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Kinesin-1 uses a membrane-induced curvature-sensitive amphipathic helix to bind directly to cargo membranes., The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxyl-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical, and cell biology approaches, we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a subset of long kinesin light-chain paralogs and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids, and it contributes to kinesin-1–dependent lysosome positioning, a canonical activity that, until now, has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a protein-lipid coincidence detection framework for kinesin-1–mediated organelle transport.

Summary Dietary leucine has been suspected to play an important role in insulin release, a hormone that controls satiety and metabolism. The mechanism by which insulin-producing cells (IPCs) sense leucine and regulate insulin secretion is still poorly understood. In Drosophila, insulin-like peptides (DILP2 and DILP5) are produced by brain IPCs and are released in the hemolymph after leucine ingestion. Using Ca2+-imaging and ex vivo cultured larval brains, we demonstrate that IPCs can directly sense extracellular leucine levels via minidiscs (MND), a leucine transporter. MND knockdown in IPCs abolished leucine-dependent changes, including loss of DILP2 and DILP5 in IPC bodies, consistent with the idea that MND is necessary for leucine-dependent DILP release. This, in turn, leads to a strong increase in hemolymph sugar levels and reduced growth. GDH knockdown in IPCs also reduced leucine-dependent DILP release, suggesting that nutrient sensing is coupled to the glutamate dehydrogenase pathway., Graphical Abstract, Highlights • IPCs directly sense extracellular leucine levels via minidiscs (MND) • MND knockdown in IPCs abolishes loss of DILP2 and DILP5 • This leads to a strong increase in hemolymph sugar levels and reduces growth • GDH knockdown in IPCs reduces leucine-dependent DILP release, Manière et al. find that leucine induces the disappearance of two DILPs in Drosophila IPCs. Minidiscs (MND) is the primary leucine sensor, and downregulation has consequences for glycemia and growth. The authors propose that the leucine/MND pathway represents a conserved mechanism for insulin release.

The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1¿ and IgG1¿ versions of three therapeutic antibodies directed against pro-inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF-a, and Ustekinumab, directed against the interleukin-12p40 subunit. We evaluated antibody yield, quality and N-glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell-based assay. However, IgA1¿-based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1¿- and IgG1¿-based Infliximab were enriched in oligomannose-type N-glycan structures. For IgG1¿-based Ustekinumab and Adalimumab, the major N-glycan type was the typical plant complex N-glycan, biantennary with terminal N-acetylglucosamine, ß1,2-xylose and core a1,3-fucose. In contrast, the major N-glycan on the IgA-based antibodies was xylosylated, but lacked core a1,3-fucose and one terminal N-acetylglucosamine. This type of N-glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant-produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N-glycan an antibody receives.

A 48-yr-old nonsmoking female presented with a 5-yr history of recurrent bronchitis with progressive exercise dyspnoea. She had been diagnosed with a small monoclonal gammopathy of undermined significance (MGUS) 6 yrs previously. Inspiratory vital capacity was 4.7 L (125% predicted) and forced expiratory volume in one second/forced vital capacity (FVC) ratio was 79%. Carbon monoxide transfer factor was markedly impaired (35% pred), and arterial oxygen tension ( P a,O2) decreased from 9.2 to 6.0 kPa at exercise (40 W, 10 min). A thoracic computed tomography (CT) scan was performed (fig. 1⇓). Fibreoptic bronchoscopy found inflammation of the bronchial mucosa with candle stain-like deposits on the trachea and large bronchi, without any purulent secretion within the respiratory tract (fig. 2⇓). The histopathological aspect of bronchial biopsies is shown in figure 3⇓. Blood tests disclosed type I cryoglobulinaemia composed of a 32 g·L−1 monoclonal immunoglobulin (Ig)Mκ serum component (at 37°C). Blood cell count was normal. Bone marrow examination showed malignant lymphoplasmacytic cells representing 30% of total cells, with a monotypic surface expression of IgMκ. The C-reactive protein level was 21 mg·L−1. Extensive renal, cardiac and hepatic evaluations were normal. As severe hypoxaemia and reduction of carbon monoxide transfer factor were unexplained, the patient underwent videothoracoscopic lung biopsies. Fig. 1— Thoracic computed tomography scan. Fig. 2— Fibreoptic bronchoscopy. Arrow shows candle stain-like deposits. Fig. 3— a) Bronchial biopsy of candle-stain like lesions stained by haematoxylin-eosin-saffron. Submucosal abnormal deposits with concentric pattern around the vessels (black arrow), arranging in cords or ribbon-like structures in the interstitium (black dashed arrow) or in an organoid, raspberry-like fashion in smooth muscle fascicles (black arrowhead). b) Bronchial biopsy; perivascular deposits with negative Congo red dye staining. c) Direct anti-κ-light chain immunofluorescence in bronchial biopsy showing positivity of perivascular (white arrow) and interstitial (white dashed arrow) deposits, and epithelial …

