Virus-like particles (VLPs) comprise one or many structural components of virions, except their genetic material. Thus, VLPs keep their structural properties of cellular recognition while being non-infectious. VLPs of Parvovirus B19 (B19V) can be produced by the heterologous expression of their structural proteins VP1 and VP2 in bacteria. These proteins are purified under denaturing conditions, refolded, and assembled into VLPs. Moreover, chimeric forms of VP2 have been constructed to harbor peptides or functional proteins on the surface of the particles without dropping their competence to form VLPs, serving as presenting nanoparticles. The in-vitro assembly approach offers exciting possibilities for the composition of VLPs, as more than one chimeric form of VP2 can be included in the assembly stage, producing multifunctional VLPs. Here, the heterologous expression and in-vitro assembly of B19V structural proteins and their chimeras are reviewed. Considerations for the engineering of the structural proteins of B19V are also discussed. Finally, the construction of multifunctional VLPs and their future potential as innovative medical tools are examined. Virus-like particles (VLPs) are composed of some or all of the structural components of viruses, excluding their genetic material. The lack of genetic material makes these particles non-infectious and safe for use. At the same time, they retain most or all of their structural properties, such as cell-tagging and antigenic properties . VLPs caMany studies have demonstrated that VLPs can be accumulated naturally in tumors because of their defective vascular structure ,10. MoreHere, the potential applications of parvovirus B19 (B19V) VLPs are discussed with an emphasis on protein engineering and the in vitro self-assembling advantages for developing potential therapeutic biocompatible materials. B19V is a human pathogen, and its potential application as an anti-B19V vaccine and for presenting heterologous epitopes is direct. In contrast, for applications outside the vaccine development field, being a human pathogen may be seen as a drawback. However, B19V offers a chance to gain access to its natural host cells and other non-permissive human cells. Moreover, any VLP can be hidden from the immune system by mutations in epitope regions or by covering it with stealth polymers ,14. ConsB19V is an icosahedral virus whose capsid comprises 60 subunits of the major protein, VP2 (~95%), and the minor protein, VP1 (~5%). These proteins are encoded in its ~5.6 kb ssDNA genome in a single ORF with two start sites ,18. For VLPs of B19V have been constructed through the heterologous expression of VP2, VP2 + VP1, and VP1 proteins in different expression systems . VP1 is The structure of the B19V-VP2 VLPs was elucidated at a resolution of 3.5 Å via X-ray crystallography A 26]. M. M26]. ME. coli have a C-terminal 6xHisTag for IMAC purification [The core of the B19V VP2 structure consists of an eight-stranded, antiparallel β-barrel consisting of two β-sheets, and the rest of the structure comprises insertions connecting the β-barrel strands. The antiparallel β-barrel is a common feature of mammalian parvoviruses and other icosahedral viruses, and the loops have different lengths and aminoacidic compositions but have homologous positions among parvoviruses . The capfication . In contB19V wildtype virions carry about 5% VP1, and CryoEM analyses of these capsids show that the N-termini of VP2 are projected to the outer side of the capsid through the 5-fold axis pore, allegedly because of the crowded interior of the capsid. VLPs, however, have no enclosed genomes; thus, the reduced steric restrictions at the internal core of the capsid may hold the VP2 protein N-termini inside the capsid . It has VP1u has been studied, and some of its roles in infection have been described, including the presence of a receptor-binding domain for cell internalization and a phospholipase-A2 (PLA2) domain for lysosomal escape ,36,37,38Several groups have taken advantage of the presence of the N-termini of VP2/VP1 on the external surface of the B19V capsid, adding heterologous peptides to the N-termini of VP2 to use the resulting VLPs as a scaffold for antigen presentation, including antigenic determinants of dengue 2 virus (DEN-2), Bacillus anthracis, herpes simplex virus 1 (HSV-1), mouse hepatitis virus (MHV), and the syncytial respiratory virus (SRV) ,41,42,43S. cerevisiae previously fused with the bio-orthogonal partner of the peptide SpyTag, the SpyCatcher domain [1/2) of 40 min for the unconjugated enzyme to 48 min for the VLP-bound α-glucosidase. Additionally, a three-fold increase in the kcat of the coupled enzyme was observed without changes in KM. The resulting particles, carrying the α-glucosidase activity, also showed activity on glycogen so that they could be used for the delivery of this activity into the lysosomes of people suffering from glycogen storage disease type II (Pompe disease) caused by a deficiency of α-1,4-glucosidase in lysosomes as a form of enzyme replacement therapy [Nevertheless, antigen presentation is not the only role these particles can play. Our group has included functional peptides and proteins fused or conjugated at the N-terminus of VP2. For example, the bioconjugating peptide SpyTag was genetically fused at the N-terminus of VP2, and the expressed protein was assembled into VLPs displaying this peptide on their external surface. Those SpyTag-VLPs were successfully decorated with an α-glucosidase from r domain . The Spyr domain . The kin therapy .21–47, derived from the hepatitis B virus (HBV) [21–47 [21–47 fused to VP2 efficiently targeted the chimeric particles to HepG2 cells. Remarkably, in this experiment, VLPs were constructed with a second chimeric form of VP2, the SpyTag-VP2 mentioned above. The hybrid particles, decorated with both peptides, PreS21–47 and SpyTag, were decorated with a GFP protein fused to the SpyCatcher domain. As a result, the VLPs harboring the PreS21–47 peptide were also fluorescent, and the efficient internalization of the particles into hepatocytes was quickly confirmed by confocal microscopy.A second example of functional peptide addition at the N-terminus of VP2 is the peptide PreSus (HBV) . The sod) [21–47 , and the) [21–47 . Therefo21–47-VP2 and SpyTag-VP2 particles. While the hybrid VP1/VP2 particles produced in eucaryotic cells were carried out by the coexpression of VP1 and VP2 genes in the same cell (E. coli in the form of inclusion bodies (IBs) that can be resuspended in guanidine hydrochloride (GuHCl) and then purified by IMAC, given that VP2 and its chimeras have a C-terminal 6x-HisTag that does not impair its competence for self-assembly [Another special feature was included in the construction of the hybrid PreSame cell , the hybassembly ,51. VP2 assembly . This stMicrococcus luteus cell wall, demonstrating the potential of B19V VLPs as a scaffold for protein presentation and as enzyme carriers. However, the authors found that the range of HEL domains is low, at between 0.5 and 5 per capsid, while the expected range was between 24 and 30 because of the transforming methods used on Sf cells. It was suggested by the authors that this is because most HEL domains remain inside the capsid.In addition to functional peptides, large domains could be added at the N-terminal of VP2. In a pioneering study by Miyamura and coworkers , it was S. cerevisiae (Ima1p), superfolder GFP (sfGFP), and Bacillus pumilus lipase A (BplA) were constructed and expressed (unpublished data). None of them could efficiently self-assemble into VLPs, suggesting that large heterologous N-terminal domains impose steric constraints on the association of proteins to form VLPs. Here, the coassembly of these particles with 75% of VP2 allowed the formation of VLPs for all N-terminal chimeras. Regrettably, only the hybrid VLPs with the heterologous domains of sfGFP and BplA showed fluorescence and lipase–esterase activity, respectively. On the other hand, the DHFR and Ima1p domains showed no significant activity. Interestingly, VP2 and the VP2 N-terminal chimeras of sfGFP and BplA were successfully coassembled to produce three-component hybrid VLPs, simultaneously displaying fluorescence and lipase–esterase activity.Remarkably, in the in vitro refolding–assembly method, not every single N-terminal chimera of VP2 can form VLPs. N-terminal chimeras of VP2 with the human dihydrofolate reductase (DHFR), α-glucosidase from E. coli as IBs. The recombinant chimera was purified under denaturing conditions and then refolded and self-assembled into VLPs. These particles were administered to mice in which 4T1 cells were previously implanted to establish tumors. The 4T1 cell line is a mammary carcinoma model, which is highly tumorigenic and invasive and spontaneously metastasizes from the mammary gland to other tissues, including the lungs and liver [Recently , multiepnd liver . As forend liver .In a different approach to fighting cancer, breast-cancer-related epitopes targeting the insulin-like growth factor receptor (IGF-1R) were used to construct chimeric VLPs . IGF-1R Hansenula polymorpha by using strong promoters and terminator sequences [Considering that the capsid of natural virions is also a hybrid particle composed of VP1 and VP2, our group applied the in vitro coassembly method to construct VP1/VP2 VLPs . Our resequences . Finallyequences .A second approach for VLP engineering was inserting heterologous sequences in the external surface loops. The pioneering work on loop modifications was carried out in 1994 , before α atoms of the structure gave insight into the loops with the largest mobilities that can be correlated with a less compromised structure. As observed in 2, which was only exceeded by three loops, 62–75, 395–399, and barely 525–533. In addition, loop 301–313, which showed no electron density in the crystallographic structure due to its high mobility, was also considered among the engineering candidates.With the elucidation of the structure of the capsid of B19V, a vast amount of information became available to resume the engineering of surface loops of B19V VLPs. First, it became possible to define the location and size of the loops, as depicted in Loop 62–75 was engineered to harbor a 64-residue peptide from the F protein of the RSV . The engE. coli as IBs, which were processed as the VP2 IBs and then refolded to produce VLPs. The resulting particles were in the diameter range of VLPs, and regrettably were not fluorescent. In contrast, the lipase–esterase BplA (181 aa), inserted in the same position and flanked again by single Gly residues, produced particles with diameters in the range of VLPs and had lipase–esterase activity. The lipase immobilized on the surface of the VLPs showed a 20-fold decrease in specific activity, which may be related to rigidity produced by its attachment to the surface of the particles, reducing the thermal fluctuations required by enzymes to work properly. On the other hand, the first-order thermal inactivation constant of the immobilized lipase was reduced 7-fold, producing a longer half-life to the immobilized enzyme at 40 °C, from 0.14 to 1.01 h. Taking into consideration the possible effects of the reduced freedom degrees imposed by the attaching of the N- and C-termini of the hosted protein to the surface of the VLP, we explored the effect of a longer linker and a fast-folding domain, the sfGFP (238 aa); this protein is a derivative of the EGFP with mutations that enhance its folding rate [While loop 301–313 was also successfully engineered to present the same 64-residue peptide of the F protein of the RSV (unpublished data), a more ambitious goal was set for engineering this loop—to display a complete and functional protein on the surface of the B19V VLPs . This pring rate . This prLoop 266–270 was also engineered to host the previously mentioned 64-residue peptide from the F protein of the RSV (unpublished data), confirming that this stem loop (262–275) can host heterologous peptides, as found by Brown and colleagues in 1994. Other loops have yet to be tested. However, the proven properties of loops 62–75, 301–313, and 266–270 are appealing enough to insert functional peptides or proteins to provide new functions to the VLPs.Spodoptera frugiperda Sf-9 insect cells to obtain different ratios of VP1 and VP2 in the produced VLPs, and it was found that the higher the VP1/VP2 ratio, the higher the titer of neutralizing antibodies produced. This approach was later scaled to a bioreactor by considering several process variables, such as the temperature, harvest time, lactate concentration of dissolved O2, cell density, and MOI [S. cerevisiae instead of insect cells, and VP1 was mutated to remove its PLA2 activity, addressing the two main possible causes for the reactogenicity found in the previous trial [S. cerevisiae were homogeneous in size and elicited a potent neutralizing response in mice. Similar VLPs were produced recently in H. polymorpha, lacking PLA2 activity and showing excellent preclinical properties [E. coli with high yields and are purified under denaturing conditions, removing most contaminants that may produce side effects in trials. To our knowledge, no currently active trials for B19V vaccines are in progress.B19V has been identified as the causal agent of several diseases in humans, including erythema infectiosum in children, aplastic crisis in sickle cell disease patients, persistent anemia in immunocompromised patients, polyarthralgias or polyarthritis, and hydrops fetalis in pregnant women ,67. Infe and MOI . This st and MOI . All vol and MOI . The autus trial . The paroperties . In-vitrB19V VLPs are an exciting platform for potential biomedical applications . As reviB19V structural proteins can be produced in different heterologous expression systems, from bacteria and yeasts to insect and mammal cells. This flexibility allows for producing VLPs to display different peptides or proteins on the external surfaces of the nanoparticles, including those that may require post-translational modifications. B19V VP2/VP1 VLPs can be modified, either in the N-terminus or in surface loops, and such modifications are not mutually exclusive, thereby allowing them to harbor more than one new property in each subunit. Moreover, the possibility to regulate the ratio of VP2 and its chimeras by mixing different amounts of each protein during the folding–assembly process exponentially permits the accumulation of new properties in the built VLPs. This feasibility to gather properties is rare in other VLPs and promises to be the basis for new tools in nanomedicine.


