Quantitative description of the phase-separation behavior of the multivalent SLP65-CIN85 complex.

Biomolecular condensates play a major role in cell compartmentalization, besides membrane-enclosed organelles. The multivalent SLP65 and CIN85 proteins are proximal B-cell antigen receptor (BCR) signal effectors and critical for proper immune responses. In association with intracellular vesicles, the two effector proteins form phase separated condensates prior to antigen stimulation, thereby preparing B lymphocytes for rapid and effective activation upon BCR ligation. Within this tripartite system, 6 proline-rich motifs (PRMs) of SLP65 interact promiscuously with 3 SH3 domains of the CIN85 monomer, establishing 18 individual SH3-PRM interactions whose individual dissociation constants we determined. Based on these 18 dissociation constants, we measured the phase-separation properties of the natural SLP65/CIN85 system as well as designer constructs that emphasize the strongest SH3/PRM interactions. By modeling these various SLP65/CIN85 constructs with the program LASSI (LAttice simulation engine for Sticker and Spacer Interactions), we reproduced the observed phase-separation properties. In addition, LASSI revealed a deviation in the experimental measurement, which was independently identified as a previously unknown intramolecular interaction. Thus, thermodynamic properties of the individual PRM/SH3 interactions allow us to model the phase-separation behavior of the SLP65/CIN85 system faithfully.

Protein degradation by human 20S proteasomes elucidates the interplay between peptide hydrolysis and splicing.

If and how proteasomes catalyze not only peptide hydrolysis but also peptide splicing is an open question that has divided the scientific community. The debate has so far been based on immunopeptidomics, in vitro digestions of synthetic polypeptides as well as ex vivo and in vivo experiments, which could only indirectly describe proteasome-catalyzed peptide splicing of full-length proteins. Here we develop a workflow-and cognate software - to analyze proteasome-generated non-spliced and spliced peptides produced from entire proteins and apply it to in vitro digestions of 15 proteins, including well-known intrinsically disordered proteins such as human tau and α-Synuclein. The results confirm that 20S proteasomes produce a sizeable variety of cis-spliced peptides, whereas trans-spliced peptides are a minority. Both peptide hydrolysis and splicing produce peptides with well-defined characteristics, which hint toward an intricate regulation of both catalytic activities. At protein level, both non-spliced and spliced peptides are not randomly localized within protein sequences, but rather concentrated in hotspots of peptide products, in part driven by protein sequence motifs and proteasomal preferences. At sequence level, the different peptide sequence preference of peptide hydrolysis and peptide splicing suggests a competition between the two catalytic activities of 20S proteasomes during protein degradation.

Autoinhibition in the Signal Transducer CIN85 Modulates B Cell Activation.

Signal transduction by the ligated B cell antigen receptor (BCR) depends on the preorganization of its intracellular components, such as the effector proteins SLP65 and CIN85 within phase-separated condensates. These liquid-like condensates are based on the interaction between three Src homology 3 (SH3) domains and the corresponding proline-rich recognition motifs (PRM) in CIN85 and SLP65, respectively. However, detailed information on the protein conformation and how it impacts the capability of SLP65/CIN85 condensates to orchestrate BCR signal transduction is still lacking. This study identifies a hitherto unknown intramolecular SH3:PRM interaction between the C-terminal SH3 domain (SH3C) of CIN85 and an adjacent PRM. We used high-resolution nuclear magnetic resonance (NMR) experiments to study the flexible linker region containing the PRM and determined the extent of the interaction in multidomain constructs of the protein. Moreover, we observed that the phosphorylation of a serine residue located in the immediate vicinity of the PRM regulates this intramolecular interaction. This allows for a dynamic modulation of CIN85's valency toward SLP65. B cell culture experiments further revealed that the PRM/SH3C interaction is crucial for maintaining the physiological level of SLP65/CIN85 condensate formation, activation-induced membrane recruitment of CIN85, and subsequent mobilization of Ca2+. Our findings therefore suggest that the intramolecular interaction with the adjacent disordered linker is effective in modulating CIN85's valency both in vitro and in vivo. This therefore constitutes a powerful way for the modulation of SLP65/CIN85 condensate formation and subsequent B cell signaling processes within the cell.

