Deletion of junctional adhesion molecule A from platelets increases early‐stage neointima formation after wire injury in hyperlipidemic mice

Platelets play an important role in the pathogenesis of vascular remodelling after injury. Junctional adhesion molecule A (JAM‐A) was recently described to regulate platelet activation. Specific deletion of JAM‐A from platelets resulted in increased reactivity and in accelerated progression of atherosclerosis. The aim of this study was to investigate the specific contribution of platelet‐derived JAM‐A to neointima formation after vascular injury. Mice with or without platelet‐specific (tr)JAM‐A‐deficiency in an apolipoprotein e (apoe^?/?) background underwent wire‐induced injury of the common carotid artery. Ex vivo imaging by two‐photon microscopy revealed increased platelet coverage at the site of injury in trJAM‐A‐deficient mice. Cell recruitment assays showed increased adhesion of monocytic cells to activated JAM‐A‐deficient platelets than to control platelets. Inhibition of α_Mβ₂ or GPIbα, but not of CD62P, suppressed those differences. Up to 4 weeks after wire injury, intimal neoplasia and neointimal cellular content were analysed. Neointimal lesion area was increased in trJAM‐A^?/? apoe^?/? mice and the lesions showed an increased macrophage accumulation and proliferating smooth muscle cells compared with trJAM‐A^+/+ apoe^?/? littermates 2 weeks, but not 4 weeks after injury. Re‐endothelialization was decreased in trJAM‐A^?/? apoe^?/? mice compared with controls 2 weeks after injury, yet it was complete in both groups after 4 weeks. A platelet gain of function by deletion of JAM‐A accelerates neointima formation only during earlier phases after vascular injury, through an increased recruitment of mononuclear cells. Thus, the contribution of platelets might become less important when neointima formation progresses to later stages.     

Alkalispirillum mobile gen. nov., spec. nov., an alkaliphilic non-phototrophic member of the Ectothiorhodospiraceae

From cultures of the anoxygenic phototroph Halorhodospira halophila SL-1, an aerobic, gram-negative spirillum was isolated. This moderately halophilic, alkaliphilic bacterium was motile by means of a single polar flagellum. It is described here as Alkalispirillum mobile gen. nov., spec. nov. Phylogenetic analysis of the Alkalispirillum mobile 16S rRNA gene led to its classification in the gamma-subclass of the Proteobacteria, as it appears closely related to phototrophic purple sulfur bacteria of the genera Ectothiorhodospira and Halorhodospira. Surprisingly, A. mobile is an obligate aerobe. The organism grows optimally with a number of carboxylic acids (such as sodium acetate) as carbon source, at 2% (i.e. approximately 0.34 M) sodium chloride, at pH 9-10, and at temperatures ranging from 35 to 38 degrees C. The dominant cellular fatty acids of Alkalispirillum mobile are C12:0, C16:0, C18:1cis11, and C18:0; its G+C content is 66.2+/-0.5 mol%.     

IDH1/2 Mutations in Cancer Stem Cells and Their Implications for Differentiation Therapy

Isocitrate dehydrogenase 1 and 2 (IDH1/2) are enzymes recurrently mutated in various types of cancer, including glioma, cholangiocarcinoma, chondrosarcoma, and acute myeloid leukemia. Mutant IDH1/2 induce a block in differentiation and thereby contribute to the stemness and oncogenesis of their cells of origin. Recently, small-molecule inhibitors of mutant IDH1/2 have been Food and Drug Administration–approved for the treatment of IDH1/2-mutated acute myeloid leukemia. These inhibitors decrease the stemness of the targeted IDH1/2-mutated cancer cells and induce their differentiation to more mature cells. In this review, we elucidate the mechanisms by which mutant IDH1/2 induce a block in differentiation and the biological and clinical effects of the release into differentiation by mutant-IDH1/2 inhibitors. (J Histochem Cytochem 70:83–97, 2022)     

Target Specificity of the E3 Ligase LUBAC for Ubiquitin and NEMO Relies on Different Minimal Requirements

The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin.     