A relationship between p38 and autophagy remains debated. The aim of the current study is to investigate whether an inhibitor of p38 prevents axon loss induced by TNF and whether it affects autophagy.Rats were given intravitreal injection of TNF, TNF plus SB203580, a p38 inhibitor, or SB203580 alone. Immunoblot analysis was performed to examine p62 expression which is a marker of autophagic flux and LC3-II expression which is an autophagy marker in optic nerves 1 week after intravitreal injection. Morphometric analysis of axons was performed to evaluate the effects of SB203580 against TNF-induced optic nerve damage 2 weeks after intravitreal injection. Immunohistochemical analysis was performed to evaluate the expressions of LC3, neurofilament, phosphorylated p38 and p62 in the optic nerve.Quantification of axon number showed that TNF-induced axon loss was significantly protected by SB203580. Immunoblot analysis showed that the increase of p62 induced by TNF was totally eliminated by SB203580, and the SB203580 alone injection decreased the expression of p62. The level of LC3-II was significantly upregulated in the TNF plus SB203580 group compared with the TNF alone group, and the SB203580 alone injection increased the expression of LC3-II. Immunohistochemical analysis showed that LC3 immunoreactivity was found in the neurofilament positive fibers and that these immunoreactivities were enhanced by SB203580. Some colocalizations of p-p38 and p62 were observed in the TNF-treated optic nerve.These results suggest that inhibition of p38 exerts axonal protection with upregulated autophagy in TNF-induced optic nerve damage. Autophagy is a cellular process that eliminates unnecessary proteins and subcellular elements through lysosome-related degradation to maintain homeostasis and be associated with differentiation, development and survival . AutophaEight-week-old male Wistar rats were used in this experiment. This experiment was approved by the Ethics Committee of the Institute of Experimental Animals of St. Marianna University Graduate School of Medicine. This study was performed according to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. The rats were housed in controlled conditions .Intravitreal injection was performed as described previously . In anes2 each; total area of 29,250 µm2 per eye) were acquired and quantified using an image processing software (Aphelion).Axon number counting was performed in optic nerves from 16 rats as described previously , 17. Theg for 15 min at 4 °C as described previously [One week after intravitreal injection, optic nerves (4 mm in length) were collected, homogenized, and centrifuged at 15,000 × The eyes were enucleated 1 week after intravitreal administration, immersed in 4% paraformaldehyde, processed, and embedded in paraffin. Transverse sections were made through the optic disc and blocked with 1% bovine serum. The primary antibodies were anti-LC3 antibody , anti-neurofilament-L antibody , p62 antibody , and anti-p-p38 antibody . FITC-labeled and rhodamine-labeled antibodies were diluted 1:5000 and used as secondary antibodies. The images were captured with a confocal microscopy system .P < 0.0001, *** for P < 0.0005, ** for P < 0.01, * for P < 0.05).Differences among groups were analyzed using one-way ANOVA, with post-hoc Tukey’s HSD test. The results were considered statistically significant when probability value of less than 0.05. Statistical significance is reported as asterisks in graphs (**** for Consistent with our previous studies , 15 and To examine the effects of SB203580 on autophagic status, we evaluated the changes in protein levels of p62, a maker of autophagy flux. Consistent with our previous studies , 15, TNFWe evaluated the effect of SB203580 on the level of LC3-II, an autophagic marker. Consistent with our previous studies , 15, theSince we previously observed that LC3-immunopositive dots were located inside neurofilament-positive fibers , the curWe previously observed that p-p38 immunoreactivity was colocalized with neurofilament-positive fibers in the TNF model . The curIn addition to the previous study showing that SB203580 protected RGC death induced by two different models such as optic nerve injury and NMDA administration , 19, theAutophagy is a dynamic process and the assessments of LC3-II and p62 are informative. Inhibition of autophagy has been linked to the augmentation of p62 and its decline is associated with autophagy flux enhancement . Our rec


Hyponatremia  is a disorder of water homeostasis due to abnormal renal diluting capacity. The body limits the degree to which serum sodium concentration falls through a mechanism called “vasopressin escape”. Vasopressin escape is a process that prevents the continuous decrease in serum sodium concentration even under conditions of sustained high plasma vasopressin levels. Previous reports suggest that aldosterone may be involved in the vasopressin escape mechanism. The abilities of aldosterone synthase (Cyp11b2) knockout and wild-type mice to escape from vasopressin were compared. Wild-type mice escaped while the aldosterone synthase knockout mice did not. Both the water channel aquaporin 2 (AQP2) and the urea transporter UT-A1 protein abundances were higher in aldosterone synthase knockout than in wild-type mice at the end of the escape period. Vasopressin escape was also blunted in rats given spironolactone, a mineralocorticoid receptor blocker. Next, the role of the phosphatase, calcineurin , in vasopressin escape was studied since aldosterone activates calcineurin in rat cortical collecting ducts. Tacrolimus, a calcineurin inhibitor, blunted vasopressin escape in rats compared with the control rats, increased UT-A1, AQP2, and pS256-AQP2, and decreased pS261-AQP2 protein abundances. Our results indicate that aldosterone regulates vasopressin escape through calcineurin-mediated protein changes in UT-A1 and AQP2. Hyponatremia  is a disorder of water homeostasis due to abnormal renal diluting capacity. It is the most common clinical electrolyte disorder in hospitalized patients, with an incidence of 15% and a prevalence of 30% . PatientVasopressin, acting through adenylyl cyclase and protein kinase A, phosphorylates the urea transporter UT-A1 and the water channel aquaporin 2 (AQP2), thereby increasing urea and water reabsorption in the kidney ,5,6. UT-Previous studies indicate that in animal models of SIADH, the very low serum sodium concentration increases systemic aldosterone ,14, suggThis study provides direct evidence that aldosterone leads to vasopressin escape by regulating AQP2 and UT-A1 activities. We first compared the changes in the protein levels of UT-A1 and AQP2 in aldosterone synthase (Cyp11b2) knockout and wild-type mice during vasopressin escape. We then confirmed aldosterone-dependent vasopressin escape by inhibiting mineralocorticoid receptors (MRs) with spironolactone. We finally examined the effect of inhibition of calcineurin on vasopressin escape and the changes in the total protein and phosphorylation levels of UT-A1 and AQP2 proteins.All animal surgical procedures were designed to adhere to the NIH standards for animal use and were approved by the Emory Institutional Animal Care and Use Committee . Both male and female rats (100–140 g) for measuring vasopressin escape were purchased from Charles River Laboratories, Wilmington, MA, USA. The wild-type mice used for these studies were sourced from our colonies maintained at Emory University School of Medicine under the supervision of the Division of Animal Resources. Aldosterone synthase (Cyp11b2) knockout mice were obtained from Dr. James Luther, Vanderbilt University  and then maintained in a colony in the Emory animal facilities under supervision of the Emory Division of Animal Resources.We used a well-described rodent model of vasopressin escape ,14,22 toOsmolality was measured in daily 24 h urine collections throughout the experiment using a Wescor vapor pressure osmometer . In preliminary experiments, serum sodium concentration was measured using a Medica EasyLyte electrolyte analyzer  and blood glucose was measured using a LifeScan One Touch Ultra glucometer . Sodium concentration correlated with serum osmolality. Blood glucose was normal and did not make a significant contribution to serum osmolality. We did not measure blood urea nitrogen levels in these animals.Vasopressin escape is indicated by a reduction in urine osmolality despite the continual presence of vasopressin from the osmotic minipump. If urine osmolality remains high, that indicates a failure to escape from vasopressin. At the end of vasopressin escape, animals were sacrificed, and inner medullas (IMs) were dissected from the kidneys for further detection of phosphorylation of AQP2 and UT-A1 proteins.To test the role of aldosterone, aldosterone synthase (Cyp11b2) knockout and wild-type litter-mate mice were placed into the vasopressin escape protocol. Aldosterone synthase (Cyp11b2) knockout mice fail to produce aldosterone . Mice weTo test whether calcineurin regulates AQP2 and UT-A1 during vasopressin escape, rats were placed into the vasopressin escape protocol. After 4 days of increased osmolality, all rats were given a gel diet to force fluid consumption, and half received daily injections of 1 mg/kg/day tacrolimus  for a further 4 days. Rats were sacrificed and protein lysates were prepared for Western blot analysis of total and phosphorylated AQP2 protein and total and phosphorylated UT-A1 protein.Inner medullary protein lysate at a concentration of 1 mg/mL was loaded (20 µg/lane) into wells of polyacrylamide gels with each well containing a different animal’s lysate. Proteins were size separated on 12.5% gels for AQP2 or 10% gels for UT-A1, then electroblotted to polyvinylidene difluoride membranes . Membranes were bathed in a 5% solution of Carnation nonfat dry milk in TBS  to block open sites. Primary antibodies were prepared in the 5% milk/TBS buffer with 0.1% Tween 20 added. Blots were incubated with primary antibodies at 4 °C overnight. All primary antibodies were prepared in rabbit. Alexa Fluor 680-linked antirabbit IgG (1:4000 dilution)  was used as the secondary antibody to identify bound primary antibodies. The Alexa fluor was detected using the LICOR Odyssey protein analysis system . Antibodies to phosphorylated AQP2 p-serine 256 or p-serine 261 (1:2000 dilution) were