Chemoselective umpolung of thiols to episulfoniums for cysteine bioconjugation.

Cysteine conjugation is an important tool in protein research and relies on fast, mild and chemoselective reactions. Cysteinyl thiols can either be modified with prefunctionalized electrophiles, or converted into electrophiles themselves for functionalization with selected nucleophiles in an independent step. Here we report a bioconjugation strategy that uses a vinyl thianthrenium salt to transform cysteine into a highly reactive electrophilic episulfonium intermediate in situ, to enable conjugation with a diverse set of bioorthogonal nucleophiles in a single step. The reactivity profile can connect several nucleophiles to biomolecules through a short and stable ethylene linker, ideal for introduction of infrared labels, post-translational modifications or NMR probes. In the absence of reactive exogenous nucleophiles, nucleophilic amino acids can react with the episulfonium intermediate for native peptide stapling and protein-protein ligation. Ready synthetic access to isotopologues of vinyl thianthrenium salts enables applications in quantitative proteomics. Such diverse applications demonstrate the utility of vinyl-thianthrenium-based bioconjugation as a fast, selective and broadly applicable tool for chemical biology.

Success and complications in lumbar punctures of pediatric patients with leukemia: a study protocol for a randomized clinical crossover trial of a bioimpedance needle system versus conventional procedure.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common malignancy diagnosed in children. At present, the long-term survival from pediatric ALL is well over 90%. However, the probability of event-free survival is reduced if the lumbar puncture (LP) procedures at the beginning of the patient's intrathecal therapy cause blood leakage into the spinal canal and blast cells contaminate the cerebrospinal fluid. According to the literature, such traumatic LP procedures concern one out of five pediatric patients with ALL. Recently, a novel medical device measuring the tissue bioimpedance at the tip of a spinal needle was found feasible in pediatric patients with ALL. The LP procedure was successful at the first attempt in 80% of procedures, and the incidence of traumatic LPs was then 11%. The purpose of the present study is to compare the bioimpedance spinal needle system with the standard clinical practice resting on a conventional spinal needle and investigate its efficacy in clinical practice. METHODS: The study is a multicenter, randomized, two-arm crossover noninferiority trial of pediatric hemato-oncology patients that will be conducted within the usual clinical workflow. Patients' LP procedures will be performed alternately either with the IQ-Tip system (study arm A) or a conventional Quincke-type 22G spinal needle (study arm B). For each enrolled patient, the order of procedures is randomly assigned either as ABAB or BABA. The total number of LP procedures will be at least 300, and the number of procedures per patient between two and four. After each study LP procedure, the performance will be recorded immediately, and 1-week diary-based and 4-week record-based follow-ups on symptoms, complications, and adverse events will be conducted thereafter. The main outcomes are the incidence of traumatic LP, first puncture success rate, and incidence of post-dural puncture headache. DISCUSSION: The present study will provide sound scientific evidence on the clinical benefit, performance, and safety of the novel bioimpedance spinal needle compared with the standard clinical practice of using conventional spinal needles in the LP procedures of pediatric patients with leukemia. TRIAL REGISTRATION: ISRCTN ISRCTN16161453. Registered on 8 July 2022.

Polyphenols-Based Nanosheets of Propolis Modulate Cytotoxic Amyloid Fibril Assembly of α-Synuclein.

