Identification of Biomarkers for Systemic Distribution of Nanovesicles From Lactobacillus johnsonii N6.2

The ability of bacterial extracellular vesicles (EV) to transport biological molecules has increased the research to determine their potential as therapeutic agents. In this study, Lactobacillus johnsonii N6.2-derived nanovesicles (NV) were characterized to identify components that may serve as biomarkers in host-microbe interactions. Comparative proteomic and lipidomic analyses of L. johnsonii N6.2 NV and cell membrane (CM) were performed. The lipidomic profiles indicated that both fractions contained similar lipids, however, significant differences were observed in several classes. LC-MS/MS proteomic analysis indicated that NV contained several unique and differentially expressed proteins when compared to the CM. Analysis of Gene Ontology (GO) terms, based on cellular component, showed significant enrichment of proteins in the cytoplasm/intracellular space category for the NV fraction. Based on these results, the proteins T285_RS00825 (named Sdp), Eno3 and LexA were selected for studies of localization and as potential biomarkers for host-microbe interactions. Immunogold staining, followed by scanning and transmission electron microscopy (SEM and TEM, respectively), revealed that Sdp was preferentially localized along the cell wall/membrane, and on NV-like structures surrounding the bacteria. These results were confirmed using immunofluorescence staining in Caco-2 cells incubated with NV. Consequently, we evaluated the potential for NV surface-exposed proteins to generate an immune response in the host. Plasma from individuals administered L. johnsonii N6.2 showed that IgA and IgG antibodies were generated against NV and Sdp domains in vivo. Altogether, these results show that L. johnsonii N6.2 NV have the potential to mediate host interactions through immune modulation.

Mitochondrial dysfunction associated with autophagy and mitophagy in cerebrospinal fluid cells of patients with delayed cerebral ischemia following subarachnoid hemorrhage

Decreased mitochondrial membrane potential in cerebrospinal fluid (CSF) was observed in patients with subarachnoid hemorrhage (SAH) accompanied by delayed cerebral ischemia (DCI). However, whether abnormal mechanisms of mitochondria are associated with the development of DCI has not been reported yet. Under cerebral ischemia, mitochondria can transfer into the extracellular space. Mitochondrial dysfunction can aggravate neurologic complications. The objective of this study was to evaluate whether mitochondrial dysfunction might be associated with autophagy and mitophagy in CSF cells to provide possible insight into DCI pathogenesis. CSF samples were collected from 56 SAH patients (DCI, n = 21; and non-DCI, n = 35). We analyzed CSF cells using autophagy and mitophagy markers (DAPK1, BNIP3L, BAX, PINK1, ULK1, and NDP52) via qRT-PCR and western blotting of proteins (BECN1, LC3, and p62). Confocal microscopy and immunogold staining were performed to demonstrate the differentially expression of markers within dysfunctional mitochondria. Significant induction of autophagic flux with accumulation of autophagic vacuoles, increased expression of BECN1, LC3-II, and p62 degradation were observed during DCI. Compared to non-DCI patients, DCI patients showed significantly increased mRNA expression levels (2−ΔCt) of DAPK1, BNIP3L, and PINK1, but not BAX, ULK1, or NDP52. Multivariable logistic regression analysis revealed that Hunt and Hess grade ≥ IV (p = 0.023), DAPK1 (p = 0.003), and BNIP3L (p = 0.039) were related to DCI. Increased mitochondrial dysfunction associated with autophagy and mitophagy could play an important role in DCI pathogenesis.

Human Platelets Take up Anti-VEGF Agents

Purpose. Growing evidence suggests different systemic exposure of anti-vascular endothelial growth factor (anti-VEGF) agents with repeated intravitreal application. Since the penetration of anti-VEGF agents through vascular barrier was reported, the interaction of anti-VEGF with nonresident platelets has become a topic of interest. The purpose of this study was to evaluate, with the help of visualization techniques, whether platelets take up the anti-VEGF agents ranibizumab, aflibercept, and bevacizumab. Methods. The uptake of anti-VEGF agents with or without VEGF treatment was investigated using immunofluorescence and immunogold staining in human platelets. The role of actin filaments and clathrin-coated vesicles in the transport of ranibizumab, aflibercept, and bevacizumab was evaluated by two pharmacologic inhibitors: staurosporine (protein kinase C inhibitor) and cytochalasin D. Results. All three anti-VEGF agents were taken up by platelets and colocalized with VEGF. Ranibizumab and aflibercept were mainly detected in alpha-granules; however, bevacizumab was equally localized in alpha-granules and in platelet vesicles. Both staurosporine and cytochalasin D completely inhibited the uptake of aflibercept into platelets. Both pharmacological inhibitors also decreased the transport of ranibizumab and bevacizumab into platelets. Bevacizumab was significantly more frequently colocalized within clathrin-coated vesicles than ranibizumab and aflibercept. Conclusion. All three anti-VEGF agents are taken up by platelets and internalized in alpha-granules, which may result in a higher local exposure of anti-VEGF after the activation of platelets, potentially contributing to arterial thromboembolic events. Clathrin-coated vesicles seem to be more prominent in the transport of bevacizumab than ranibizumab and aflibercept. Nevertheless, whether the different localization and transport of bevacizumab are truly related to specific differences of receptor-mediated endocytosis has to be revealed by further research.