purchased from Cell Signaling Technology, Danvers, MA, USA and used at a dilution of 1:2000. Antibodies to AQP2 (1:2000) or UT-A1 (1:1000) were made in our laboratory . Antibodt-test for two groups. The criterion for statistical significance is p < 0.05.All data are presented as mean ± s.e. Urine osmolalities from vasopressin escape studies were analyzed using a longitudinal analysis. The data from the protein analysis studies were analyzed using a Student’s 2O  knockout and wild-type litter-mate mice were implanted with 14-day osmotic minipumps delivering 5 ng/h ddAVP  agonist). After 2 days of increased urine osmolality, mice were given a gel diet to force additional fluid consumption. Urine osmolality of the wild-type mice was significantly decreased from 4181 ± 283 to 2499 ± 277 mosmol/kg H2O  vs. 76 ± 14 , n = 6, p < 0.05; In contrast, the urine osmolality of the aldosterone synthase knockout mice was only slightly reduced from 4479 ± 868 to 3435 ± 221 mosmol/kg HO n = 6; . The cha2O and from 2710 ± 93 to 2112 ± 188 mosmol/kg H2O, respectively .To confirm that aldosterone regulates vasopressin escape, spironolactone was used to block MRs in rats. After 6 days of increased urine osmolality, rats were given a gel diet to force additional fluid consumption. The urine osmolality of vehicle-treated rats and spironolactone-treated rats was decreased from 2938 ± 154 to 1846 ± 248 mosmol/kg H2O in the tacrolimus-treated rats while it dropped from 5675 ± 866 to 2638 ± 104 mosmol/kg H2O in the vehicle-treated rats  through a process that counters the water-retaining action of vasopressin . IncreasNielsen’s group showed that aldosterone reduces whole cell  AQP2 expression in the presence of desmopressin acetate (ddAVP) . When inAldosterone is a mineralocorticoid hormone that maintains water and electrolyte balance by modulating sodium channels and sodium transport. Aldosterone interacts with its intracellular MR, and the combination of aldosterone and MRs activates genomic pathways to control sodium channel and sodium transporter gene transcription ,16. The Tacrolimus is a widely used antirejection medication that inhibits calcineurin, which is also named phosphatase 2B (PP2B). The mechanism by which hyponatremia results from tacrolimus is unknown. Proposed mechanisms include SIADH and aldosterone resistance. The reduced vasopressin escape in tacrolimus-treated rats can be attributed to the increase in osmotic water permeability in the collecting duct resulting from the higher phosphorylation state of AQP2 when calcineurin is inhibited by tacrolimus . TacroliIt is worth mentioning that calcineurin inhibition with tacrolimus attenuated vasopressin escape in rats  but did In this study, we analyzed the changes in urine osmolality during vasopressin escape using a longitudinal analysis. A longitudinal analysis allows one to observe the changes in a variable over a period of time, during which repeated measurements for the variable on the same individuals are performed. We observed urine osmolalities for 14 days by measuring the urine osmolality of each animal on each experimental day. Therefore, our osmolality data were subject to a longitudinal analysis. A longitudinal analysis takes into account the values at all time points and provides an inference for the effect of treatment.In conclusion, the present study shows that aldosterone acts through calcineurin to mediate vasopressin escape. Vasopressin escape is prevented if aldosterone or its receptor is not available, or calcineurin is inhibited. Increased protein abundance of AQP2 or UT-A1, or pS256-AQP2, and decreased expression of pS261-AQP2 contribute to deficient vasopressin escape in the absence of aldosterone. This study provides a new understanding of vasopressin escape in terms of phosphatase-regulated water channel activities, suggesting a new direction for therapies for hyponatremia.


A subset of B-cells with tolerogenic functions, termed B-regulatory cells or Bregs, is characterized by the expression of anti-inflammatory/tolerogenic cytokines, namely IL-10, TGF-β, and IL-35, that contribute to their regulatory functions. Breg regulation favors graft acceptance within a tolerogenic milieu. As organ transplantation invariably triggers inflammation, new insights into the crosstalk between cytokines with dual properties and the inflamed milieu are needed to tailor their function toward tolerance. Using TNF-α as a proxy of dual-function cytokines involved in immune-related diseases and transplantation settings, the current review highlights the multifaceted role of TNF-α. It focuses on therapeutic approaches that have revealed the complexity of TNF-α properties tested in clinical settings where total TNF-α inhibition has proven ineffective and often detrimental to clinical outcomes. To improve the efficacy of current TNF-α inhibiting therapeutics, we propose a three-prong strategy to upregulate the tolerogenic pathway engaging the TNFR2 receptor while simultaneously inhibiting the inflammatory mechanisms associated with TNFR1 engagement. When combined with additional administrations of Bregs-TLR that activate Tregs, this approach may become a potential therapeutic in overcoming transplant rejection and promoting graft tolerance. Although B cells may be most well-known as antibody producers, they also display a gamut of other properties, including the presentation of antigens to T cells. This function is modulated by the differential expression of costimulatory molecules, leading to the regulation of T-cell activation. Furthermore, B cells tailor their cellular environment via the secretion of cytokines and chemokines . In tranBregs’ immunomodulatory functions are often associated with the production of anti-inflammatory cytokines, notably interleukin-10 (IL-10) –4. BregsThus, depending on the local pro-tolerogenic or -inflammatory environment, the TNF-α receptor binding duality can result in tolerogenic or inflammatory responses to transplants. The potential to manipulate receptor binding has not yet been closely examined and could improve graft acceptance. Given that TLRs are recruited during graft reperfusion and transplantation, it appears essential to understand the modalities of preserving a pro-tolerogenic graft milieu favorable to immune regulation via Bregs and other regulatory cells and cytokines. Furthermore, accounting for the pro-inflammatory properties of Bregs will allow for better optimization of transplant tolerance strategies through awareness of potential pro-inflammatory cytokine-induced complications post-transplant. This review examines the immune-suppressing and-activating functions of the Breg-produced cytokine TNF-α and its synergistic role with IL-10, ultimately leading to transplantation tolerance. Using examples from outside the transplantation field, we also evaluate the translational potential of TNF-α therapies that had promising clinical outcomes in alleviating autoimmune-driven inflammation , 12. We The suppressive functions of B cells and postulation on the existence of a subpopulation of regulatory B cells were first explored in the 1970s following observation by Ruth and Salvin on the suppression of delayed hypersensitivity reactions in guinea pigs after lymphoid cell transfer into antigen-sensitized recipients . The effTransforming growth factor β (TGF-β) activates several pathways that regulate various cellular functions . Under sIL-35 is another immunosuppressive and anti-inflammatory cytokine mainly secreted by regulatory T cells but is also expressed by Bregs . Bregs’ + B cells co-express the pro-inflammatory cytokines IL-6 and TNF-α (Upregulation of the immunoregulatory Nucleoside triphosphate diphosphohydrolases (NTPDase), CD39, and the transferrin receptor CD71 expression was found to be strongly associated with IL-10 expression by B cells . Other rnd TNF-α , 29. A snd TNF-α .+CD39hi B cells, are enriched within IL-10-producing B cells and are abundant among cells secreting TNF-α   . As apprTumor necrosis factor alpha (TNF-α) is a member of the TNF superfamily, which is involved in the regulation of immune cell functions but also in inducing fever, apoptotic cell death, muscle loss, inflammation, inhibition of tumorigenesis, and viral replication . CytokinThis cytokine appears in two molecular forms that each engage a different cell surface receptor. The soluble TNF-α (sTNF-α) binds to the type 1 receptor (TNFR1), whereas the membrane-bound form of TNF-α (tmTNF-α) is recognized by the type 2 receptor (TNFR2) , 11, 28.+ B cells substantially decreased in number, while the IL-10+ B cell population remained significant , compared with control transplant recipients under maintenance immunosuppression (6% IL-10+ TNF-α+B cells) . Breg involvement in tolerance was suggested by the fact that operationally graft-tolerant liver transplant recipients and transplant control patients had a significantly higher proportion of IL-10B cells) . There wolerance .in vitro. The study also found a reduced TrB IL-10:TNF-α ratio predictive of poorer clinical outcomes in allogeneic renal transplants  determined that those who exhibited overexpression of both anti- and pro-inflammatory cytokines were still able to maintain a balanced inflammatory response to surgery . SurveysThe predictive value of the IL-10:TNF-α ratio has also been seen in fields other than that of transplantation. In a study of severe burn injury, IL-10:TNF-α plasma levels measured shortly after burn trauma were directly correlated with burn size, the severity of the injury, and the susceptibility to repeat infections (≥3) during recovery . In the Collectively, these studies document the value of the IL-10:TNF-α ratio as a relevant indicator of an anti- to pro-inflammation shift and a potential predictor for clinical treatment outcomes. The significance of this metric has contrasting implications for the role of each cytokine, as it appears that a balance pro-inflammatory and anti-inflammatory cytokines are required for positive clinical results , 31, 35.