While it was long held that T cells were the primary mediators of multiple sclerosis (MS) pathogenesis, the beneficial effects observed in response to treatment with Rituximab (RTX), a monoclonal antibody (mAb) targeting CD20, shed light on a key contributor to MS that had been previously underappreciated: B cells. This has been reaffirmed by results from clinical trials testing the efficacy of subsequently developed B cell-depleting mAbs targeting CD20 as well as studies revisiting the effects of previous disease-modifying therapies (DMTs) on B cell subsets thought to modulate disease severity. In this review, we summarize current knowledge regarding the complex roles of B cells in MS pathogenesis and current and potential future B cell-directed therapies.

Napsin A is an intracellular aspartic protease and biomarker of various malignancies like lung adenocarcinoma and ovarian clear cell carcinoma, but its detection is usually limited to immunohistochemical techniques gaining excellent information on its distribution but missing information about posttranslational modifications (e.g. maturation state) of the protein. We present a protocol for specific enrichment of napsin A from clinical or biological specimens, that facilitates detailed analysis of the protein. By using the exceptionally broad pH range under which napsin A binds to its inhibitor pepstatin A we achieve highly selective binding of napsin A while other aspartic proteases have negligible affinity. Using this method we demonstrate that lung napsin A in many mammals is a heterogeneous enzyme with a characteristic ladder-like appearance in SDS-PAGE that might be caused by proteolytically processed N- and/or C-termini, in contrast to the more homogeneous form found in kidneys and primary lung adenocarcinoma.

Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%-20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations.

Defects in mitotic spindle orientation (MSO) disrupt the organization of stem cell niches impacting tissue morphogenesis and homeostasis. Mutations in centrosome genes reduce MSO fidelity, leading to tissue dysplasia and causing several diseases such as microcephaly, dwarfism, and cancer. Whether these mutations perturb spindle orientation solely by affecting astral microtubule nucleation or whether centrosome proteins have more direct functions in regulating MSO is unknown. To investigate this question, we analyzed the consequences of deregulating Plk4 (the master centriole duplication kinase) activity in Drosophila asymmetrically dividing neural stem cells. We found that Plk4 functions upstream of MSO control, orchestrating centriole symmetry breaking and consequently centrosome positioning. Mechanistically, we show that Plk4 acts through Spd2 phosphorylation, which induces centriole release from the apical cortex. Overall, this work not only reveals a role for Plk4 in regulating centrosome function but also links the centrosome biogenesis machinery with the MSO apparatus.

Endothelial cells (ECs), which line blood and lymphatic vessels, are generally described to come from the lateral plate mesoderm despite experimental evidence for a broader source of origin, including the paraxial mesoderm (PXM). Current dogma suggests that following specification from mesoderm, local environmental cues establish the distinct molecular and functional characteristics of ECs in different vascular beds. Here we present evidence to challenge this view, showing that lymphatic EC fate is imprinted during transition through the PXM lineage. We show that PXM-derived cells form the lymphatic endothelium of multiple organs and tissues, with a more restricted contribution to blood vessel endothelium. By deleting Prox1 specifically in PXM-derived cells, we show that this lineage is indispensable for lymphatic vessel development. Collectively, our data establish lineage history as a critical determinant of EC specialization, a finding with broad implications for our understanding of vascular development and heterogeneity.

Growth factor signaling is initiated at the plasma membrane and propagated through the cytoplasm for eventual relay to intracellular organelles such as lysosomes. The serine/threonine kinase mTOR participates in growth factor signaling as a component of two multi-subunit complexes, mTORC1 and mTORC2. mTORC1 associates with lysosomes, and its activity depends on the positioning of lysosomes within the cytoplasm, although there is no consensus regarding the exact effect of perinuclear versus peripheral distribution. mTORC2 and its substrate kinase AKT have a widespread distribution, but they are thought to act mainly at the plasma membrane. Using cell lines with knockout of components of the lysosome-positioning machinery, we show that perinuclear clustering of lysosomes delays reactivation of not only mTORC1, but also mTORC2 and AKT upon serum replenishment. These experiments demonstrate the existence of pools of mTORC2 and AKT that are sensitive to lysosome positioning.