The t(11;14)(q13;q32) is the most com- mon translocation in multiple myeloma (MM), resulting in up-regulation of cyclin D1. We used a segregation fluorescence in situ hybridization (FISH) assay to de- tect t(11;14) breakpoints in primary MM cases and real-time reverse transcriptase– polymerase chain reaction (RT-PCR) to quantify cyclin D1 and MYEOV (myeloma overexpressed) expression, another puta- tive oncogene located on chromosome 11q13. High levels of cyclin D1 mRNA (cyclin D1/TBP [TATA box binding pro- tein] ratio > 95) were found exclusively in the presence of a t(11;14) translocation (11/48 cases; P < .00001). In addition, a subgroup of MM cases (15/48) with inter- mediate to low cyclin D1 mRNA (cyclin D1/TBP ratio between 2.3 and 20) was identified. FISH analysis ruled out a t(11; 14) translocation and 11q13 amplification in these cases; however, in 13 of 15 patients a chromosome 11 polysomy was demonstrated ( P < .0001). These results indicate an effect of gene dosage as an alternative mechanism of cyclin D1 de- regulation in MM. The absence of chromo- some 11 abnormalities in 2 of 15 patients with intermediate cyclin D1 expression supports that there are presumably other mechanism(s) of cyclin D1 deregulation in MM patients. Our data indicate that deregulation of MYEOV is not favored in MM and further strengthens the role of cyclin D1 overexpression in lymphoid ma- lignancies with a t(11;14)(q13;q32) trans- location.

Aims—To investigate whether the analysis of immunoglobulin (Ig)/T cell receptor (TCR) rearrangements is useful in the diagnosis of lymphoproliferative disorders. Methods—In a series of 107 consecutive cases with initial suspicion of non-Hodgkin's lymphoma (NHL), Southern blot (SB) analysis of Ig/TCR rearrangements was performed. Results—In 98 of 100 histopathologically conclusive cases, Ig/TCR gene results were concordant. In one presumed diffuse large B cell lymphoma (DLCL) and one follicular lymphoma (FL) case no clonality could be detected by SB analysis, or by polymerase chain reaction (PCR) at second stage. In the DLCL, sampling error might have occurred; the FL was revised after an initial diagnosis of reactivity. In many of the histopathologically inconclusive cases Ig/TCR gene SB analysis was helpful, giving support for the histopathological suspicion. However, because of a lack of (clinical) follow up data this could not be confirmed in a few cases. Conclusions—Experienced haematopathologists or a pathologist panel can diagnose malignant versus reactive lesions in most cases without the need for Ig/TCR gene analysis and can select the 5–10% of cases that might benefit from molecular clonality studies. Key Words: B cell lymphoma • immunoglobulin and T cell receptor genes • clonality analysis • Southern blotting

Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of He La cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.

In order to define the most suitable instrumental protocol for the diagnosis of multiple sclerosis (MS), 41 patients with definite (D = 14), probable (P = 14) and suspected (S = 13) MS were examined with CSF immunology, brain MRI and multimodal evoked potentials. The central motor tracts were also tested. The following alteration rates were found: MRI = 78%, CSF = 63.6%, VEP = 70.0%, median nerve SEP = 50%, peroneal nerve SEP = 68.0%, BAEPs = 35.7%, motor-evoked potentials (MEPs) = 74.0%. Altogether, EPs were abnormal in 90% of cases. Normal MRI with altered EPs were found in 22% of cases, whilst a normal EP battery with defective CSF or MRI findings were found in 7%. Twenty-six out of 27 patients with P or S forms were reclassified into a D one when considering EPs and MRI features.

Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies.