Reconstitution of the phosphodiesterase 6 maturation process important for photoreceptor cell function.

The sixth family phosphodiesterases (PDE6) are principal effector enzymes of the phototransduction cascade in rods and cones. Maturation of nascent PDE6 protein into a functional enzyme relies on a coordinated action of ubiquitous chaperone HSP90, its specialized cochaperone aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), and the regulatory Pγ-subunit of PDE6. Deficits in PDE6 maturation and function underlie severe visual disorders and blindness. Here, to elucidate the roles of HSP90, AIPL1, and Pγ in the maturation process, we developed the heterologous expression system of human cone PDE6C in insect cells allowing characterization of the purified enzyme. We demonstrate that in the absence of Pγ, HSP90, and AIPL1 convert the inactive and aggregating PDE6C species into dimeric PDE6C that is predominantly misassembled. Nonetheless, a small fraction of PDE6C is properly assembled and fully functional. From the analysis of mutant mice that lack both rod Pγ and PDE6C, we conclude that, in contrast to the cone enzyme, no maturation of rod PDE6AB occurs in the absence of Pγ. Co-expression of PDE6C with AIPL1 and Pγ in insect cells leads to a fully mature enzyme that is equivalent to retinal PDE6. Lastly, using immature PDE6C and purified chaperone components, we reconstituted the process of the client maturation in vitro. Based on this analysis we propose a scheme for the PDE6 maturation process.

Oral administration of S-nitroso-L-glutathione (GSNO) provides anti-inflammatory and cytoprotective effects during ocular bacterial infections.

Bacterial endophthalmitis is a severe complication of eye surgeries that can lead to vision loss. Current treatment involves intravitreal antibiotic injections that control bacterial growth but not inflammation. To identify newer therapeutic targets to promote inflammation resolution in endophthalmitis, we recently employed an untargeted metabolomics approach. This led to the discovery that the levels of S-nitroso-L-glutathione (GSNO) were significantly reduced in an experimental murine Staphylococcus aureus (SA) endophthalmitis model. In this study, we tested the hypothesis whether GSNO supplementation via different routes (oral, intravitreal) provides protection during bacterial endophthalmitis. Our results show that prophylactic administration of GSNO via intravitreal injections ameliorated SA endophthalmitis. Therapeutically, oral administration of GSNO was found to be most effective in reducing intraocular inflammation and bacterial burden. Moreover, oral GSNO treatment synergized with intravitreal antibiotic injections in reducing the severity of endophthalmitis. Furthermore, in vitro experiments using cultured human retinal Muller glia and retinal pigment epithelial (RPE) cells showed that GSNO treatment reduced SA-induced inflammatory mediators and cell death. Notably, both in-vivo and ex-vivo data showed that GSNO strengthened the outer blood-retinal barrier during endophthalmitis. Collectively, our study demonstrates GSNO as a potential therapeutic agent for the treatment of intraocular infections due to its dual anti-inflammatory and cytoprotective properties.

In-depth investigations into symmetrical labyrinthine acoustic metamaterial with two micro-slit entries for low-frequency sound absorption.

Sound absorption below 1000 Hz has been extremely difficult through traditional barriers and absorbers, but it is required for noise control of appliances and machineries. Existing passive acoustic metamaterials attenuate low-frequency noise but with narrow bandwidths and bulky sizes. Hence, this paper proposes an acoustic metamaterial with enclosed symmetrical labyrinthine air channels and two micro-slits (configuration 1, identical slits; configuration 2, unequal length slits) at the end channels. Its theoretical model is established by acoustic impedance analysis using electro-acoustic analogy and validated numerically and experimentally. Sound absorption is found to happen as a result of impedance matching, Fabry-Perot-like labyrinthine resonances, and thermo-viscous losses in micro-slits. Parametric investigations reveal that increase in the number of channels, channel length, total height, and outer panel thickness shifts sound absorption peak to lower frequency but also decreases the magnitude and frequency range of absorption. Decreasing the channel width and slit width increases the sound absorption magnitude without changing absorption frequencies. Interestingly, unequal slit lengths perform better than equal slits by giving a lower frequency sound absorption with increased magnitude and frequency range, which is unlike that in existing labyrinthine metamaterials. Therefore, the proposed unequal slit metamaterial has enhanced low-frequency sound absorption and can be applied to appliances and machineries.