MO057REGIONAL IMPORTANCE OF FIBRINOGEN A Α-CHAIN MUTATION ON RENAL AMYLOIDOSIS

Background and Aims Renal amyloidosis include amyloid A (AA) and light chain (AL) as well as amyloidogenic leukocyte chemotactic factor 2 (ALECT2) and numerous hereditary forms. After identifying amyloidosis by its suggestive pale pink amorphous appearance in optic microscopy (OM) and Congo red positivity, a correct diagnosis of the amyloidogenic precursor protein is determinant to establish prognosis and treatment. Immunohistochemistry (IHC) and immunofluorescence (IF) studies use a limited number of antibodies to detect specific epitopes and may be difficult to interpret. The gold standard has become proteomics but laser microdissection and mass spectroscopy are not routinely available. Other options include electron microscopy with immunogold staining and complementary exams such as scintigraphy with 99mTc-DPD to detect transthyretin-related amyloidosis. Since the cause of amyloidosis vary among regions, analyzing local patterns can help establish a diagnostic procedure. We aimed at describing cases of kidney amyloidosis identified by biopsy during a 4-year interval and discuss possible implication for future diagnosis. Method We analyzed our kidney biopsy database and selected all cases of renal amyloidosis collecting clinical, laboratory and imaging data. Results From January 2016 until December 2019, 194 kidney biopsies were performed at the Hospital of Braga in the Portuguese province of Minho. Among these, 8 (4.1%) revealed amyloidosis. Mean age was 63.8±9.2 years of age, 5 were female, 6 were referred for nephrotic syndrome and 2 for what seemed like acute kidney injury. Mean creatinine at presentation was 3.2±2.3mg/dL. Among them, 2 had AL amyloidosis with light chain restriction by IF, 1 had AA amyloidosis with intense IHC stain and 5 patients had non-AL and non-AA forms of amyloidosis. Of these, 3 had probable fibrinogen A alpha-chain (AFib) amyloidosis, after a heterozygous mutation FGA p.Glu545Val was detected, 1, who did not have IHC performed, was assumed as having AA amyloidosis due to a history of serious recurrent infections and 1 is still under study. Four performed scintigraphy with 99mTc-DPD which was negative. The 2 patients with AL amyloidosis had, by OM, in one case glomerular and tubulointerstitial and on the other, glomerular and vascular involvement and, by IF, both had k light chain restriction. Both had additional cardiac and neurovegetative involvement, were treated with cyclophosphamide-bortezomib-dexamethasone and oral doxycycline with complete hematologic response and stabilization of kidney function. In 1 case, proteinuria only showed a slow decline 2 years after treatment. The 2 patients with AA amyloidosis had glomerular, vascular and tubular deposits. One had bronchiectasis and allergic bronchopulmonary aspergillosis and the other had common variable immunodeficiency with recurrent gastrointestinal and urinary infections with frequent bacteremia. None of them had confirmed extra-renal involvement, although the latter had hepatic fibrosis awaiting biopsy. Both progressed to dialysis soon after diagnosis. The 3 patients with AFib amyloidosis all had glomerular amyloidosis with additional amyloid deposition at tubular, vascular and both respectively. Two had had an increased creatinine and subnephrotic proteinuria for some years whereas 1 had kidney function decline and nephrotic syndrome in the course of few months. All were hypertensive and none had evident extra-renal deposits. Conclusion Identifying the amyloidogenic precursor may be difficult. Algorithms for diagnosis may vary according to local prevalence of specific types and available resources. AFib amyloidosis was very significant in our series. It was also described in 4.5% of hemodialysis patients in the district of Braga making it one of the first causes of amyloidosis in our region. This high prevalence may justify early genetic testing for the specific mutation in non-AL and non-AA forms.