+ T cell cytotoxic response  –38. TNF-response , therebyin vitro and analyzed for TNF-α-producing CD14+CD16+ cell populations  performed syngeneic and allogeneic hepatocyte transplantation in dipeptidyl-peptidase-deficient (DPP) F344 rats, treated with mild lymphocyte immunosuppression (mofetil and tacrolimus) . As expeTNF-α has become increasingly recognized for its potent role in the pathogenesis of chronic inflammatory diseases leading to the emergence of various anti-TNF-α therapies that assert their effects through different pathways and mechanisms that should be understood as precautionary measures for potential complications when applied to other fields such as transplantation. When TNF-α is dysfunctional, diverse immunopathology can result, including rheumatoid arthritis, inflammatory bowel disease, Wegener granulomatosis, sarcoidosis, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile chronic arthritis, atherosclerosis, and sepsis . Of the The clinical application of cytokine inhibitors, including those that antagonize the effects of TNF-α or IL-6, is often done with caution due to the complex nature of their signaling pathways, where alterations to one aspect may result in a myriad of unforeseen downstream effects such as infection or developing cancer , 42. TheStudies on the application of anti-TNF-α treatment in transplantation demonstrate the risks of using cytokine inhibitors to eliminate the function of cytokines that seem solely inflammatory yet are intricately intertwined in the signaling and function of complementary anti-inflammatory cytokines. Therapies have recently focused on targeting TNFR1 with antagonists and TNFR2 with agonists, which may alleviate the unwanted, severe side effects associated with anti-TNF-α treatment without disturbing the IL-10/TNF-α co-expression balance that contributes to a tolerogenic milieu .+ Tregs, CD8+ Tregs, Myeloid-derived suppressor cells (MDSCs), and Bregs  originating from the thymus. The study found that TNF-α exposure to TNFR1-/- mice promoted naïve CD4+ T cells to differentiate into iTregs while TNFR2-/- mice iTreg induction was hampered indicating TNF-α’s ability to induce Tregs and the important role of TNFR2 in Treg induction.Although it is known that TNF-α disrupts both differentiation and function of TGF-β-induced Tregs in autoimmune diseases via the Akt and Smad3 signaling pathways, TNF-α has also been shown to expand murine natural Tregs both in vitro . Furtherresearch . A studyTo demonstrate the dichotomous relationship between TNFR1 and TNFR2 function, Richter et al. (2021) used an experimental autoimmune EAE animal model of multiple sclerosis to show that mice deficient in TNFR1 developed a much less severe form of the disease. In contrast, mice without TNFR2 had significantly more severe signs of EAE disease . Beyond Since TNFR1 is thought to be the primary promoter of inflammation and cell death in the TNF signaling pathway, it may be possible to use a therapeutic blockade of TNFR1 signaling while keeping homeostatic TNFR2 signaling intact as a treatment for TNF-mediated diseases , 49. ThiClinical research in TNF-mediated disease where complete TNF inhibition is contradictory and has shown to have severe unwanted side effects is required to fully confirm the therapeutic benefits of TNFR1 blockade fully. Preclinical studies using dominant-negative inhibitors of soluble TNF (DN-TNF) to block soluble TNF binding to TNFR1 while sparing the binding of transmembrane TNF to TNFR2 have shown promising results , 44.Patients receiving allogeneic hematopoietic cell transplantation (HCT) often have GvHD maintained largely by TNF-α release . Increasin vivo was also shown to inhibit TNF-induced release of inflammatory cytokine IL-6, as well as TNF-induced weight loss. In a murine model of rheumatoid arthritis, low-dose Atrosimab treatment slowed disease progression, and high-dose treatment improved therapeutic activity  as a modified anti-TNFR1 antibody, Atrosimab. This molecule can selectively block TNFR1 signaling while leaving the anti-inflammatory TNFR2 pathways unaffected. Its therapeutic efficacy was demonstrated in murine models of acute and chronic inflammation, including non-alcoholic steatohepatitis (NASH) and EAE . Use of activity . Though + and CD8+ T cells  expansion and sustain cell survival, which can be leveraged to promote tolerance of allogeneic transplants and protect against GvHD . As disc T cells . The in eatments , 54. Amoal rates , 54. Ano in vivo .Here we discuss the role TNF-α plays in the pathogenesis of many autoimmune diseases and their relation to Breg suppressive function with the ultimate goal of designing a combinatorial therapy involving blocking the TNFR1 receptor associated with inflammatory TNF-α signaling, activation of TNFR2 signaling involved with an anti-inflammatory response, and TLR-Bregs to suppress effector T cell proliferation and induce regulatory T cells. Regulatory B cells are primarily known for their ability to suppress inflammation, promote immunological tolerance, and downregulate pro-inflammatory cytokine production via the immune modulating cytokines IL-10, TGF-β, and IL-35. We highlight studies detailing the expression of TNF-α in IL-10 producing B cells that initially appears antithetical to their function, but after further investigation of TNF-α binding dynamics to its two receptors (TNFR1 and TNFR2) that allow for its pleiotropic, opposing signaling pathways, the potential inhibition of the inflammatory pathway while maintaining the homeostatic signaling of the other becomes an apparent solution to many of the autoimmune diseases mediated by TNF-α dysregulation. Furthermore, we discuss the clinical applications of the IL-10:TNF-α ratio, using it as an predictor of pro- and anti-inflammatory treatment outcomes in autoimmune diseases and transplantation.in vivo graft-specific T cell proliferation while inducing Tregs via the TGF-β pathway  with either TNFR1 antagonists (to block pro-inflammatory responses of TNF-α), TNFR2 agonists (to stimulate tolerogenic responses of TNF-α), or both Figure 2 pathway . A TNFR1SP and MY generated the figure, wrote, and edited manuscript. KD, and QF provided critical reading and edited of the manuscript CL and JM provided critical reading, edited, and wrote the manuscript. All authors contributed to the article and approved the submitted version.


TNF-α is of interest in oral squamous cell carcinoma (OSCC), with its demonstrated presence affecting both tumour and stromal inflammatory cells to enhance proliferation and facilitate invasion. TNF-α gene polymorphisms have also been associated with an increased risk for both oral pre-cancer and cancer development. Here we present a review of the current knowledge of the role of TNF-α in the aetiology, pathogenesis, and potential therapy of OSCC. Uncovering the inflammatory mechanisms underpinning initiation, progression, and promotion of oral squamous cell carcinoma (OSCC) development is fundamental to the rational pursuit of targeted therapeutics. Here we present a review of the current knowledge of the role of TNF-α in the aetiology, pathogenesis, and potential therapies with regards to OSCC. TNF-α is worthy of particular attention in OSCC, with its presence demonstrated to enhance cell proliferation and its downregulation demonstrated to inhibit proliferation and migration in other carcinomas in both in vitro and in vivo models and oral cancer patients. Increased TNF-α in the OSCC tumour microenvironment has been demonstrated to favour invasion through promotion of firstly the pro-inflammatory, pro-invasive phenotypes of OSCC cells and secondly its paracrine mechanism mediating recruitment and activation of inflammatory cells. Polymorphisms affecting the gene expression of TNF-α have been strongly associated with an increased risk for oral squamous cell carcinoma. A number of studies have considered TNF-α within biofluids, including saliva and serum, as a potential biomarker for the early detection of OSCC, as well as its staging, differentiation, and prognosis. The broad and multifaceted role that TNF-α plays in many inflammatory states presents an obvious confounder, particularly with demonstrated increased TNF-α levels in common oral disease states. Lastly, biologic agents targeting TNF-α are currently in clinical use for immune-mediated inflammatory rheumatological and gastrointestinal diseases. There is the potential that these biological agents might have an adjunctive role in OSCC prevention and treatment.  Oral squamous cell carcinomas (OSCC) are the most common mucosal cancers of the head and neck . OSCC caThere is increasing evidence of an association between OSCC and chronic inflammation. Particularly relevant is an observed progressive increase in inflammatory infiltrate alongside increasing grades of epithelial dysplasia to OSCC . The micTNF-α  has been implicated as a large player in several inflammatory, infectious, metabolic, and neoplastic diseases. TNF-α plays a central role in the onset of immune response. It is predominantly secreted by macrophages but is also produced by other immune and non-immune cells, including fibroblasts, muscle, and endothelial cells ,9. It afTNF-α is a trimeric cytokine and exists in two isoforms—a transmembrane form and a soluble form  ,12. The TNFα has been highlighted as a crucial mediator of cancer-related inflammation and thus its targeting may be a key to oncological therapy resistance. Expression and induction of TNFR1 and TNFR2 on immune stromal cells within the tumour microenvironment has been shown to promote tumour growth and progression. Further, their induction of immune suppressor cell differentiation also leads to evasion of tumour surveillance ,15.TNF-α has been linked to several human cancers, including breast , gastricThe discordant effects that TNFα can exert in cancer depend on a multitude of factors that include its concentration and the ratio of its two isoforms , among others. For example, one of the main characteristics of TNF superfamily members is their duality. Being able to act as both ligands and receptors creates a peculiar phenomenon known as “reverse signalling” . TmTNFα Other factors that can modify the role of TNFα in cancer include caspase activation, the variable expression of adaptor proteins, and proteins from the Bcl-2 family .TNF-α can therefore act either as a pro-tumoral cytokine, favouring cell proliferation, cell migration, angiogenesis, and diseases progression in a number of cancer types, or as an anti-tumorigenic biomolecule. A summary of TNFα effects in several cancer types can be found in the work by Mercogliano et al. .TNF-α plays a critical role in the pathobiology of cancer. It has also shown to be