Transcriptome Analysis of Dermal Fibroblasts Derived From Visceral Leishmaniasis and Post-Kala-Azar Dermal Leishmaniasis Patients Reveal Disease-Specific Gene Expression and Pathological Regulation.

BACKGROUND: Post-kala-azar dermal leishmaniasis (PKDL), a dermal form of the disease, occurs in some visceral leishmaniasis (VL) patients following treatment. The PKDL disease mechanism is not yet clearly understood. Here we have studied the role of dermal fibroblasts in VL and PKDL disease mechanism. METHODS: Dermal fibroblasts were grown from skin biopsy explants collected from individual VL and PKDL patients and healthy controls. Fibroblasts from the third passage were subjected to RNA sequencing to analyze differentially expressed genes (DEGs). Significantly important genes were further validated by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: Transcriptome analysis of PKDL versus VL identified 516 DEGs (263 were overrepresented and 253 were underrepresented in PKDL). Among the top hub genes, MMP2, IL1B, CXCL8, IFIH1, NFKB1A, IL6, ISG15, and EGFR were underexpressed and ACTB, HSP90AA1, RAB7A, and RPS27A were overexpressed in PKDL compared to VL. CONCLUSIONS: Our data indicate that PKDL fibroblasts may present antigens through the MHC I pathway activating CD8+ T-cell mediated response, while VL fibroblasts express nuclear factor-κB (NFκB)-mediated chemokines, IL1B, IL6, and IL8, resulting in the recruitment of natural killer (NK)-cells and monocytes to the site of infection, leading to the clearance of parasite from the skin and visceralization of the disease.

Fingolimod (FTY720), an FDA-approved sphingosine 1-phosphate (S1P) receptor agonist, restores endothelial hyperpermeability in cellular and animal models of dengue virus serotype 2 infection.

Extensive vascular leakage and shock is a major cause of dengue-associated mortality. At present, there are no specific treatments available. Sphingolipid pathway is a key player in the endothelial barrier integrity; and is mediated through the five sphingosine-1-phosphate receptors (S1PR1-S1PR5). Signaling through S1PR2 promotes barrier disruption; and in Dengue virus (DENV)-infection, there is overexpression of this receptor. Fingolimod (FTY720) is a specific agonist that targets the remaining barrier-protective S1P receptors, without targeting S1PR2. In the present study, we explored whether FTY720 treatment can alleviate DENV-induced endothelial hyperpermeability. In functional assays, in both in vitro systems and in AG129 animal models, FTY720 treatment was found effective. Upon treatment, there was complete restoration of the monolayer integrity in DENV serotype 2-infected human microvascular endothelial cells (HMEC-1). At the molecular level, the treatment reversed activation of the S1P pathway. It significantly reduced the phosphorylation of the key molecules such as PTEN, RhoA, and VE-Cadherin; and also, the expression levels of S1PR2. In DENV2-infected AG129 mice treated with FTY720, there was significant improvement in weight gain, in overall clinical symptoms, and in survival. Whereas 100% of the DENV2-infected, untreated animals died by day-10 post-infection, 70% of the FTY720-treated animals were alive; and at the end of the 15-day post-infection observation period, 30% of them were still surviving. There was a significant reduction in the Evan's-blue dye permeability in the organs of FTY720-treated, DENV-2 infected animals; and also improvement in the hemogram, with complete restoration of thrombocytopenia and hepatic function. Our results show that the FDA-approved molecule Fingolimod (FTY720) is a promising therapeutic intervention in severe dengue.

Advent of phytobiologics and nano-interventions for bone remodeling: a comprehensive review.