Natural compounds with anti-aggregation capacity are increasingly recognized as viable candidates against neurodegenerative diseases. Recently, the polyphenolic fraction of propolis (PFP), a complex bee product, has been shown to inhibit amyloid aggregation of a model protein especially in the nanosheet form. Here, we examine the aggregation-modulating effects of the PFP nanosheets on α-synuclein (α-syn), an intrinsically disordered protein involved in the pathogenesis of Parkinson's disease. Based on a range of biophysical data including intrinsic and extrinsic fluorescence, circular dichroism (CD) data, and nuclear magnetic resonance spectroscopy, we propose a model for the interaction of α-syn with PFP nanosheets, where the positively charged N-terminal and the middle non-amyloid component regions of α-syn act as the main binding sites with the negatively charged PFP nanosheets. The Thioflavin T (ThT) fluorescence, Congo red absorbance, and CD data reveal a prominent dose-dependent inhibitory effect of PFP nanosheets on α-syn amyloid aggregation, and the microscopy images and MTT assay data suggest that the PFP nanosheets redirect α-syn aggregation toward nontoxic off-pathway oligomers. When preformed α-syn amyloid fibrils are present, fluorescence images show co-localization of PFP nanosheets and ThT, further confirming the binding of PFP nanosheets with α-syn amyloid fibrils. Taken together, our results demonstrate the binding and anti-aggregation activity of PFP nanosheets in a disease-related protein system and propose them as potential nature-based tools for probing and targeting pathological protein aggregates in neurodegenerative diseases.

Efficient spontaneous site-selective cysteine-mediated toxin attachment within a structural loop of antibodies.

BACKGROUND: Site-specific coupling of toxin entities to antibodies has become a popular method of synthesis of antibody-drug conjugates (ADCs), as it leads to a homogenous product and allows a free choice of a convenient site for conjugation. METHODS: We introduced a short motif, containing a single cysteine surrounded by aromatic residues, into the N-terminal FG-loop of the CH2 domain of two model antibodies, cetuximab and trastuzumab. The extent of conjugation with toxic payload was examined with hydrophobic interaction chromatography and mass spectrometry and the activity of resulting conjugates was tested on antigen-overexpressing cell lines. RESULTS: Antibody mutants were amenable for rapid coupling with maleimide-based linker endowed toxin payload and the modifications did not impair their reactivity with target cell lines or negatively impact their biophysical properties. Without any previous reduction, up to 50% of the antibody preparation was found to be coupled with two toxins per molecule. After the isolation of this fraction with preparative hydrophobic interaction chromatography, the ADC could elicit a potent cytotoxic effect on the target cell lines. CONCLUSION: By fine-tuning the microenvironment of the reactive cysteine residue, this strategy offers a simplified protocol for production of site-selectively coupled ADCs. GENERAL SIGNIFICANCE: Our unique approach allows the generation of therapeutic ADCs with controlled chemical composition, which facilitates the optimization of their pharmacological activity. This strategy for directional coupling could in the future simplify the construction of ADCs with double payloads ("dual warheads") introduced with orthogonal techniques.

Cln5 represents a new type of cysteine-based S-depalmitoylase linked to neurodegeneration.

Genetic CLN5 variants are associated with childhood neurodegeneration and Alzheimer's disease; however, the molecular function of ceroid lipofuscinosis neuronal protein 5 (Cln5) is unknown. We solved the Cln5 crystal structure and identified a region homologous to the catalytic domain of members of the N1pC/P60 superfamily of papain-like enzymes. However, we observed no protease activity for Cln5; and instead, we discovered that Cln5 and structurally related PPPDE1 and PPPDE2 have efficient cysteine palmitoyl thioesterase (S-depalmitoylation) activity using fluorescent substrates. Mutational analysis revealed that the predicted catalytic residues histidine-166 and cysteine-280 are critical for Cln5 thioesterase activity, uncovering a new cysteine-based catalytic mechanism for S-depalmitoylation enzymes. Last, we found that Cln5-deficient neuronal progenitor cells showed reduced thioesterase activity, confirming live cell function of Cln5 in setting S-depalmitoylation levels. Our results provide new insight into the function of Cln5, emphasize the importance of S-depalmitoylation in neuronal homeostasis, and disclose a new, unexpected enzymatic function for the N1pC/P60 superfamily of proteins.