Stroma is a poorly defined non-parenchymal component of virtually every organ with key roles in organ development, homeostasis, and repair. Studies of the bone marrow stroma have defined individual populations in the stem cell niche regulating hematopoietic regeneration and capable of initiating leukemia. Here, we use single-cell RNA sequencing (scRNA-seq) to define a cellular taxonomy of the mouse bone marrow stroma and its perturbation by malignancy. We identified seventeen stromal subsets expressing distinct hematopoietic regulatory genes spanning new fibroblastic and osteoblastic subpopulations including distinct osteoblast differentiation trajectories. Emerging acute myeloid leukemia impaired mesenchymal osteogenic differentiation and reduced regulatory molecules necessary for normal hematopoiesis. These data suggest that tissue stroma responds to malignant cells by disadvantaging normal parenchymal cells. Our taxonomy of the stromal compartment provides a comprehensive bone marrow cell census and experimental support for cancer cell crosstalk with specific stromal elements to impair normal tissue function and thereby enable emergent cancer.

Zika virus (ZIKV) infection during pregnancy may cause major congenital defects, including microcephaly, ocular, articular and muscle abnormalities, which are collectively defined as Congenital Zika Syndrome. Here, we performed an in-depth characterization of the effects of congenital ZIKV infection (CZI) in immunocompetent mice.

Murine norovirus (MNoV) infects a low percentage of enteric tuft cells and can persist in these cells for months following acute infection. Both tuft-cell tropism and resistance to interferon-λ (IFN-λ)-mediated clearance during persistent infection requires the viral nonstructural protein 1/2 (NS1/2). We show that processing of NS1/2 yields NS1, an unconventionally secreted viral protein that is central for IFN-λ resistance. MNoV infection globally suppresses intestinal IFN-λ responses, which is attributable to secreted NS1. MNoV NS1 secretion is triggered by caspase-3 cleavage of NS1/2, and a secreted form of human NoV NS1 is also observed. NS1 secretion is essential for intestinal infection and resistance to IFN-λ in vivo. NS1 vaccination alone protects against MNoV challenge, despite the lack of induction of neutralizing anti-capsid antibodies previously shown to confer protection. Thus, despite infecting a low number of tuft cells, NS1 secretion allows MNoV to globally suppress IFN responses and promote persistence.

Down syndrome (DS) is a common neurodevelopmental disorder, and cognitive defects in DS patients may arise from imbalances in excitatory and inhibitory neurotransmission. Understanding the mechanisms underlying such imbalances may provide opportunities for therapeutic intervention. Here, we show that human induced pluripotent stem cells (hiPSCs) derived from DS patients overproduce OLIG2+ ventral forebrain neural progenitors. As a result, DS hiPSC-derived cerebral organoids excessively produce specific subclasses of GABAergic interneurons and cause impaired recognition memory in neuronal chimeric mice. Increased OLIG2 expression in DS cells directly upregulates interneuron lineage-determining transcription factors. shRNA-mediated knockdown of OLIG2 largely reverses abnormal gene expression in early-stage DS neural progenitors, reduces interneuron production in DS organoids and chimeric mouse brains, and improves behavioral deficits in DS chimeric mice. Thus, altered OLIG2 expression may underlie neurodevelopmental abnormalities and cognitive defects in DS patients.

Two optical isomers, +/- gelsemine (1, 2), together with one known compound were isolated from the whole plant of G. elegans. The structures of the separated constituents were elucidated on 1D and 2D (1H-1H COSY, HMBC, HSQC) NMR spectroscopy and high-resolution mass spectrometry (HRMS). The isolated alkaloids were tested in vitro for cytotoxic potential against PC12 cells by the MTT assay. As a result, (+) gelsemine (compound 1) exhibited cytotoxic activity against PC12 cells with an IC50 value of 31.59 μM, while (-) gelsemine (compound 2) was not cytotoxic.

P-glycoprotein (P-gp) is a transmembrane protein that actively transports a wide variety of chemically diverse compounds out of the cell. It is highly associated with the ADMET (absorption, distribution, metabolism, excretion and toxicity) properties of drugs/drug candidates and contributes to decreasing toxicity by eliminating compounds from cells, thereby preventing intracellular accumulation. Therefore, in the drug discovery and toxicological assessment process it is advisable to pay attention to whether a compound under development could be transported by P-gp or not. In this study, an in silico multiclass classification model capable of predicting the probability of a compound to interact with P-gp was developed using a counter-propagation artificial neural network (CP ANN) based on a set of 2D molecular descriptors, as well as an extensive dataset of 2512 compounds (1178 P-gp inhibitors, 477 P-gp substrates and 857 P-gp non-active compounds). The model provided a good classification performance, producing non error rate (NER) values of 0.93 for the training set and 0.85 for the test set, while the average precision (AvPr) was 0.93 for the training set and 0.87 for the test set. An external validation set of 385 compounds was used to challenge the model's performance. On the external validation set the NER and AvPr values were 0.70 for both indices. We believe that this in silico classifier could be effectively used as a reliable virtual screening tool for identifying potential P-gp ligands.