a key player in head and neck SCC (HNSCC), where it can stimulate the expression of programmed death-ligand 1 (PD-L1), a ligand for PD-1 that is primarily expressed on activated T/B cells, monocytes, and a small percentage of thymocytes . PD-L1 iIn contrast, a clear role in oesophageal SCC for tumour necrosis factor alpha-induced protein 8 (TNFAIP8) has been demonstrated. TNFAIP8 is a suppressor of TNF-α-mediated apoptosis, and its expression is induced by NF-κB activation. A retrospective study by Sun et al. showed that TNFAIP8 overexpression correlated with lymphatic recurrence in ESCC patients . TNF-α hPolymorphisms affecting gene expression of cytokines related to inflammation, such as IL-4, IL-6, IL-8, IL-10, and TNF-α, have been strongly associated with an increased risk for OSCC . AlthougRegarding the ability of OSCC to invade and metastasize or be enhanced by the presence of inflammatory mediators ,47, TNF-Although the mechanism of action through which TNF-α promotes these phenomena has not yet been elucidated, there is evidence to demonstrate that the NF-kB signalling pathway, activated by TNF-α, may be key to the promotion of invasion and migration . In vitrA further mechanism of action through which TNF-α can be considered as a promoter of OSCC cell invasion and migration has been demonstrated in an in vitro study showing that TNF-α is able to activate the transcriptional repressor Snail that down-regulates E-cadherin and induces the mechanism of epithelial-connective tissue transition . AnalysiThe aforementioned mechanisms discussed clearly link the role of TNF-α with enhanced metastatic potential and highlight the ability of TNF-α to induce the malignant cell epithelial-connective tissue transition in OSCC. In contrast to the above, a recent study has demonstrated cross-talk between the macrophage-related inflammatory cytokines and the migration of cancer cells . IL-6, IPrognosis is defined as the predicted course of a disease process and the anticipated outcome expected from treatment. In OSCC, TNM staging (tumour-node-metastasis), cancer staging, and certain conventional histological tumour grading have been utilized for therapeutic decision making but have been found to be imperfect predictors of prognosis. Several biomarkers have been discovered that appear to correlate with tumour aggressiveness and potentially prognosis of OSCC. These include epidermal growth factor, p53, cyclooxygenase-2 (COX-2), and matrix metalloproteinase-9 (MMP-9) ,55,56,57An indirect way of determining the importance of TNF-α in the prognosis of cancer is by measuring TNF-α converting enzyme (TACE) activity. TACE belongs to a large group of type I integral membrane proteins known as ADAMs . These proteins are involved in a variety of cellular processes, including conversion of TNF-α as well as several other membrane-anchored proteins, including both epidermal cell growth factors and TNF family receptors, to their active form ,60. TACEIdentifying more specific genotypic expression of TNF-α from serum samples of head and neck cancer patients may also provide another avenue to guide prognosis. In a study by Santana et al., 2021, blood collected from 163 oral and oropharyngeal SCCs and 143 healthy controls was genotyped for PON1, TNF-α (two different genotypes), and TGF-β single nucleotide polymorphisms . They foRecent studies have also demonstrated interesting findings when investigating the potential role of TNF-α and its family proteins to predict prognosis, tumour immune characteristics, and immunotherapy response in several cancer types. Although these studies have not been replicated in OSCC, they pose an interesting avenue for future research. The family proteins include the TNF receptor superfamilies that are composed of 19 ligands and 29 receptors. The communication between these pathways orchestrates inflammation and controls cell survival, proliferation, and differentiation. Gene expression, overall survival rate, and somatic mutation data were collected for 516 colorectal cancer cases from the National Cancer institute GDC data Portal (TCGA) to identify the TNF family genes that are risk factors for poor overall survival and the genes that are protective . Out of Studies have considered TNF-α within biofluids  and tissWhole saliva produced from the major and minor salivary glands are secreted fluid transported from serum as well as from surrounding glandular tissues. Saliva is composed of the secretions from salivary glands as well as oral mucosa, periodontium, and oral microflora that contribute to the final content of whole saliva . The hetInterestingly, cytokines in salivary levels increased regularly when compared with well differentiated to poorly differentiated oral squamous cell carcinoma. Levels of IL-6, IL-8, TNF-α, IL-1β, TNF-α, and IFN-γ were raised in early stage OSCC when compared to healthy controls . SalivarTNF-α serum levels for the correlation of OSCC for diagnosis remains inconsistent. Schiegnitz et al., 2017, carried out a prospective cohort study investigating serum biomarkers  in patients with OSCC, oral lichen planus (OLP), and healthy controls and found no significant differences in TNF-α serum levels between controls, OLP, and OSCC patients . Similarp = 0.0012), IL-8 (p = 0.0000), and TNF-α (p = 0.0492) were higher in patients with OSCC compared to healthy controls and patients with oral leukoplakia without associated dysplasia. The results of the above study are promising as they represent a positive correlation with IL-8 and TNF-α for tumour expression and salivary expression. Further studies are required to corroborate these results but constitute a step forward towards a non-invasive bedside/chairside adjunct to differentiate dysplasia or OSCC from suspicious oral lesions.There are two studies investigating TNF-α levels in tissue samples and peripheral blood or saliva looking for correlation. Healthy matched controls were compared with clinically and histopathologically confirmed cases of SCC in a case control cohort study (n = 75 per group) . ResultsThere are over 770 bacterial species in the oral cavity , in addiP. gingivalis and F. nucleatum) and are considered responsible for immune related periodontal tissue damage [P. gingivalis showed an overexpression of TLR2, which in turn promoted the overexpression of MMP9, TNF-α, and IL-6 that augmented the proliferation and growth of infected cells. Further, P. gingivalis infection associated increased transcription of TNF-α coincided with fibroblastic phenotypic shifts, the expression of stem-cell like properties, and higher migratory invasive capacity of OSCC cells [F. nucleatum and P. gingivalis in a murine model of periodontitis-associated oral tumorigenesis demonstrated stimulation of OSCC induction and proliferation through direct interaction with oral epithelial cells via toll-like receptors [The production of pro-inflammatory cytokines, such as interleukin IL-1b, IL-6, and TNF-α, is enhanced by common lipopolysaccharides and bacterial endotoxin of the pathogens associated with periodontal and endodontic disease  are still unknown. However, it is postulated that chronic inflammation, consisting of various types of proinflammatory mediators, such as activated cytokines and chemokines, likely plays a role in this process .TNF-α can influence the different stages of etiopathogenesis of OLP, divided into specific and non-specific mechanisms. However, the specific mechanisms involved in OLP are poorly understood, and it is proposed that an altered or a foreign antigen in the basal cell layer is a trigger to provoke a T cell-mediated autoimmune response against these cells. Some genes of heat shock protein (HSP) have been found in the TNF-α gene region that increase the expression of this protein on the keratinocyte surface and it heralds the role of TNF-α in OLP ; oral keA meta-analysis demonstrated that TNF-α levels in OLP were significantly increased in various tissue types, suggesting that this increased level may be effective in initiating the disease and activating auto-inflammatory mechanisms . In partp = 0.0102) was found, which may support its role in progressive pre-malignancy [Cytokines may be involved in anti-tumour mechanisms or enhance the malignant transformation and tumour growth. Immunoreactivity using IHC analysis has shown significantly higher expression of TNF-α in OSCCs and OPMDs with and without dysplasia compared to normal controls. Interestingly, comparing leukoplakias with dysplasia to OSCC, significantly less TNF-α expression in the stroma  and significantly elevated salivary TNF-α levels in patients with OSCC compared to normal tissue, as well as oral dysplasia with or without dysplasia (ANOVA p < 0.001) [Evidence has suggested that the NF-κB–dependent cytokine levels  are elevated in both the saliva as well as the tissue specimens from patients with oral premalignant lesions  Table 2Table 2. < 0.001) . These cBiologic agents targeting TNF-α are currently in clinical use for immune-mediated inflammatory rheumatological and gastrointestinal disease. Current FDA approved drugs are monoclonal antibodies  or receptor fusion proteins (etanercept). There is accumulating evidence that TNF-α may be a target for solid tumour therapy, with clinical trials of etanercept showing disease stabilization or partial improvement in patients with metastatic breast cancer . There aSmall animal studies such as these are encouraging, but many therapeutic targets have fallen short of the clinical bedside and remained on the lab bench or fallen victim to the translational canyon. As such, further studies in larger animal models/clinical trials are required to establish if TNF-α antagonists have a role to play in the management and prevention of OSCC clinically.TNF-α has proven to be an important cytokine in the inflammatory cascade and may yet be a potential target in OSCC detection, prevention, and treatment. TNF-α has been observed in a participant role in OSCC but additionally in pre-malignant mucosal and microbiome-related oral disease, complicating its allocation to a biomarker role. Despite some promising results, further work is required in larger clinical populations to correlate TNF-α with prognosis and suitable utility as a biomarker, and ultimately if the TNF-α antagonism can play a novel adjunctive therapeutic role in the prevention or treatment of OSCC.


The maxillary midline diastema (MMD) has experienced a renaissance in popular culture over the last 10 years. Current literature states that MMDs should be eliminated where possible or be reduced to 1 mm or less in width. This literature review explores the evidence for this in a West African population. A systematic search of the literature (Medline and Google Scholar) followed by a hand search was carried out to identify all papers evaluating perceptions of MMDs. In all, 209 papers were identified in the literature search, 8 of which met the inclusion criteria. The review revealed that midline diastemas are desirable in this specific population with a width of 2 to 4 mm deemed as attractive. As many as 95% of participants found the MMD beautiful, and as many as 95% would consider having one created artificially. The literature does not support the position that MMDs larger than 1 mm should always be treated to be perceived as attractive in this population. MMDs are accepted as attractive and should be considered a key feature in smile design for this population. A maxillary midline diastema (MMD) is a space between the 2 central incisors irrespective of its size, and it is considered a normal and naturally occurring phenomenon . In someMMDs occur for many reasons and may be developmental or acquired . McVay a,Whilst perceptions about midline diastemas are multifactorial and complex, racial and national variation in these perceptions have been noted.In West Africa, perceptions about midline diastemas and spacing in general differ significantly from those often expressed in the literature. A “gap” is often considered a sign of beautyAs dentists, it is common for us to provide treatment in the aesthetic zone that results in a dramatic change in a patient's smile. Black triangles and other spaces in the smile are frequently seen as problematic for patients and dentists alikeWhen planning a smile design case, it is important that we consider cultural and racial factors as they pertain to our patients. This understanding could help inform our treatment plans and ensure that our patients are making informed decisions. Whilst for some patients, having aesthetic dental treatments is a status symbol, it is important that we recognise that treatment options are patient-specific. Providing a smile that is culturally and racially unusual may cause an imbalance.1.Are MMDs desirable/aesthetic in a Black West African population?2.Should MMD design be considered as an integral part of smile design in a Black West African patient?3.How can the quality of the evidence in this region be improved?A literature review was carried out to answer following questions:A literature search was conducted using the Medline database to search for papers that looked at perceptions about diastemas using keywords and subject headings . This seHand-searching was then conducted by looking at reference lists of these papers and conducting a Google Scholar search. Screening reference lists yielded a further 82 papers that were not on the original list, and Google Scholar yielded a further 12.The total number of papers identified was 208. The titles and abstracts of these papers were screened for relevance. Those papers that passed screening were subsequently retrieved, read, and scored against the inclusion criteria for eligibility . One pap,,53,The final number of papers included in the literature review was 8All the papers that met the inclusion criteria except oneIn all cases, the lead researchers were affiliated with dental departments in government hospitals. In Nigeria, all these universities were based in South Nigeria. The study from GhanaBased on the data available, this review speaks predominantly to opinions within the Nigerian population at best and, perhaps more selectively, the Southern coast of Nigeria.The papers represent 3553 participants, all of whom are aged 16 years or older, who identify as Black West Africans. The prevalence of MMDs in the sample ranged from 16.9%,,,,In the case of 4 papers, the participants for the study were taken from the institutions where the researchers were based.Akinboboye et alThe demographic information available for the samples studied varied considerably . The hetThe Witt and Flores-Mir,,,,,The Akinbobye et al papers,,,,Whilst all the researchers used a structured questionnaire, there was some variation in the content. The evidence can be split into 2 groups. Group A papersAll 8 papers show that MMDs are a regular occurrence in this population . These f,,The evidence suggests that the MMD is considered beautiful within a Nigerian and Ghanaian population,,Minimal differences in opinion were noted across ethnic,,A diastema of 2 to 3 mmMMDs were preferred on females,Although the evidence supports an MMD as an aesthetic feature in a West African smile, the findings are not robust, lack validity (as 0 mm was not included as an option in Group A studies), and have limited representation of the West African region. It is likely that the absence of a 0-mm and 1-mm diastema in these surveys however, is due to the overall acceptability of diastemas in the region.Despite having reasonable sample numbers, the existing data is biased. The sample population does not provide a balanced representation of the region geographically (with the exception of one of the Akinboboye studiesThe information on the questionnaires themselves lacks detail. For the first Akinbobye study,Further research in this area should take the Witt and Flores-MirThe papers included in In West Africa, the commonly described midline diastema ideals did not hold true in pre-Colonial and Colonial times when a diastema was not perceived as a hinderance to beauty. The impact of colonialisation affected all levels of society, and there has been an instilled belief that the closer one can get to looking like “a White man,” the more successful and revered one will be. This transition can still be seen today in how clothing, hair, and diet are used as status symbols. In the same way, there has been an increased interest and availability of cosmetic dental treatments available, with some individuals being willing to travel abroad to obtain treatment. This may be in part the pursuit of a Western standard of beauty,Recently, there has been a shift in how women in particular are presented in the fashion and beauty industries, pivoting away from the pursuit of White standards of beauty as the accepted norm and embracing the diversity that exists in normal life.The aim of this study was to explore perceptions in West Africa; however, given that papers were only identifiable from 2 West African countries, the data is limited. It is possible that there is more research into this topic in the region that remains unpublished, in addition to those papers that did not meet the inclusion criteria. Given that this study only examined peer-reviewed papers at evidence levels 1 through 3, such information could not be included. A further study with broader inclusion criteria would allow more data from the region to be captured.Given the density of data from Nigeria, and the lack of it from elsewhere, the findings of this study are most generalisable to that specific population. Moreover, the heterogeneity of the data collected by the various authors and the variations in their study designs limited the ways in which that data could be combined and prevents further statistical analysis.,,,The interaction between perceived attractiveness and diastema width is incredibly complex, and it is impacted by more than just diastema width. This literature review shows that MMDs are desirable and that they should be kept between 2 and 4 mm wide in this population. The practice of artificial diastema creation in some parts of NigeriaNone disclosed.


Alzheimer disease (AD) is the most common progressive neurodegenerative disorder. AD causes enormous personal and economic burden to society as currently only limited palliative therapeutic options are available. The pathological hallmarks of the disease are extracellular plaques, composed of fibrillar amyloid-β (Aβ), and neurofibrillary tangles inside neurons, composed of Tau protein. Until recently, the search for AD therapeutics was focussed more on the Aβ peptide and its pathology, but the results were unsatisfying. As an alternative, Tau might be a promising therapeutic target as its pathology is closely correlated to clinical symptoms. In addition, pathological Tau aggregation occurs in a large group of diseases, called Tauopathies, and in most of them Aβ aggregation does not play a role in disease pathogenesis. The formation of Tau aggregates is triggered by two hexapeptide motifs within Tau; PHF6* and PHF6. Both fragments are interesting targets for the development of Tau aggregation inhibitors (TAI). Peptides represent a unique class of pharmaceutical compounds and are reasonable alternatives to chemical substances or antibodies. They are attributed with high biological activity, valuable specificity and low toxicity, and often are developed as drug candidates to interrupt protein–protein interactions. The preparation of peptides is simple, controllable and the peptides can be easily modified. However, their application may also have disadvantages. Currently, a few peptide compounds acting as TAI are described in the literature, most of them developed by structure-based design or phage display. Here, we review the current state of research in this promising field of AD therapy development. Tauopathies are a variety of progressive neurodegenerative disorders, characterized by the deposition of the abnormally aggregated microtubule-associated protein Tau. They include about 20 diseases, e.g. Huntington disease (HD), progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), primary age-related tauopathy (PART), chronic traumatic encephalopathy (CTE), and, most abundant, Alzheimer disease (AD) , and neurofibrillary tangles inside of neurons, composed of Tau, as already described by Alois Alzheimer in 1907  gene, located on chromosome 17q21, six isoforms are generated in the central nervous system (CNS). These can be divided into 4R Tau, containing 4 repeats (31–32 amino acids each), and 3R Tau, containing three repeats   have toxic effects, but also that small, soluble protein oligomers play a fundamental role in AD pathology, as they cause synaptic and mitochondrial dysfunction. They might cause neurodegeneration a long time before protein fibrilization and deposition starts  is strongly driven by two hexapeptide fragments within Tau: PHF6* (275-VQIINK-280) and PHF6 (306-VQIVYK-311). The PHF6 sequence is located at the beginning of the third repeat (R3) and can be found in all Tau isoforms. In contrast, the PHF6* sequence is placed at the beginning of the second repeat (R2) and is only present in four-repeat (4R) Tau isoforms. Both hexapeptide motifs show the highest predicted β-structure potential within the Tau sequence, and point mutations in the hexapeptide regions can change β-propensity, leading to an increase or decrease of aggregation  imaging studies reveal that amyloid accumulation may predict the onset of Tau accumulation. However, Tau accumulation only predicts the onset of cognitive impairment, while the onset of Tau pathology occurs at the same time as the symptoms appear .In order to reduce Tau pathology in AD, a variety of small molecules, including modulators of post-translational modifications and aggregation inhibitors of Tau, have been described. Most of them are in the preclinical stage  amino acids, represent a unique class of pharmaceutical compounds and are reasonable alternatives to chemical substances. Physiologically, they act as key regulators of biological functions and are attributed with high biological activity, valuable specificity, and in most cases, low toxicity  as a template to design peptide aggregation inhibitors  aggregation was not demonstrated in this article, as only shortened K12 and K19 Tau constructs missing R2 with enhanced aggregation propensity were used. Therefore, the very similar PHF6* sequence is missing in both constructs. Later, the group reported that D-tlkivw was not able to inhibit TauFL aggregation, suggesting that TAI compounds based on the PHF6 sequence might be less effective in aggregation inhibition of TauFL  containing segment, forming steric zippers, as determined by the cryo EM method micro electron diffraction (MicroED)  assays. However, while WINK showed no detectable self-aggregation, MINK was found to self-aggregate to a certain extent. Both inhibitors could reduce the ability of exogenous TauFL fibrils to seed intracellular Tau in HEK293 biosensor cells expressing Tau-K18-(P301S)-EYFP in a dose-dependent way, whereas the VQIVYK (PHF6) targeted inhibitor D-tlkivw inhibited seeding poorly .Two inhibitors derived from the PHF6* sequence, MINK and WINK, were found to inhibit TauFL fibril seeding even more successfully than MINK, with IC50 = 1.1 µM. Together, the results indicate that VQIINK (PHF6*) is, in comparison to PHF6, a superior target for TAI -2,5-didiphenyltetrazolium bromide), but only one inhibitor, designated D1, eliminated toxic effects of Aβ1-42 on Neuro-2a (N2a) cells at a 10 molar excess.A Rosetta-based strategy was used to design capping peptide inhibitors on a search model of Aβ1-42 toxicity on N2a cells efficiently in a tenfold excess and equimolar ratio. D1, D1b and D1d elicited a dose-dependent response, and the IC50 value was estimated to be less than 1 µM. D1b and D1d were more effective than D1 in reducing fibril formation. The L-amino acid form of D1 did not reduce cell toxicity, as judged by MTT assay. The reduction of Aβ toxicity could be explained by a dose-dependent inhibition of Aβ aggregation, as demonstrated by ThT assays and transmission electron microscope (TEM) analysis. Antibody studies indicated that the inhibitors reduced Aβ oligomers as well as Aβ fibrils. In addition, the reduction of pre-formed Aβ aggregate toxicity by inhibitors D1b and D1d could be demonstrated with D1d being more effective, and there was evidence that pre-formed fibrils were either capped or coated.In a second round of design, six new inhibitors, designated D1a-D1f, were selected and tested. Two of them, D1b and D1d, reduced AβFL fibril seeding in Tau biosensor cells, D1b being the most effective inhibitor . Tau mutant experiments in Tau biosensor cells lead to the conclusion that D1b acted on the PHF6 and PHF6* segments of Tau. The authors hypothesized that PHF6 and PHF6* might share common structural features with the Aβ core. They could also demonstrate that seeding in Tau biosensor cells by amyloid species in brain-derived tissue from patients with AD or with progressive nuclear palsy could be prevented by D1b  EYFP, suggesting that there is an interaction between Tau and Aβ in the microtubule-binding domain. The inhibitor peptides were able to reduce seeding of Tau by aggregated Aβ, D1b being most effective. In addition, D1, D1b and D1d were able to reduce aggregation of Tau monomers, as demonstrated by ThT assays. They were not general amyloid inhibitors, as aggregation of human islet amyloid polypeptide (IAPP) and α-Synuclein was not inhibited. The inhibitors also prevented TauFL fibrils, and modulated the aggregation of the respective Tau form, as demonstrated by ThT-tests and dynamic light scattering (DLS) . The selected D-amino acid peptides bound to PHF6 fibrils, Tau isoform fibrils such as 3RD-Tau (K19), as well as to TauFL in vitro, as demonstrated by Thioflavin assays. DLS, pelleting assays and atomic force microscopy (AFM) demonstrated that MMD3 prevented the formation of Tau fibrils rich in β-sheets by an interesting mechanism, partitioning Tau into large amorphous aggregates. Enzyme-Linked Immunosorbent Assay (ELISA) data demonstrated binding of MMD3 and MMD3rev to PHF6*- and TauFL fibrils, while NMR measurements suggested that they bound to monomeric TauFL only with rather low affinity . The D-peptides were characterized with respect to their specificity for Tau conformers (monomers and aggregates) and their therapeutic potential. Using ELISA and fluorescein amidite (FAM)-labelled peptides, binding of the peptides to both TauFL and TauFL fibrils was demonstrated. The aggregation-prone hexapeptide motif within Tau, PHF6, was identified as a possible binding site of the most promising D-peptide ISAD1. ISAD1 inhibited fibrillization of TauFL and a wide variety of disease-relevant Tau isoforms . Similar to the D-peptide binding PHF6* described above, it was found that the D-peptides reduced regular Tau fibril formation by forming large non-fibrillar, non-toxic aggregates, which were ThT negative. They were examined in more detail with respect to their particle size in DLS, pelleting assay and western blot. ISAD1 and ISAD1rev were tested in cell culture, where it was evident that the peptides were taken up by neuronal Tau expressing cells and accumulated in the cytosol. The peptides were non-toxic to cells and prevented Tau fibril-mediated cell toxicity of externally added and internally expressed Tau -cells, (TAT)-7H inhibited Tau phosphorylation at Ser202 and Thr205 , which unexpectedly inhibited R3-Tau aggregation in a dose-dependent way, as shown by DLS and EM. 7H was originally investigated as an inhibitor on Ku70 and Huntingtin protein interaction. The peptide transiently contacted Tau at multiple sites with a possible preference for PHF6*. Addition of the trans-activator of transcription (TAT)-sequence to 7H increased its cell permeability. In human neurons differentiated from homozygous TauPeptides, especially D-amino acid peptides, can be interesting alternatives to small chemical molecules or antibodies. Cell and blood–brain barrier permeability are possible advantages of at least some peptides over therapeutic antibodies, which might have a higher affinity towards their target molecules but cross membranes only poorly (Matsson et al. As peptides are produced synthetically, they can easily be modified and optimized to fulfil the demands of the pharmaceutical industry. Using methods such as alanine-scans, one can investigate which of the amino acids are essential to perform the desired biological functions (Morrison and Weiss Recently, it was demonstrated that therapeutic D-amino acid peptides can be absorbed systematically after oral or intranasal administration (Pappenheimer et al. The D3 and RD2 sequences contain arginines, so do the sequences of the D-peptidic TAI p-NH described by Zhang and colleagues (Zhang et al. Most of the peptides described as TAI target the PHF6 or the PHF6* sequence motifs of Tau. It remains to be seen which of the sites is the more potent driver for Tau aggregation and therefore the more interesting target for TAI. Seidler and colleagues suggested that PHF6* is the most promising target for the development of TAI (Seidler et al. Fitzpatrick and colleagues have also shown that Tau filaments extracted from patients with different tauopathies differ in structure (Fitzpatrick et al. In recent years, a variety of peptide compounds acting as Tau aggregation inhibitors were developed as a promising therapeutic approach towards Alzheimer disease. In particular. the suitability of D-amino acid peptides for possible in vivo applications has already been demonstrated. Most of the peptides already described in the literature were developed by structure-based design or phage display selections. The therapeutic properties of those peptides were summarized in this manuscript. Molecular insight into the binding mode of different peptides was gained by in silico modelling. Some of the identified Tau-targeting peptides were able to penetrate neuronal cells, reduce Tau phosphorylation or improved cognitive abilities of Tau transgenic mice, making them interesting for AD therapy. Future studies are needed to demonstrate which of the peptides can be developed into a therapeutic compound for the treatment of AD.


Pseudomonas and Hymenobacter. Among fungi, Aureobasidium and Rhodotorula were differentially associated with GTD escape vines, while the GTD pathogen, Eutypa, was associated with the diseased vines. This is the first report of the link between the GTD escape phenotype and the grapevine microbiome.Grapevine trunk diseases (GTDs) are a substantial challenge to viticulture, especially with a lack of available control measures. The lack of approved fungicides necessitates the exploration of alternative controls. One promising approach is the investigation of disease escape plants, which remain healthy under high disease pressure, likely due to their microbiome function. This study explored the microbiome of grapevines with the disease escape phenotype. DNA metabarcoding of the ribosomal internal transcribed spacer 1 (ITS1) and 16S ribosomal RNA gene was applied to trunk tissues of GTD escape and adjacent diseased vines. Our findings showed that the GTD escape vines had a significantly different microbiome compared with diseased vines. The GTD escape vines consistently harbored a higher relative abundance of the bacterial taxa  The rel, Pseudomonas fluorescens and Achromobacter xylosoxidans, were inversely correlated with the presence of the pathogen, Xylella fastidiosa. Pseudomonas fluorescens was identified as a potential key player in the disease escape phenotype. Burkholderia territorii, Burkholderia metallica, Pseudomonas geniculata, and Bacillus pumilus, displayed antimicrobial properties against the surrogate bacterium for the HLB pathogen, Candidatus Liberibacter asiaticus, and the citrus root pathogen Phytophthora nicotianae. Similarly, Lactobacillus sp., Exophiala sp., Aureobasidium sp., and Angustimassarina, from HLB survivor (or escape) citrus trees. The researchers suggested that these taxa played a significant role in preserving the health of the HLB escape citrus trees. These findings indicate that harnessing the microbial components of disease escape plants could contribute to the development of innovative strategies for managing plant diseases and promoting plant health.Recently, there has been an increased interest in studying the microbiomes of plants displaying a disease escape phenotype . DiseaseEutypa lata, Phaeomoniella chlamydospora, and members of the Botryosphaeriaceae family have been widely studied as GTD pathogens. Moreover, the diseases progress slowly, and there is a potential for symptoms to overlap with other GTDs or abiotic stresses . In this study, we applied a combination of DNA metabarcoding and microbial isolation to characterize the microbiomes of these plants and contrast them with diseased plants growing in the vicinity. The research was built around two pivotal questions: Do the microbiomes of candidate GTD escape vines differ from those of diseased vines? If so, are there microbial taxa in the GTD escape vines that can be linked to the observed GTD escape phenotype? We aimed to uncover microbial contributors to the expression of the GTD escape phenotype, which could help in the development of effective strategies for managing GTDs.2.2.1.We visited nine vineyards in Hawke’s Bay and Canterbury between November 2019 and February 2020 to identify candidate GTD escape vines. Hawke’s Bay and Canterbury, situated in North Island and South Island of New Zealand, respectively, are two of the major winegrowing regions in New Zealand. The vineyards in the Hawke’s Bay region have loamy soil, a mean air temperature of 13.2°C, and an annual rainfall of 716 mm  and visualized under UV illumination using the Gel Doc EZ System .Genomic DNA was extracted from the woody trunk tissue samples using a cetyltrimethylammonium bromide (CTAB) method, as outlined in the study of 2.4.The fungal metabarcoding was as in ™ mix (Bioline), 1 μL of 10 μM of each primer, and 7 μL of molecular grade water. The PCR amplification was carried out in a T100 thermal cycler (Bio-Rad) with the following program: initial denaturation at 94°C for 3 min, 35 cycles (30 cycles for bacteria) of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, elongation at 72°C for 60 s and a final extension phase at 72°C for 5 min, before holding at 12°C. All PCRs included a negative, no template control. The mock DNA community, ZymoBIOMICS microbial standard , was included as a positive control. All PCR products were visualized in 1% agarose (at 100 V for 30 min). The PCR products were purified using Agencourt® AMPure® XP magnetic beads (Beckman Coulter). Sample-specific barcodes were added to the purified amplicons in a second PCR reaction