Autophagy mediates the clearance of oligodendroglial SNCA/alpha-synuclein and TPPP/p25A in multiple system atrophy models.

Accumulation of the neuronal protein SNCA/alpha-synuclein and of the oligodendroglial phosphoprotein TPPP/p25A within the glial cytoplasmic inclusions (GCIs) represents the key histophathological hallmark of multiple system atrophy (MSA). Even though the levels/distribution of both oligodendroglial SNCA and TPPP/p25A proteins are critical for disease pathogenesis, the proteolytic mechanisms involved in their turnover in health and disease remain poorly understood. Herein, by pharmacological and molecular modulation of the autophagy-lysosome pathway (ALP) and the proteasome we demonstrate that the endogenous oligodendroglial SNCA and TPPP/p25A are degraded mainly by the ALP in murine primary oligodendrocytes and oligodendroglial cell lines under basal conditions. We also identify a KFERQ-like motif in the TPPP/p25A sequence that enables its effective degradation via chaperone-mediated autophagy (CMA) in an in vitro system of rat brain lysosomes. Furthermore, in a MSA-like setting established by addition of human recombinant SNCA pre-formed fibrils (PFFs) as seeds of pathological SNCA, we thoroughly characterize the contribution of CMA and macroautophagy in particular, in the removal of the exogenously added and the seeded oligodendroglial SNCA pathological assemblies. We also show that PFF treatment impairs autophagic flux and that TPPP/p25A exerts an inhibitory effect on macroautophagy, while at the same time CMA is upregulated to remove the pathological SNCA species formed within oligodendrocytes. Finally, augmentation of CMA or macroautophagy accelerates the removal of the engendered pathological SNCA conformations further suggesting that autophagy targeting may represent a successful approach for the clearance of pathological SNCA and/or TPPP/p25A in the context of MSA.Abbreviations: 3MA: 3-methyladenine; ACTB: actin, beta; ALP: autophagy-lysosome pathway; ATG5: autophagy related 5; AR7: atypical retinoid 7; CMA: chaperone-mediated autophagy; CMV: cytomegalovirus; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; Epox: epoxomicin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCIs: glial cytoplasmic inclusions; GFP: green fluorescent protein; HMW: high molecular weight; h: hours; HSPA8/HSC70: heat shock protein 8; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mcherry: monomeric cherry; MFI: mean fluorescence intensity; mRFP: monomeric red fluorescent protein; MSA: multiple system atrophy; OLN: oligodendrocytes; OPCs: oligodendroglial progenitor cells; PBS: phosphate-buffered saline; PC12: pheochromocytoma cell line; PD: Parkinson disease; PFFs: pre-formed fibrils; PIs: protease inhibitors; PSMB5: proteasome (prosome, macropain) subunit, beta type 5; Rap: rapamycin; RFP: red fluorescent protein; Scr: scrambled; SDS: sodium dodecyl sulfate; SE: standard error; siRNAs: small interfering RNAs; SNCA: synuclein, alpha; SQSTM1: sequestosome 1; TPPP: tubulin polymerization promoting protein; TUBA: tubulin, alpha; UPS: ubiquitin-proteasome system; WT: wild type.

Protease-Activation of Fc-Masked Therapeutic Antibodies to Alleviate Off-Tumor Cytotoxicity.