The replacement of synthetic compounds by natural products witnesses an increasing demand from the pharmaceutical, cosmetic, food and nutraceutical industries. Included in the set of natural raw materials that are poorly explored are the macroalgae. Despite the detailed characterization and identification of most relevant biomolecules that are present in the main macroalgae species, there remains a lack of efficient and economically viable processes available to meet the needs of the markets. In this work, an efficient and single-step process, based on aqueous solutions of Tween 20, to recover carotenoids from Sargassum muticum, an invasive brown macroalgae species present in the Portuguese coast, is proposed and optimized allowing an extraction yield of 2.78 ± 0.4 mgcarotenoids.gdried mass-1, which is shown to increase the extraction efficiency by 38% when compared with traditional methods.

An understanding of the interaction between the antibody and its targeted antigen and knowing of the epitopes are critical for the development of monoclonal antibody drugs. Complement factor H (CFH) is implied to play a role in tumor growth and metastasis. An autoantibody to CHF is associated with anti-tumor cell activity. The interaction of a human monoclonal antibody Ab42 that was isolated from a cancer patient with CFH polypeptide (pCFH) antigen was analyzed by molecular docking, molecular dynamics (MD) simulation, free energy calculation, and computational alanine scanning (CAS). Experimental alanine scanning (EAS) was then carried out to verify the results of the theoretical calculation. Our results demonstrated that the Ab42 antibody interacts with pCFH by hydrogen bonds through the Tyr315, Ser100, Gly33, and Tyr53 residues on the complementarity-determining regions (CDRs), respectively, with the amino acid residues of Pro441, Ile442, Asp443, Asn444, Ile447, and Thr448 on the pCFH antigen. In conclusion, this study has explored the mechanism of interaction between Ab42 antibody and its targeted antigen by both theoretical and experimental analysis. Our results have important theoretical significance for the design and development of relevant antibody drugs.

In an earlier study, signs of commencing degeneration of spinal motor neurons were induced in mice with short-term intraperitoneal injections of immunoglobulin G (IgG) taken from patients with amyotrophic lateral sclerosis (ALS). Since in that study, neither weakness nor loss of motor neurons was noted, to test whether the ALS IgG in this paradigm has the potential to evoke relentless degeneration of motor neurons, treatment with repeated injections over a longer period was carried out. Mice were systematically injected intraperitoneally with serum taken from ALS patients over a 75-day period. At selected time points, the isometric force of the limbs, number of spinal motor neurons and their intracellular calcium levels were determined. Furthermore, markers of glial activation and the motoneuronal uptake of human IgG were monitored. During this period, gliosis and progressive motoneuronal degeneration developed, which led to gradual loss of spinal motor neurons, more than 40% at day 21, along with decreasing muscle strength in the limbs. The inclusion-like accumulation of IgG appeared in the perikarya with the increase of intracellular calcium in the cell bodies and motor nerve terminals. Our results demonstrate that ALS serum can transfer motor neuron disease to mice.

Norovirus is the most common cause of non-bacterial gastroenteritis and is a burden worldwide. The increasing norovirus diversity is currently categorized into at least 10 genogroups which are further classified into more than 40 genotypes. In addition to humans, norovirus can infect a broad range of hosts including livestock, pets, and wild animals, e.g., marine mammals and bats. Little is known about norovirus infections in most non-human hosts, but the close genetic relatedness between some animal and human noroviruses coupled with lack of understanding where newly appearing human norovirus genotypes and variants are emerging from has led to the hypothesis that norovirus may not be host restricted and might be able to jump the species barrier. We have systematically reviewed the literature to describe the diversity, prevalence, and geographic distribution of noroviruses found in animals, and the pathology associated with infection. We further discuss the evidence that exists for or against interspecies transmission including surveillance data and data from in vitro and in vivo experiments.