Bone metabolism constitutes the intricate processes of matrix deposition, mineralization, and resorption. Any imbalance in these processes leads to traumatic bone injuries and serious disease conditions. Therefore, bone remodeling plays a crucial role during the regeneration process maintaining the balance between osteoblastogenesis and osteoclastogenesis. Currently, numerous phytobiologics are emerging as the new therapeutics for the treatment of bone-related complications overcoming the synthetic drug-based side effects. They can either target osteoblasts, osteoclasts, or both through different mechanistic pathways for maintaining the bone remodeling process. Although phytobiologics have been widely used since tradition for the treatment of bone fractures recently, the research is accentuated toward the development of osteogenic phytobioactives, constituent-based drug designing models, and efficacious delivery of the phytobioactives. To achieve this, different plant extracts and successful isolation of their phytoconstituents are critical for osteogenic research. Hence, this review emphasizes the phytobioactives based research specifically enlisting the plants and their constituents used so far as bone therapeutics, their respective isolation procedures, and nanotechnological interventions in bone research. Also, the review enlists the vast array of folklore plants and the newly emerging nano-delivery systems in treating bone injuries as the future scope of research in the phytomedicinal orthopedic applications.

Characterization of a recombinant factor IX molecule fused to coagulation factor XIII-B subunit.

INTRODUCTION AND AIM: Severe haemophilia B (HB) is characterized by spontaneous bleeding episodes, mostly into joints. Recurrent bleeds lead to progressive joint destruction called haemophilic arthropathy. The current concept of prophylaxis aims at maintaining the FIX level >3-5 IU/dL, which is effective at reducing the incidence of haemophilic arthropathy. Extended half-life FIX molecules make it easier to achieve these target trough levels compared to standard FIX concentrates. We previously reported that the fusion of a recombinant FIX (rFIX) to factor XIII-B (FXIIIB) subunit prolonged the half-life of the rFIX-LXa-FXIIIB fusion molecule in mice and rats 3.9- and 2.2-fold, respectively, compared with rFIX-WT. However, the mechanism behind the extended half-life was not known. MATERIALS AND METHODS: Mass spectrometry and ITC were used to study interactions of rFIX-LXa-FXIIIB with albumin. Pharmacokinetic analyses in fibrinogen-KO and FcRn-KO mice were performed to evaluate the effect of albumin and fibrinogen on in-vivo half-life of rFIX-LXa-FXIIIB. Finally saphenous vein bleeding model was used to assess in-vivo haemostatic activity of rFIX-LXa-FXIIIB. RESULTS AND CONCLUSION: We report here the key interactions that rFIX-LXa-FXIIIB may have in plasma are with fibrinogen and albumin which may mediate its prolonged half-life. In addition, using the saphenous vein bleeding model, we demonstrate that rFIX-FXIIIB elicits functional clot formation that is indistinguishable from that of rFIX-WT.

Genome-wide identification of bZIP transcription factor family in Artemisia annua, its transcriptional profiling and regulatory role in phenylpropanoid metabolism under different light conditions.

The basic leucine zipper (bZIP) protein transcription factors are known to modulate development, plant growth, metabolic response, and resistance to several biotic and abiotic stressors and have been widely studied in the model plant Arabidopsis thaliana. However, no comprehensive information about the bZIP transcription factor family in Artemisia annua has been explored to date. In this genome-wide study, we identified 61 bZIP TFs after removing false positives and incomplete sequences from Artemisia annua. Seven highly expressed homolog AabZIP TF genes under UV-B and differential light conditions in different tissues were identified from the publicly available microarray dataset as having their cis-regulatory elements involved in, flavonoids biosynthesis, seed-specific gene regulation, stress responses, and metabolic regulation. In-silico analysis and electrophoretic mobility shift assay (EMSA) confirmed the interaction of AabZIP19 TF over the AaPAL1 promoter in order to regulate the phenolics and flavonoid biosynthesis via the phenylpropanoid pathway. Further, RT-PCR analysis has been carried out to validate the transcript levels of selected AabZIP genes under white light, red light, blue light (45 min), and UV-B exposure (12 and 24 h). These genes have their highest expression levels under UV-B and blue light exposure, in contrast with white light. Therefore, the detection of ROS through staining confirms the accumulation of superoxide radicals and H2O2, and in addition to reducing ROS accumulation under UV-B and blue light irradiation, total phenols and flavonoids are significantly enhanced. This study laid the groundwork for deciphering the possible role of AabZIP TFs under different light stress-responsive conditions and in the regulation of secondary metabolism. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01338-0.