The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these approaches result in permanently Fc-silenced antibody derivates which partially or completely lack antibody-mediated effector functions. Nevertheless, for a majority of antibody-based drugs, Fc-mediated effector functions, like antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) as well as complement-dependent cytotoxicity (CDC), represent the most substantial modes of action. We argued that a new strategy combining the beneficial properties of Fc-silencing and controlled activation of effector functions can pave the way to potent antibody therapeutics, reducing the FcγRs-mediated off-target toxicity. We present a novel Fc-tamed antibody format, where the FcγR-binding sites of antibodies are blocked by anti-isotypic masking units, hindering the association of FcγR and complement component 1 (c1q) to the Fc domain. The masking units were genetically fused to trastuzumab, including a protease-addressable peptide-liker. Our Fc-tamed antibodies demonstrated completely abolished interaction to soluble high-affinity Fcγ-Receptor I and c1q. In reporter cell-based ADCC assays, our Fc-tamed antibodies exhibited a 2,700 to 7,100-fold reduction in activation, compared to trastuzumab. Upon demasking by a tumor-associated protease, the Fc-activated antibodies demonstrated restored FcγR-binding, c1q-binding and the ability to induce potent ADCC activation. Furthermore, cell killing assays using donor-derived NK cells were performed to validate the functionality of the Fc-tamed antibody variants. To our knowledge, this approach represents the first non-permanently Fc-silenced antibody, which can be re-activated by a tumor-associated protease, eventually extending the field of novel antibody formats.

Brain iron enrichment attenuates α-synuclein spreading after injection of preformed fibrils.

Regional iron accumulation and α-synuclein (α-syn) spreading pathology within the central nervous system are common pathological findings in Parkinson's disease (PD). Whereas iron is known to bind to α-syn, facilitating its aggregation and regulating α-syn expression, it remains unclear if and how iron also modulates α-syn spreading. To elucidate the influence of iron on the propagation of α-syn pathology, we investigated α-syn spreading after stereotactic injection of α-syn preformed fibrils (PFFs) into the striatum of mouse brains after neonatal brain iron enrichment. C57Bl/6J mouse pups received oral gavage with 60, 120, or 240 mg/kg carbonyl iron or vehicle between postnatal days 10 and 17. At 12 weeks of age, intrastriatal injections of 5-µg PFFs were performed to induce seeding of α-syn aggregates. At 90 days post-injection, PFFs-injected mice displayed long-term memory deficits, without affection of motor behavior. Interestingly, quantification of α-syn phosphorylated at S129 showed reduced α-syn pathology and attenuated spreading to connectome-specific brain regions after brain iron enrichment. Furthermore, PFFs injection caused intrastriatal microglia accumulation, which was alleviated by iron in a dose-dependent way. In primary cortical neurons in a microfluidic chamber model in vitro, iron application did not alter trans-synaptic α-syn propagation, possibly indicating an involvement of non-neuronal cells in this process. Our study suggests that α-syn PFFs may induce cognitive deficits in mice independent of iron. However, a redistribution of α-syn aggregate pathology and reduction of striatal microglia accumulation in the mouse brain may be mediated via iron-induced alterations of the brain connectome.

Natural Glycoforms of Human Interleukin 6 Show Atypical Plasma Clearance.

A library of glycoforms of human interleukin 6 (IL-6) comprising complex and mannosidic N-glycans was generated by semisynthesis. The three segments were connected by sequential native chemical ligation followed by two-step refolding. The central glycopeptide segments were assembled by pseudoproline-assisted Lansbury aspartylation and subsequent enzymatic elongation of complex N-glycans. Nine IL-6 glycoforms were synthesized, seven of which were evaluated for in vivo plasma clearance in rats and compared to non-glycosylated recombinant IL-6 from E. coli. Each IL-6 glycoform was tested in three animals and reproducibly showed individual serum clearances depending on the structure of the N-glycan. The clearance rates were atypical, since the 2,6-sialylated glycoforms of IL-6 cleared faster than the corresponding asialo IL-6 with terminal galactoses. Compared to non-glycosylated IL-6 the plasma clearance of IL-6 glycoforms was delayed in the presence of larger and multibranched N-glycans in most cases.

Opportunities in the cloud or pie in the sky? Current status and future perspectives of telemedicine in nephrology.