In September 2015, a massive occurrence of the Ostreopsis species was recorded in central Adriatic Kaštela Bay. In order to taxonomically identify the Ostreopsis species responsible for this event and determine their toxin profile, cells collected in seawater and from benthic macroalgae were analyzed. Conservative taxonomic methods (light microscopy and SEM) and molecular methods (PCR-based assay) allowed the identification of the species Ostreopsis cf. ovata associated with Coolia monotis. The abundance of O. cf. ovata reached 2.9 × 104 cells L-1 in seawater, while on macroalgae, it was estimated to be up to 2.67 × 106 cells g-1 of macroalgae fresh weight and 14.4 × 106 cells g-1 of macroalgae dry weight. An indirect sandwich immunoenzymatic assay (ELISA) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) were used to determine the toxin profile. The ELISA assay revealed the presence of 5.6 pg palytoxin (PLTX) equivalents per O. cf. ovata cell. LC-HRMS was used for further characterization of the toxin profile, which showed that there were 6.3 pg of the sum of ovatoxins (OVTXs) and isobaric PLTX per O. cf. ovata cell, with a prevalence of OVTXs (6.2 pg cell-1), while the isobaric PLTX concentration was very low (0.1 pg cell-1). Among OVTXs, the highest concentration was recorded for OVTX-a (3.6 pg cell-1), followed by OVTX-b (1.3 pg cell-1), OVTX-d (1.1 pg cell-1), and OVTX-c (0.2 pg cell-1).

Freely-dissolved silicon quantum dots were prepared by thermal hydrosilation of 1-undecene at high-porosity porous silicon under reflux in toluene. This reaction produces a suspension of alkyl-capped silicon quantum dots (alkyl SiQDs) with bright orange luminescence, a core Si nanocrystal diameter of about 2.5 nm and a total particle diameter of about 5 nm. Previous work has shown that these particles are rapidly endocytosed by malignant cell lines and have little or no acute toxicity as judged by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for viability and the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis. We have extended this work to the CACO-2 cell line, an established model for the human small intestinal mucosa, and demonstrate that neither acute nor chronic (14 days) toxicity is observed as judged by cell morphology, viability, ATP production, ROS production and DNA damage (single cell gel electrophoresis) at doses of 50-200 μ g mL - 1 . Quantitative assessment of the extent of uptake of alkyl SiQDs by CACO-2, HeLa, HepG2, and Huh7 cell lines by flow cytometry showed a wide variation. The liver cell lines (HepG2 and Huh7) were the most active and HeLa and CACO-2 showed comparable activity. Previous work has reported a cholesterol-sensitivity of the endocytosis (HeLa), which suggests a caveolin-mediated pathway. However, gene expression analysis by quantitative real-time polymerase chain reaction (RT-PCR) indicates very low levels of caveolins 1 and 2 in HepG2 and much higher levels in HeLa. The data suggest that the mechanism of endocytosis of the alkyl SiQDs is cell-line dependent.

The increasing prevalence of drug resistant and/or high-risk cancers indicate further drug discovery research is required to improve patient outcome. This study outlines a simplified approach to identify lead compounds from natural products against several cancer cell lines, and provides the basis to better understand structure activity relationship of the natural product cephalotaxine. Using high-throughput screening, a natural product library containing fractions and pure compounds was interrogated for proliferation inhibition in acute lymphoblastic leukemia cellular models (SUP-B15 and KOPN-8). Initial hits were verified in control and counter screens, and those with EC50 values ranging from nanomolar to low micromolar were further characterized via mass spectrometry, NMR, and cytotoxicity measurements. Most of the active compounds were alkaloid natural products including cephalotaxine and homoharringtonine, which were validated as protein synthesis inhibitors with significant potency against several cancer cell lines. A generated BODIPY-cephalotaxine probe provides insight into the mode of action of cephalotaxine and further rationale for its weaker potency when compared to homoharringtonine. The steroidal natural products (ecdysone and muristerone A) also showed modest biological activity and protein synthesis inhibition. Altogether, these findings demonstrate that natural products continue to provide insight into structure and function of molecules with therapeutic potential against drug resistant cancer cell models.

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA. Here, we report an expansion of the zebrafish genetic code to incorporate the UNAAs, azido-lysine (AzK), bicyclononyne-lysine (BCNK), and diazirine-lysine (AbK) into green fluorescent protein (GFP) and glutathione-s-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of AzK, BCNK, and AbK introduction into proteins as a means to investigate and engineer their function in zebrafish.