Increased Mitochondrial Dysfunction Associated with Autophagy and Mitophagy in Cerebrospinal Fluid Cells Following Subarachnoid Hemorrhage in Patients with Delayed Cerebral Ischemia

Decreased mitochondrial membrane potential in cerebrospinal fluid (CSF) was observed in patients with subarachnoid hemorrhage (SAH) accompanied with delayed cerebral ischemia (DCI); however, the underlying mechanism remains unclear. We evaluated the mitochondrial dysfunction associated with autophagy and mitophagy in CSF cells for possible insight into DCI pathogenesis. CSF samples were collected from 56 SAH patients (DCI, n=21; and non-DCI, n=35). We analyzed CSF cells using autophagy and mitophagy markers (DAPK1, BNIP3L, BAX, PINK1, ULK1, and NDP52) via qRT-PCR and western blotting of proteins (BECN1, LC3, and p62). Confocal microscopy and immunogold staining were performed to demonstrate the differentially expression of markers within dysfunctional mitochondria. Significant induction of autophagic flux with accumulation of autophagic vacuoles, increased expression of BECN1, LC3-II, and p62 degradation were observed during DCI. DCI patients showed a significantly increased mRNA expression (2-ΔCt) than non-DCI patients: DAPK1, 0.279 (0.220–0.297) in DCI vs. 0.043 (0.021–0.086) in non-DCI; BNIP3L, 0.134 (0.060–0.202) in DCI vs. 0.045 (0.020–0.101) in non-DCI; and PINK1, 0.064 (0.044–0.810) in DCI vs. 0.045 (0.012–0.063) in non-DCI. Other markers including BAX, ULK1, and NDP52 did not differ significantly. The vWF-positive CSF cells showed a colocalization with antibodies for DAPK1, BNIP3L/NIX, PINK1, and BECN1 with dysfunctional mitochondria. Increased dysfunctional mitochondria associated with autophagy and mitophagy are closely associated with DCI pathogenesis.

mTOR Modulates Methamphetamine-Induced Toxicity through Cell Clearing Systems

Methamphetamine (METH) is abused worldwide, and it represents a threat for public health. METH exposure induces a variety of detrimental effects. In fact, METH produces a number of oxidative species, which lead to lipid peroxidation, protein misfolding, and nuclear damage. Cell clearing pathways such as ubiquitin-proteasome (UP) and autophagy (ATG) are involved in METH-induced oxidative damage. Although these pathways were traditionally considered to operate as separate metabolic systems, recent studies demonstrate their interconnection at the functional and biochemical level. Very recently, the convergence between UP and ATG was evidenced within a single organelle named autophagoproteasome (APP), which is suppressed by mTOR activation. In the present research study, the occurrence of APP during METH toxicity was analyzed. In fact, coimmunoprecipitation indicates a binding between LC3 and P20S particles, which also recruit p62 and alpha-synuclein. The amount of METH-induced toxicity correlates with APP levels. Specific markers for ATG and UP, such as LC3 and P20S in the cytosol, and within METH-induced vacuoles, were measured at different doses and time intervals following METH administration either alone or combined with mTOR modulators. Western blotting, coimmunoprecipitation, light microscopy, confocal microscopy, plain transmission electron microscopy, and immunogold staining were used to document the effects of mTOR modulation on METH toxicity and the merging of UP with ATG markers within APPs. METH-induced cell death is prevented by mTOR inhibition, while it is worsened by mTOR activation, which correlates with the amount of autophagoproteasomes. The present data, which apply to METH toxicity, are also relevant to provide a novel insight into cell clearing pathways to counteract several kinds of oxidative damage.

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane

ABSTRACT Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins, such as HA and M2, inherently cocluster within the membrane, although M2 is found mostly at the periphery of regions of HA, consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza virus proteins, such as M2 and NP, appears to be brokered by additional influenza virus proteins, in this case M1. HA and NA, while raft associated, reside in distinct domains, reflecting their distributions in the viral membrane.

Cell Division by Longitudinal Scission in the Insect EndosymbiontSpiroplasma poulsonii

ABSTRACTSpiroplasmabacteria are highly motile bacteria with no cell wall and a helical morphology. This clade includes many vertically transmitted insect endosymbionts, includingSpiroplasma poulsonii, a natural endosymbiont ofDrosophila melanogaster.S. poulsoniibacteria are mainly found in the hemolymph of infected female flies and exhibit efficient vertical transmission from mother to offspring. As is the case for many facultative endosymbionts,S. poulsoniican manipulate the reproduction of its host; in particular,S. poulsoniiinduces male killing inDrosophila melanogaster. Here, we analyze the morphology ofS. poulsoniiobtained from the hemolymph of infectedDrosophila. This endosymbiont was not only found as long helical filaments, as previously described, but was also found in a Y-shaped form. The use of electron microscopy, immunogold staining of the FtsZ protein, and antibiotic treatment unambiguously linked the Y shape ofS. poulsoniito cell division. Observation of the Y shape in anotherSpiroplasma,S. citri, and anecdotic observations from the literature suggest that cell division by longitudinal scission might be prevalent in theSpiroplasmaclade. Our study is the first to report the Y-shape mode of cell division in an endosymbiotic bacterium and addsSpiroplasmato the so far limited group of bacteria known to utilize this cell division mode.IMPORTANCEMost bacteria rely on binary fission, which involves elongation of the bacteria and DNA replication, followed by splitting into two parts. Examples of bacteria with a Y-shape longitudinal scission remain scarce. Here, we report thatSpiroplasma poulsonii, an endosymbiotic bacterium living inside the fruit flyDrosophila melanogaster, divide with the longitudinal mode of cell division. Observations of the Y shape in anotherSpiroplasma,S. citri, suggest that this mode of scission might be prevalent in theSpiroplasmaclade.Spiroplasmabacteria are wall-less bacteria with a distinctive helical shape, and these bacteria are always associated with arthropods, notably insects. Our study raises the hypothesis that this mode of cell division by longitudinal scission could be linked to the symbiotic mode of life of these bacteria.

