtypes found that the typical CIDP subtype was the most common among patients with moderate QALB levels and moderate-to-severe QALB levels. Shimizu et al. showed that compared with MADSAM or distal acquired demyelinating symmetric (DADS) patients, typical CIDP patients had a more severely damaged BCB. In serum obtained from typical CIDP patients, the levels of claudin-5 protein and transendothelial electrical resistance (TEER) in peripheral nerve microvascular endothelial cells (PnMECs) were significantly reduced , 21. ThiThe IgG SR and IgG index are more indicative than the absolute concentration of IgG in CSF and are important indicators for the determination of abnormal intrathecal humoural immune responses. The IgG index can exclude the effect of S-IgG on the C-IgG level and can determine whether C-IgG is the result of serum infiltration or intrathecal synthesis in the CNS. The IgG SR can exclude the impact of serum and BCB factors on the C-IgG level and can determine the level of endogenous IgG in CSF. However, the IgG SR and IgG index are used under restricted conditions. The calculation of the IgG SR assumes that the ability of ALB and IgG to breach the BCB is not related to damage to BCB, implying that the formulas used to calculate the IgG index and IgG SR are only applicable to patients with no or minor damage to the BCB . The speIntrathecal synthesis in GBS and CIDP remains controversial. One study found that the response of serum anti-ganglioside in GBS was associated with an increased intrathecal IgG SR, while no such finding was observed in CIDP . An intrFrom linear regression analysis, the IgG SR and QALB were linearly positive correlated in the GBS and CIDP groups. Studies have shown that MMP-9 is negatively correlated with the IgG SR in CNS diseases with evident intrathecal synthesis . CombineThis study was a retrospective cross-sectional study that did not collect or detect gangliosides in each patient and did not reveal the correlations between gangliosides and the IgG SR, IgG index and QALB in GBS and CIDP. In addition, since this was a cross-sectional study, patients could not be followed to dynamically observe changes in related indicators. Prospective case-control studies and cohort studies should be performed in the future to improve the examination and follow-up of patients and produce more detailed and accurate studies.In summary, our study indicated that an elevated QALB was associated with CIDP, while a QALB higher than 57.37 or lower than 0.60 had high specificity for differentiating between GBS and CIDP. In CIDP, the BCB was generally moderately to severely damaged; in GBS, the BCB was generally moderately damaged. Future work should attempt to repair damaged BCB early to prevent deterioration and progression in GBS and CIDP.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Beijing Tiantan Hospital Institutional Ethics Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.YT managed and collected data and wrote the manuscript. XG researched the data and wrote the manuscript. XY conceived and designed the study and contributed to the manuscript. YZ, JP, and WZ researched the data, contributed to the discussion, and reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.The present study was supported by the Zhuhai Science and Technology Plan Project (No. 20191208E030034).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.