Association between CYP metabolizer phenotypes and selective serotonin reuptake inhibitors induced weight gain: a retrospective cohort study.

BACKGROUND: Prescription medications such as selective serotonin reuptake inhibitors (SSRIs), commonly used to treat depression, are associated with weight gain. The role of pharmacogenomics in predicting SSRI-induced weight gain is unclear. METHODS: In this retrospective cohort study from participants in the Mayo Clinic RIGHT study who were prescribed citalopram, paroxetine, sertraline, or fluoxetine, our aim was to evaluate the association of metabolizer phenotype and total body weight after 6 months of SSRIs initiation. We evaluated the metabolizer phenotypes (poor/intermediate, normal, and rapid/ultra-rapid) of the cytochromes P450 enzymes genes: CYP2C9, CYP2C19, and CYP2D6 known to influence the metabolism of SSRI medications: CYP2C19 for citalopram, CYP2D6 for paroxetine, CYP2D6 and CYP2C19 for sertraline, and CYP2D6 and CYP2C9 fluoxetine. In addition, we assessed the association of metabolizer phenotype and total body weight change at six months following SSRI prescription using parametric analysis of covariance adjusted for baseline body weight and multivariate regression models. RESULTS: CYP2C19 poor/intermediate metabolizers prescribed citalopram gained significantly more weight than normal or rapid/ultra-rapid metabolizers at 6 months (TBWG %: 2.6 [95% CI 1.3-4.1] vs. 0.4 [95% CI -0.5 - 1.3] vs. -0.1 [-95% CI -1.5-1.1]; p = 0.001). No significant differences in weight outcomes at six months of treatment with paroxetine, sertraline, or fluoxetine were observed by metabolizer status. CONCLUSIONS: Weight gain observed with citalopram may be mediated by CYP2C19 metabolizer status.

Multimodal Potentials of Gold Nanoparticles for Bone Tissue Engineering and Regenerative Medicine: Avenues and Prospects.

Osseous tissue repair has advanced due to the introduction of tissue engineering. The key elements required while engineering new tissues involve scaffolds, cells, and bioactive cues. The macrostructural to the nanostructural framework of such complex tissue has engrossed the intervention of nanotechnology for efficient neo-bone formation. Gold nanoparticles (GNPs) have recently gained interest in bone tissue regeneration due to their multimodal functionality. They are proven to modulate the properties of scaffolds and the osteogenic cells significantly. GNPs also influence different metabolic functions within the body, which directly or indirectly govern the mechanism of bone regeneration. Therefore, this review highlights nanoparticle-mediated osteogenic development, focusing on different aspects of GNPs ranging from scaffold modulation to cellular stimulation. The toxic aspects of gold nanoparticles studied so far are critically explicated, while further insight into the advancements and prospects of these nanoparticles in bone regeneration is also highlighted.

AMP-Activated Protein Kinase Restricts Zika Virus Replication in Endothelial Cells by Potentiating Innate Antiviral Responses and Inhibiting Glycolysis.