consisting of 10 μL MyFi™ mix (Bioline), 1 μL each of 5 μM barcoding primers, 1 μL template from the first round of PCR, and 7 μL molecular grade water . The PCR reaction cycle was 95°C for 3 min; five cycles of 95°C for 20 s, 55°C for 15 s, and 72°C for 30 s; and a final 72°C extension for 60 s before holding at 12°C. The indexed PCR products were purified with Agencourt® AMPure® XP magnetic beads and quantified with a NanoDrop® ND-1000 spectrophotometer. Amplicons were pooled equimolarly and sequenced on the Illumina MiSeq platform using 2 × 300 bp paired-end reads with the MiSeq reagent kit v3 chemistry .The DNA metabarcoding was performed in a two-step PCR. The first PCRs were performed in duplicate in a total volume of 20 μL containing 1 μL of template DNA, 10 μL of MyFi2.5.trimLeft function and truncated at 260 bp for the forward and reverse reads. The taxonomic assignment of the ASVs was conducted using the assignTaxonomy function in DADA2. The UNITE (10.05.2021) database  using the DADA2 (v1.16.0) R package . The ITSdatabase  and the database  were usedatabase . For furdatabase  or made database  on R . The results were visualized with ggplot2. The principal coordinate analysis (PCoA) using the Bray-Curtis distance method was carried out with the ordinate function, and the results were visualized with the plot_ordination function in the phyloseq package. The permutational multivariate analysis of variance (PERMANOVA) test with 999 permutations was used to analyze the statistical effect of the grapevine condition on the fungal and bacterial community structures of GTD escape and diseased vines, using the adonis function from the vegan package within the R software environment  and statistically significant  Pearson’s correlations were accepted. Only ASVs with a mean relative abundance >0.01% were used for the network construction. Within kingdom analysis was conducted with the network.2 function, while bipartite network analysis between fungi and bacteria was conducted with the corBionetwork function. The networks were visualized with the model.maptree2 function. The topological roles of taxa were revealed by calculating the within-module connectivity (Zi) and among-module connectivity (Pi). Taxa that belong to the connector , module , and network hubs    can be c ≥ 0.62) .2.6.™, United States) and malt extract agar , amended with 50 mg/L of streptomycin for the isolation of fungi. For the selective isolation of basidiomycetes, separate MEA plates were further amended with benomyl (4 mg/L) to retard the growth of ascomycetes  plates amended with cycloheximide at 40 μg/mL to retard fungal growth . The ITS region of fungal isolates was amplified by PCR with the primers ITS5 and ITS26  and directly Sanger-sequenced at Macrogen Inc. . The raw sequences obtained from Macrogen Inc. were checked and cleaned in Geneious Prime (v2022.0.1). Taxonomic assignment of the isolates was performed by aligning their sequences with the NCBI ‘nt’ database using the BLAST algorithm  were unusable due to DNA extraction failure. The final number of wood samples processed for DNA sequencing was 70 (16 candidate GTD escape vine samples and 54 diseased vine samples). From the fungal dataset, 1,431,513 reads remained out of 3,632,192 raw reads after filtering and chimera removal. For the bacterial dataset, 2,856,153 reads remained out of 5,271,359 raw paired reads after the filtering and chimera removal steps in DADA2. Only samples with at least 1,000 reads were retained for further downstream analyses. Because of this, three and six samples were eliminated from the fungal and bacterial datasets, respectively. Overall, 754 ASVs were identified from 68 samples in the fungal dataset, while 3,716 ASVs were identified from 65 samples in the bacterial dataset. Twelve fungal ASVs, including  dataset .3.2.3.2.1.Phaeomoniella (40%), Epicoccum (13%), Seimatosporium (10%), Aureobasidium (10%), and Alternaria (8%) had the highest relative abundance (Phaeomoniella (35%), Eutypa (31%), Epicoccum (15%), Alternaria (4%), and Seimatosporium (3%) were the genera with the highest relative abundance. Among these fungal genera, Eutypa (GTD escape 0.3% – diseased 31%), Aureobasidium (GTD escape 10% – diseased 1%), Seimatosporium (GTD escape 10% – diseased 3%) and Cladosporium (GTD escape 2% – diseased 0.4%) showed the highest variation in relative abundance between the GTD escape and diseased vines. Many of the substantial differences in the relative abundance of these fungi between GTD escape and diseased vines were consistent across all the vineyards .Overall, the fungi were assigned to 76 genera. Among the GTD escape vines, bundance . In the ineyards . The hig3.2.2.Pseudomonas (24%) had the highest relative abundance in GTD escape vines, followed by Sphingomonas (9%), Hymenobacter (7%), Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (ANPR) (5%) and Hafnia_Obesumbacterium (5%) (Pantoea (10%), Acinetobacter (9%), Pseudomonas (9%), Sphingomonas (8%), and ANPR (6%). The higher relative abundance of Hymenobacter in the GTD escape vines was consistent across all the vineyards, while the trend for Pseudomonas was observable only in vineyards 1 and 4. Pantoea and Curtobacterium also had a higher relative abundance in diseased vines across all four vineyards . Other phyla represented were Actinobacteriota (GTD escape 14% – diseased 20%), and Bacteroidota (GTD escape 15% – diseased 20%). Firmicutes had a lower representation with 1.5% ASVs in diseased vines and less than 1% ASVs in GTD escape vines. Overall, the bacteria were assigned to 252 genera. ium (5%) . The taxineyards . Due to ineyards ,B. The rineyards . The hig3.2.3.p < 0.02), but the Shannon and Simpson diversity indexes were similar between the two conditions  and bacterial (p = 0.008) communities, explaining 2.99 and 2.47% of the variation observed in the fungal and bacterial community structures, respectively.The Bray-Curtis clustering of both fungal and bacterial communities demonstrated that GTD escape vines grouped together ,B. Furth3.3.Cladosporium, Seimatosporium, Rhodotorula, and Clonostachys, were statistically associated with GTD escape vines, while Eutypa was the major genus that was associated with diseased vines  was identified by DESeq2 in the GTD escape vines. In contrast a Pseudomonas ASV 7, which was prominent in vineyard 4, was associated with the diseased vines.In the bacterial community, edgeR showed the highest differential abundance in the GTD escape vines was from the genera nobacter . Acinetoed vines . Only on3.4.Eutypa was the major genus responsible for the fungal structural differences observed in the trunk microbiome of diseased vines. However, in the GTD escape vines, the fungal community structure was driven by fungal genera such as Aureobasidium, Cladosporium, Rhodotorula, and Seimatosporium , Pantoea (17 isolates), Curtobacterium (15 isolates), and Erwinia (9 isolates)  were obtained. The fungal isolates were placed into 20 morphotypes . Followiicillium . For thesolates) . The phyiability . Some is4.In this study, we applied high-throughput sequencing methods to investigate the microbial community structure of grapevines that escape GTDs. Utilizing DNA metabarcoding, we examined the fungal and bacterial community structures within the trunk endosphere of candidate GTD escape vines and compared them with nearby diseased vines. The findings revealed significant variations in the microbiome structure between the GTD escape and diseased vines, with specific microbial taxa driving these distinctions. These microbial taxa may contribute to the expression of the GTD escape phenotype.Eutypa. This fungus showed a significant increase in relative abundance in diseased samples and was consistently discriminant for the samples. Eutypa lata is a known GTD pathogen  compared with grapevines showing GTD symptoms (up to 29% relative abundance). They further demonstrated that some Pseudomonas strains showed biological activity against GTD pathogens. Similarly, Pseudomonas was identified as the driver of disease escape from Pierce’s disease, where there was a negative correlation between P. fluorescens and the presence of the pathogen, Xylella fastidiosa . Although a previous study detected Hymenobacter sequences on grapevine foliage and grape berries . The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. DA received a writing scholarship from the Faculty of Agriculture and Life Science, Lincoln University. This publication has been partially financed by the Lincoln University Open Access Fund.HR, DM, and SB are employed by The New Zealand Institute for Plant and Food Research Limited. BV was employed by The New Zealand Institute for Plant and Food Research Limited and is currently employed by Bragato Research Institute.The remaining 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.


Zoonotic infections with influenza A viruses (IAVs) of avian origin can cause severe disease with high fatality in humans, as in the case of the avian IAV subtypes H5N1 and H7N9. Fortunately, such spillover events from the avian reservoir to humans are rare and mostly limited to single individuals due to a robust species barrier. The molecular basis for this species barrier is the intrinsically poor adaptation of avian IAVs to humans . NumerouMany different host factors that are essential for efficient viral replication of IAV in human cells have been described to also contribute to the human species barrier preventing zoonotic IAV infections (for a review see ). Here, Most known anti-IAV restriction factors do not differentiate between IAVs of human and animal origin . A notabEscape from restriction factors of the species barrier is a prerequisite for zoonotic IAVs to establish a new lineage in the human population . Thus, aPartial escape from anti-IAV restriction factors can also be achieved by genetic changes that enhance viral fitness overall which, in turn, increases replication in the human host and may facilitate the acquisition of additional, true escape mutations . The virAcquiring the necessary set of MxA escape mutations appears to be particularly challenging because the concomitant fitness loss needs to be compensated. Consequently, without the strong selective pressure of MxA, the MxA escape phenotype will gradually be lost. The 2009 pandemic H1N1 strain, for example, lost some of the MxA escape-mediating mutations in NP after reintroduction into the swine population due to the reduced antiviral pressure by porcine Mx1 ,13. On tAnother way for IAVs to gain MxA escape is their reassortment with MxA-adapted IAVs in intermediate hosts, such as pigs. Interestingly, the majority of reassortants that circulate in pigs carry NP segments with MxA escape mutations. These genomic segments were derived from 2009 pandemic H1N1 viruses that were accidentally reintroduced into pig herds . The panIFITM3 gene have been shown to be associated with the severity of seasonal and pandemic IAV infections [MX1 gene [MX1 gene is unclear. One explanation might be defects in IFN induction and signaling hampering the expression of MxA and BTN3A3 (Among the best-studied anti-IAV restriction factors associated with IAV susceptibility is the IFN-induced transmembrane protein 3 (IFITM3), an entry inhibitor of avian and human IAVs. Genetic variations within the fections –33. In afections ,35. ThusMX1 gene . About 6d BTN3A3 . Inborn Anti-IAV restriction factors such as the MxA protein are major components of the human species barrier against transmission of zoonotic IAVs. Most likely, numerous additional host restriction factors are involved and need to be discovered. Such antiviral barriers can be overcome through the acquisition of escape mutations in the viral genome, host genetic defects that compromise innate antiviral defenses, and reassortment events. Preadaptation, including reassortment, in intermediate hosts can also increase the viral fitness of nonhuman IAVs, thereby increasing their zoonotic and pandemic potential. It will be important to monitor the capacity of IAVs originating from diverse animal reservoirs to escape human restriction factors. It will be equally important to identify and protect highly susceptible individuals carrying inactive variants of these essential antiviral host factors.