A Novel β3 Intracytoplasmic Domain Mutation Associated with Macrothrombocytopenia: Structural Analysis in Comparison with Previously Reported Cases

Background: Glanzmann thrombasthenia (GT) is a moderate to severe inherited bleeding syndrome associating a lack of platelet aggregation with quantitative or qualitative deficiencies of the αIIbβ3 integrin. Rare patients with mutations in ITGA2B or ITGB3 genes show a variable GT-like phenotype associated with macrothrombocytopenia (MTP). Gain-of-function mutations that localize to the integrin cytoplasmic domains cause autosomal dominant MTP. We now report a novel heterozygous ΔLeu in ITGB3 causing a structural modification of the cytoplasmic domain of β3 in a family with moderate MTP. In silico modeling was performed to obtain structural insights into this and other known mutations affecting cytoplasmic domains of αIIb and β3 and impacting megakaryopoiesis. Aim: To help define structural changes in αIIbβ3 linked to MTP. Results: A novel in-frame heterozygous 3bp deletion (c.2230_2232delCTC) was identified by exome sequencing performed as part of the BRIDGE-BPD consortium project in a French pedigree with lifelong mucocutaneous bleeding. The consequence is the loss of β3 Leu718 (ΔL718) the second L of a LLITIHD motif of the mature protein (nomenclature used throughout). Platelets of the proband have (i) moderate expression of αIIbβ3, (ii) a major reduction in fibrinogen (Fg) and PAC-1 binding after ADP stimulation yet (iii) spontaneous Fg or PAC-1 binding to platelets was not seen. Western blotting showed a slightly slower migration of β3 and normal αIIb. The proband had a moderate MTP with platelet size heterogeneity and frequent enlarged α-granules regrouping several of normal size with Fg bound to the membrane of the α-granules as revealed by immunogold staining and electron microscopy. Platelet aggregation was much reduced with all physiologic agonists including TRAP with a reduced surface expression of P-selectin and diminished secretion. The same 3bp del in ITGB3 co-segregates with the platelet phenotype and bleeding across 3 generations in the family. We then performed molecular modeling (PyMol Molecular Graphics System version 1.3) to compare the structural effects of this mutation on αIIbβ3 with other cytoplasmic tail mutations described in the literature giving GT-like phenotypes and/or MTP. Firstly αIIb-R995Q/W or β3-D723H were confirmed to disrupt a salt bridge between the integrin subunits and induce the separation of their tails. PyMol modeling showed that the DL718 results in D723 being no longer in phase with R995 thereby disrupting the salt bridge. Significantly, introduction of a β-turn by an activating β3 L718P substitution again results in the spontaneous separation of the tails, once more through a conformational change of the mutated β3. In contrast, a previously reported ITGB3 C2268T variant resulting in Arg724*, a truncation coming after the deletion in our patient, or a β3 S752P substitution that completely blocks platelet aggregation and PAC-1 binding (and specific loss of kindlin-3 binding) despite a capacity to secrete a storage pool of Fg bound α-granule αIIbβ3 failed to give MTP despite resulting in a GT-like syndrome. Conclusions: The novel ITGB3 ΔL718 of β3 showed an intermediate phenotype with MTP, a much decreased platelet aggregation, a reduced activity of αIIbβ3 at the platelet surface but a progressive fusion of α-granules. This is despite the theoretical retention of the talin and kindlin-3 binding sites suggesting that transmembrane signaling is the principle defect. Our studies highlight how considerable phenotypic heterogeneity exists in patients with genetic variants affecting αIIb and β3 cytoplasmic domains and suggest different signaling mechanisms for surface and intracellular αIIbβ3 pools. *PN and ET; WHO and ATN made equal contributions, On behalf of the BRIDGE-Bleeding and Platelet Disorder Consortium Disclosures No relevant conflicts of interest to declare.