Viruses are known to perturb host cellular metabolism to enable their replication and spread. However, little is known about the interactions between Zika virus (ZIKV) infection and host metabolism. Using primary human retinal vascular endothelial cells and an established human endothelial cell line, we investigated the role of AMP-activated protein kinase (AMPK), a master regulator of energy metabolism, in response to ZIKV challenge. ZIKV infection caused a time-dependent reduction in the active phosphorylated state of AMPK and of its downstream target acetyl-CoA carboxylase. Pharmacological activation of AMPK using 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), metformin, and a specific AMPKα activator (GSK621) attenuated ZIKV replication. This activity was reversed by an AMPK inhibitor (compound C). Lentivirus-mediated knockdown of AMPK and the use of AMPKα-/- mouse embryonic fibroblasts provided further evidence that AMPK has an antiviral effect on ZIKV replication. Consistent with its antiviral effect, AMPK activation potentiated the expression of genes with antiviral properties (e.g., IFNs, OAS2, ISG15, and MX1) and inhibited inflammatory mediators (e.g., TNF-α and CCL5). Bioenergetic analysis showed that ZIKV infection evokes a glycolytic response, as evidenced by elevated extracellular acidification rate and increased expression of key glycolytic genes (GLUT1, HK2, TPI, and MCT4); activation of AMPK by AICAR treatment reduced this response. Consistent with this, 2-deoxyglucose, an inhibitor of glycolysis, augmented AMPK activity and attenuated ZIKV replication. Thus, our study demonstrates that the anti-ZIKV effect of AMPK signaling in endothelial cells is mediated by reduction of viral-induced glycolysis and enhanced innate antiviral responses.

Cell type-specific transcriptomics identifies neddylation as a novel therapeutic target in multiple sclerosis.

Multiple sclerosis is an autoimmune disease of the CNS in which both genetic and environmental factors are involved. Genome-wide association studies revealed more than 200 risk loci, most of which harbour genes primarily expressed in immune cells. However, whether genetic differences are translated into cell-specific gene expression profiles and to what extent these are altered in patients with multiple sclerosis are still open questions in the field. To assess cell type-specific gene expression in a large cohort of patients with multiple sclerosis, we sequenced the whole transcriptome of fluorescence-activated cell sorted T cells (CD4+ and CD8+) and CD14+ monocytes from treatment-naive patients with multiple sclerosis (n = 106) and healthy subjects (n = 22). We identified 479 differentially expressed genes in CD4+ T cells, 435 in monocytes, and 54 in CD8+ T cells. Importantly, in CD4+ T cells, we discovered upregulated transcripts from the NAE1 gene, a critical subunit of the NEDD8 activating enzyme, which activates the neddylation pathway, a post-translational modification analogous to ubiquitination. Finally, we demonstrated that inhibition of NEDD8 activating enzyme using the specific inhibitor pevonedistat (MLN4924) significantly ameliorated disease severity in murine experimental autoimmune encephalomyelitis. Our findings provide novel insights into multiple sclerosis-associated gene regulation unravelling neddylation as a crucial pathway in multiple sclerosis pathogenesis with implications for the development of tailored disease-modifying agents.

Exploring Diverse Coagulation Factor XIII Subunit Expression Datasets: A Bioinformatic Analysis.

Coagulation factor XIII (FXIII) circulates in plasma as a pro-transglutaminase heterotetrameric complex (FXIIIA2B2), which upon activation by thrombin and calcium covalently crosslinks preformed fibrin polymers. The heterotetrameric complex is composed of a catalytic FXIIIA2 subunit and a protective/regulatory FXIII-B2 subunit coded by F13A1 and F13B genes, respectively. The catalytic FXIIIA2 subunit is encoded by the F13A1 gene, expressed primarily in cells of mesenchymal origin, whereas the FXIIIB subunit encoded by the F13B gene is expressed and secreted from hepatocytes. The plasma FXIIIA2 subunit, which earlier was believed to be secreted from cells of megakaryocytic lineage, is now understood to result primarily from resident macrophages. The regulation of the FXIII subunits at the genetic level is still poorly understood. The current study adopts a purely bioinformatic approach to analyze the temporal, time-specific expression array-data corresponding to both the subunits in specific cell lineages, with respect to the gene promoters. We analyze the differentially expressed genes correlated with F13A1 and F13B expression levels in an array of cell types, utilizing publicly available microarray data. We attempt to understand the regulatory mechanism underlying the variable expression of FXIIIA2 subunit in macrophages (M0, M1, M2 and aortic resident macrophages). Similarly, the FXIIIB2 subunit expression data from adult, fetal hepatocytes and embryonic stem cells derived hepatoblasts (hESC-hepatoblast) was analyzed. The results suggest regulatory dependence between the two FXIII subunits at the transcript level. Our analysis also predicts the involvement of the FXIIIA2 subunit in macrophage polarization, plaque stability, and inflammation.