Quantitative resistance differences between and within natural populations of Solanum chilense against the oomycete pathogen Phytophthora infestans

The wild tomato species Solanum chilense is divided into geographically and genetically distinct populations that show signs of defense gene selection and differential phenotypes when challenged with several phytopathogens, including the oomycete causal agent of late blight Phytophthora infestans. To better understand the phenotypic diversity of this disease resistance in S. chilense and to assess the effect of plant genotype versus pathogen isolate, respectively, we evaluated infection frequency in a systematic approach and with large sample sizes. We studied 85 genetically distinct individuals representing nine geographically separated populations of S. chilense. This showed that differences in quantitative resistance can be observed between but also within populations at the level of individual plants. Our data also did not reveal complete immunity in any of the genotypes. We further evaluated the resistance of a subset of the plants against P. infestans isolates with diverse virulence properties. This confirmed that the relative differences in resistance phenotypes between individuals were mainly determined by the plant genotype under consideration with modest effects of pathogen isolate used in the study. Thus, our report suggests that the observed quantitative resistance against P. infestans in natural populations of a wild tomato species S. chilense is the result of basal defense responses that depend on the host genotype and are pathogen isolate‐unspecific.     

Evaluating Contextual Processing in Diffusion MRI: Application to Optic Radiation Reconstruction for Epilepsy Surgery

Diffusion MRI and tractography allow for investigation of the architectural configuration of white matter in vivo, offering new avenues for applications like presurgical planning. Despite the promising outlook, there are many pitfalls that complicate its use for (clinical) application. Amongst these are inaccuracies in the geometry of the diffusion profiles on which tractography is based, and poor alignment with neighboring profiles. Recently developed contextual processing techniques, including enhancement and well-posed geometric sharpening, have shown to result in sharper and better aligned diffusion profiles. However, the research that has been conducted up to now is mainly of theoretical nature, and so far these techniques have only been evaluated by visual inspection of the diffusion profiles. In this work, the method is evaluated in a clinically relevant application: the reconstruction of the optic radiation for epilepsy surgery. For this evaluation we have developed a framework in which we incorporate a novel scoring procedure for individual pathways. We demonstrate that, using enhancement and sharpening, the extraction of an anatomically plausible reconstruction of the optic radiation from a large amount of probabilistic pathways is greatly improved in three healthy controls, where currently used methods fail to do so. Furthermore, challenging reconstructions of the optic radiation in three epilepsy surgery candidates with extensive brain lesions demonstrate that it is beneficial to integrate these methods in surgical planning.     

Hinge-Region O-Glycosylation of Human Immunoglobulin G3 (IgG3)

Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out.For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes.Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and represents approximately three-quarters of the total serum immunoglobulin content (1). As the main mediator of humoral immunity and an important link between the adaptive and innate immune system, IgG is a fundamental component of the immune system. IgG consists of two heavy and light chains, linked by disulfide bonds. The protein can be subdivided into the antigen-binding (Fab) and the receptor-binding (Fc) region. There are four subclasses of IgG, all of which share an overall structure homology but differ slightly in their amino acid sequence; the quantity of the subclasses in human serum is as follows: IgG1 > 2 > 3 > 4 (2).IgG3 represents ∼8% of the total amount of IgG in human serum (2), and stands out from the other IgG subclasses for a number of reasons. First of all, IgG3 contains an elongated hinge region with up to a triple repeat sequence (the actual number ranging from one to three depending on the allotype (3)), which is responsible for the increased flexibility between the Fab and the Fc part, as well as the wider and more flexible angle between the two Fab arms (4, 5). This flexibility is likely the cause of the increased affinity of IgG3, compared with the other subclasses, for divalent binding to certain types of antigens (4, 6, 7). Second, IgG3 has a higher affinity for C1q, which initiates the classical complement pathway (5, 8). The interaction between IgG3 and C1q is not due to the elongated hinge region, as demonstrated by studies showing that recombinant IgG3 with an IgG1- or IgG4-like hinge sequence exhibited even greater binding affinity for C1q than wild-type IgG3 (8–10). Third, IgG3 has a higher overall affinity for the Fcγ receptors (FcγRs), through which it can influence effector cells of the innate immune system (11). The CH2 domain and hinge region of IgG3 were shown to be instrumental in binding to the high affinity FcγRI receptor (12). Finally, IgG3 generally has a shorter half-life compared with the other IgG subclasses (1 versus 3 weeks) (2). This difference was traced back to an H435R mutation that confers a positive charge at physiological pH, resulting in a decreased binding to the neonatal Fc receptor (FcRn), which is involved in recycling IgG targeted for lysosomal degradation (13). The low-efficiency FcRn-mediated transport also gives rise to decreased levels of IgG3 in mucosal tissue and impaired transport of IgG3 across the placenta (14). These properties do not hold true for all types of IgG3 since a large number of IgG3 allotypes have been described, some of which lack the H435R substitution and have a half-life and placental transport rates similar to IgG1 (13–16). IgG3 is more polymorphic than the other IgG subclasses, as evidenced by the high number of known allotypes (16). Most of the polymorphisms reside in the CH2 or CH3 domain, but the length of the hinge region can also display a high degree of variation. Depending on the number of sequence repeats, the hinge region can vary from 27 to 83 amino acid residues between different IgG3 allotypes (3, 16, 17).An N-linked complex type glycan is highly conserved and found in the CH2 domain of all IgG subclasses and allotypes. The type of glycan present at this site has been shown to influence the effector functions of IgG (18). N-glycans that lack a core fucose cause IgG to have an enhanced proinflammatory capacity through stronger binding to FcγRIIIa and FcγRIIIb (18–20). In contrast, IgG carrying sialylated N-glycans exhibits anti-inflammatory properties, likely due to increased binding affinity to C-type lectins and/or reduced binding to FcγR (18, 21, 22).O-linked glycosylation has been reported for various immunoglobulins. O-glycans are present on the hinge region of human IgA1 and IgD and mouse IgG2b (23–25). IgA1 contains nine potential sites for O-glycosylation (serine and threonine) in the hinge region, of which 3–5 are occupied, while IgD has been reported to carry between four and seven O-glycans (24–26). The O-glycosylation in the hinge of murine IgG2b was observed to protect against proteolytic digestion (23). Likewise, IgA1 was found to be more susceptible to degradation by Streptococci proteases after neuraminidase treatment (27).In this study, we report partial O-glycosylation of the human IgG3 hinge. We obtained both poly- and monoclonal IgG3 from various sources and performed proteolytic digestion with trypsin or proteinase K. NanoLC-reverse phase (RP)-ESI-ion trap (IT)-MS/MS was used to examine the resulting (glyco)peptides, revealing core 1-type O-glycans on multiple sites within the IgG3 hinge region.     

Abnormal actomyosin assembly in proliferating and differentiating myoblasts upon expression of a cytosolic DMPK isoform

DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine-threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast-myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells.     

Year-round prevalence of norovirus in the environment of catering companies without a recently reported outbreak of gastroenteritis

Food handlers play an important role in the transmission of norovirus (NoV) in food-borne outbreaks of gastroenteritis (GE). In a year-round prevalence study, the prevalence of NoV in catering companies without recently reported outbreaks of GE was investigated and compared to the observed prevalence in catering companies with recently reported outbreaks. Swab samples were collected from surfaces in the kitchens and (staff) bathrooms in 832 randomly chosen companies and analyzed for the presence of NoV RNA. In total, 42 (1.7%) out of 2,496 environmental swabs from 35 (4.2%) catering companies tested positive. In contrast, NoV was detected in 147 (39.7%) of the 370 samples for 44 (61.1%) of the 72 establishments associated with outbreaks of gastroenteritis. NoV-positive swabs were more frequently found in winter, in specific types of companies (elderly homes and lunchrooms), and in establishments with separate bathrooms for staff. We found a borderline association with population density but no relation to the number of employees. Sequence analysis showed that environmental strains were interspersed with strains found in outbreaks of illness in humans. Thus, the presence of NoV in catering companies seemed to mirror the presence in the population but was strongly increased when associated with food-borne GE. Swabs may therefore serve as a valuable tool in outbreak investigations for the identification of the causative agent, although results should be interpreted with care, taking into account all other epidemiological data.