The use of telehealth to support, enhance or substitute traditional methods of delivering healthcare is becoming increasingly common in many specialties, such as stroke care, radiology and oncology. There is reason to believe that this approach remains underutilized within nephrology, which is somewhat surprising given the fact that nephrologists have always driven technological change in developing dialysis technology. Despite the obvious benefits that telehealth may provide, robust evidence remains lacking and many of the studies are anecdotal, limited to small numbers or without conclusive proof of benefit. More worryingly, quite a few studies report unexpected obstacles, pitfalls or patient dissatisfaction. However, with increasing global threats such as climate change and infectious disease, a change in approach to delivery of healthcare is needed. The current pandemic with coronavirus disease 2019 (COVID-19) has prompted the renal community to embrace telehealth to an unprecedented extent and at speed. In that sense the pandemic has already served as a disruptor, changed clinical practice and shown immense transformative potential. Here, we provide an update on current evidence and use of telehealth within various areas of nephrology globally, including the fields of dialysis, inpatient care, virtual consultation and patient empowerment. We also provide a brief primer on the use of artificial intelligence in this context and speculate about future implications. We also highlight legal aspects and pitfalls and discuss the 'digital divide' as a key concept that healthcare providers need to be mindful of when providing telemedicine-based approaches. Finally, we briefly discuss the immediate use of telenephrology at the onset of the COVID-19 pandemic. We hope to provide clinical nephrologists with an overview of what is currently available, as well as a glimpse into what may be expected in the future.

Tripartite phase separation of two signal effectors with vesicles priming B cell responsiveness.

Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and the proper engagement of its intracellular signal effector proteins. Src homology (SH) 2 domain-containing leukocyte protein of 65 kDa (SLP65) is the key scaffold protein mediating BCR signaling. In resting B cells, SLP65 colocalizes with Cbl-interacting protein of 85 kDa (CIN85) in cytoplasmic granules whose formation is not fully understood. Here we show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles into droplets via vesicle binding of SLP65 and promiscuous interactions between nine SH3 domains of the trimeric CIN85 and the proline-rich motifs (PRMs) of SLP65. Vesicles are clustered and the dynamical structure of SLP65 persists in the droplet phase in vitro. Our results demonstrate that phase separation driven by concerted transient interactions between scaffold proteins and vesicles is a cellular mechanism to concentrate and organize signal transducers.

A One-Step Process for the Construction of Phage Display scFv and VHH Libraries.

In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes. We show that single chain variable fragment (scFv) libraries with adequate qualities can readily be cloned in a 'scar-less' manner and that the isolation of antigen-specific antibodies from immunized chickens is feasible within three selection rounds. Moreover, we demonstrate the general applicability of this method by rapidly constructing and panning VHH single domain antibody phage display libraries from immunized Llama repertoires.

Plant species-specific recognition of long and short ß-1,3-linked glucans is mediated by different receptor systems.

Plants survey their environment for the presence of potentially harmful or beneficial microbes. During colonization, cell surface receptors perceive microbe-derived or modified-self ligands and initiate appropriate responses. The recognition of fungal chitin oligomers and the subsequent activation of plant immunity are well described. In contrast, the mechanisms underlying ß-glucan recognition and signaling activation remain largely unexplored. Here, we systematically tested immune responses towards different ß-glucan structures and show that responses vary between plant species. While leaves of the monocots Hordeum vulgare and Brachypodium distachyon can recognize longer (laminarin) and shorter (laminarihexaose) ß-1,3-glucans with responses of varying intensity, duration and timing, leaves of the dicot Nicotiana benthamiana activate immunity in response to long ß-1,3-glucans, whereas Arabidopsis thaliana and Capsella rubella perceive short ß-1,3-glucans. Hydrolysis of the ß-1,6 side-branches of laminarin demonstrated that not the glycosidic decoration but rather the degree of polymerization plays a pivotal role in the recognition of long-chain ß-glucans. Moreover, in contrast to the recognition of short ß-1,3-glucans in A. thaliana, perception of long ß-1,3-glucans in N. benthamiana and rice is independent of CERK1, indicating that ß-glucan recognition may be mediated by multiple ß-glucan receptor systems.