Dynamic coupling analysis on plant sesquiterpene synthases provides leads for the identification of product specificity determinants.

Sesquiterpene synthases catalyse cyclisation of farnesyl pyrophosphate to produce diverse sesquiterpenes. Despite utilising the same substrate and exhibiting significant sequence and structural homology, these enzymes form different products. Previous efforts were based on identifying the effect of divergent residues present at the catalytic binding pocket on the product specificity of these enzymes. However, the rationales deduced for the product specificity from these studies were not generic enough to be applicable to other phylogenetically distant members of this family. To address this problem, we have developed a novel approach combining sequence, structural and dynamical information of plant sesquiterpene synthases (SSQs) to predict product modulating residues (PMRs). We tested this approach on the SSQs with known PMRs and also on sesquisabinene synthase 1 (SaSQS1), a SSQ from Indian sandalwood. Our results show that the dynamical sectors of SSQs obtained from molecular dynamics simulation and their hydrophobicity and vicinity indices together provide leads for the identification of PMRs. The efficacy of the technique was tested on SaSQS1 using mutagenesis. To the best of our knowledge, this is a first technique of this kind which provides cues on PMRs of SSQs, with divergent phylogenetic relationship.

Resveratrol isomeric switching during bioreduction of gold nanoparticles: a gateway for cis-resveratrolArchita.

Resveratrol, a polyphenolic and biocompatible molecule, exhibits significant pharmacological effects but has poor bioavailability and metabolic stability. It appears in two isomeric forms trans-(E)-resveratrol (tRes) and cis-(Z)-resveratrol (cRes). Many pharmacological activities studied so far are of tRes and is the most stable, predominant, and natural form. cRes is not commercially available due to difficulty in its purification and hence not explored much for its biological activities. Therefore, our study focuses on investigating the stability and therapeutic potential of cRes through its bio-conjugation to nanomaterial. In this study, tRes reduces gold ions to gold nanoparticles (GNPs) and itself gets oxidized to its isomeric form cRes. The isomeric switching was evidenced through cRes characteristic spectral differences and chromatographic elution pattern. The monodispersed GNPs of 25.6 ± 0.4 nm size was formed having zeta potential of -19 ± 3.82 mV confirming it to be a stable formulation. The stability studies were further extended to be tested under different physiological fluids. The cRes loaded GNPs (cRGNPs) reflecting the biological activity of cRes presented equivalent antioxidant property to that of tRes even at low concentrations. Also, cRGNPs showed the hemocompatibility by presenting no hemotoxicity and simultaneous in vitro anti-hemolytic activity. Therefore, the stability provided to cRes upon conjugating to GNPs can further be exploited to study the biological activities of cRes through its nano-conjugated delivery.

Gut bacteria from multiple sclerosis patients modulate human T cells and exacerbate symptoms in mouse models.

The gut microbiota regulates T cell functions throughout the body. We hypothesized that intestinal bacteria impact the pathogenesis of multiple sclerosis (MS), an autoimmune disorder of the CNS and thus analyzed the microbiomes of 71 MS patients not undergoing treatment and 71 healthy controls. Although no major shifts in microbial community structure were found, we identified specific bacterial taxa that were significantly associated with MS. Akkermansia muciniphila and Acinetobacter calcoaceticus, both increased in MS patients, induced proinflammatory responses in human peripheral blood mononuclear cells and in monocolonized mice. In contrast, Parabacteroides distasonis, which was reduced in MS patients, stimulated antiinflammatory IL-10-expressing human CD4+CD25+ T cells and IL-10+FoxP3+ Tregs in mice. Finally, microbiota transplants from MS patients into germ-free mice resulted in more severe symptoms of experimental autoimmune encephalomyelitis and reduced proportions of IL-10+ Tregs compared with mice "humanized" with microbiota from healthy controls. This study identifies specific human gut bacteria that regulate adaptive autoimmune responses, suggesting therapeutic targeting of the microbiota as a treatment for MS.

Role of Somatostatin in the Regulation of Central and Peripheral Factors of Satiety and Obesity.

Obesity is one of the major social and health problems globally and often associated with various other pathological conditions. In addition to unregulated eating behaviour, circulating peptide-mediated hormonal secretion and signaling pathways play a critical role in food intake induced obesity. Amongst the many peptides involved in the regulation of food-seeking behaviour, somatostatin (SST) is the one which plays a determinant role in the complex process of appetite. SST is involved in the regulation of release and secretion of other peptides, neuronal integrity, and hormonal regulation. Based on past and recent studies, SST might serve as a bridge between central and peripheral tissues with a significant impact on obesity-associated with food intake behaviour and energy expenditure. Here, we present a comprehensive review describing the role of SST in the modulation of multiple central and peripheral signaling molecules. In addition, we highlight recent progress and contribution of SST and its receptors in food-seeking behaviour, obesity (orexigenic), and satiety (anorexigenic) associated pathways and mechanism.

Exploring the structural similarity yet functional distinction between coagulation factor XIII-B and complement factor H sushi domains.

Coagulation factor XIII (FXIII) covalently crosslinks pre-formed fibrin clots preventing their premature fibrinolysis. In plasma, FXIII circulates as a zymogenic heterotetramer composed of catalytic FXIII-A subunits, and carrier/regulatory FXIII-B subunits. FXIII-A is a well characterized component of this complex, and has been associated with several pleiotropic roles outside coagulation as well. In comparison only protective/regulatory roles towards the FXIII-A subunit have been identified for FXIII-B. Strong homology between FXIII-B and complement regulator Complement factor H suggests a putative role of FXIII-B in complement activation. In the current study we have analyzed the similarities and yet functional divergence of these two proteins using in silico sequence alignment and structural analysis. We have evaluated complement activation post reconstitution of FXIII components into FXIII deficient and CFH deficient plasma. We have also transiently expressed FXIII-B in SH-SY5Y cell lines and evaluated its effect on the endogenous complement activation. Our investigations show no effect of FXIII-B subunit on the rate of complement activation. Therefore we conclude that at a physiological level, FXIII-B subunit plays no role in the complement system, although a vestigial function in altered pathological states might still exist.

Disruption of Structural Disulfides of Coagulation FXIII-B Subunit; Functional Implications for a Rare Bleeding Disorder.

Congenital FXIII deficiency is a rare bleeding disorder in which mutations are detected in F13A1 and F13B genes that express the two subunits of coagulation FXIII, the catalytic FXIII-A, and protective FXIII-B. Mutations in FXIII-B subunit are considerably rarer compared to FXIII-A. Three mutations in the F13B gene have been reported on its structural disulfide bonds. In the present study, we investigate the structural and functional importance of all 20 structural disulfide bonds in FXIII-B subunit. All disulfide bonds were ablated by individually mutating one of its contributory cysteine's, and these variants were transiently expressed in HEK293t cell lines. The expression products were studied for stability, secretion, the effect on oligomeric state, and on FXIII-A activation. The structural flexibility of these disulfide bonds was studied using classical MD simulation performed on a FXIII-B subunit monomer model. All 20 FXIII-B were found to be important for the secretion and stability of the protein since ablation of any of these led to a secretion deficit. However, the degree of effect that the disruption of disulfide bond had on the protein differed between individual disulfide bonds reflecting a functional hierarchy/diversity within these disulfide bonds.

Identification of Potential Novel Interacting Partners for Coagulation Factor XIII B (FXIII-B) Subunit, a Protein Associated with a Rare Bleeding Disorder.

Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.