Constant domain-exchanged Fab enables specific light chain pairing in heterodimeric bispecific SEED-antibodies.

BACKGROUND: Bispecific antibodies promise to broadly expand the clinical utility of monoclonal antibody technology. Several approaches for heterodimerization of heavy chains have been established to produce antibodies with two different Fab arms, but promiscuous pairing of heavy and light chains remains a challenge for their manufacturing. METHODS: We have designed a solution in which the CH1 and CL domain pair in one of the Fab fragments is replaced with a CH3-domain pair and heterodimerized to facilitate correct modified Fab-chain pairing in bispecific heterodimeric antibodies based on a strand-exchange engineered domain (SEED) scaffold with specificity for epithelial growth factor receptor and either CD3 or CD16 (FcγRIII). RESULTS: Bispecific antibodies retained binding to their target antigens and redirected primary T cells or NK cells to induce potent killing of target cells. All antibodies were expressed at a high yield in Expi293F cells, were detected as single sharp symmetrical peaks in size exclusion chromatography and retained high thermostability. Mass spectrometric analysis revealed specific heavy-to-light chain pairing for the bispecific SEED antibodies as well as for one-armed SEED antibodies co-expressed with two different competing light chains. CONCLUSION: Incorporation of a constant domain-exchanged Fab fragment into a SEED antibody yields functional molecules with favorable biophysical properties. GENERAL SIGNIFICANCE: Our results show that the novel engineered bispecific SEED antibody scaffold with an incorporated Fab fragment with CH3-exchanged constant domains is a promising tool for the generation of complete heterodimeric bispecific antibodies with correct light chain pairing.

MMP-10 and TIMP-1 as indicators of severe sepsis in adult hematological patients with febrile neutropenia.

Commonly used indicators of sepsis are nonspecific and insufficient for predicting the course of febrile neutropenia (FN) in hematological patients. We analyzed data from 91 adult FN patients who received intensive chemotherapy for acute myeloid leukemia or autologous stem cell transplantation. Compared to patients with non-severe sepsis, patients with severe sepsis had significantly higher serum levels of tissue inhibitor of metalloproteinases-1 on the day of first occurrence of fever (day 0: 172 vs. 112 µg/L, p= .002) and for the two following days (day 1: 219 vs. 128 µg/L, p< .001; day 2: 443 vs. 128 µg/L, p= .001), and significantly higher serum levels of matrix metalloproteinase-10 on day 1 (1975 vs. 876 ng/L, p= .001) and day 2 (2020 vs. 841 ng/L, p< .001). We conclude that the measurement of these biomarkers may be useful in predicting the severity of sepsis in FN patients.

The potential of zwitterionic nanoliposomes against neurotoxic alpha-synuclein aggregates in Parkinson's Disease.

The protein α-synuclein (αSN) aggregates to form fibrils in neuronal cells of Parkinson's patients. Here we report on the effect of neutral (zwitterionic) nanoliposomes (NLPs), supplemented with cholesterol (NLP-Chol) and decorated with PEG (NLP-Chol-PEG), on αSN aggregation and neurotoxicity. Both NLPs retard αSN fibrillization in a concentration-independent fashion. They do so largely by increasing lag time (formation of fibrillization nuclei) rather than elongation (extension of existing nuclei). Interactions between neutral NLPs and αSN may locate to the N-terminus of the protein. This interaction can even perturb the interaction of αSN with negatively charged NLPs which induces an α-helical structure in αSN. This interaction was found to occur throughout the fibrillization process. Both NLP-Chol and NLP-Chol-PEG were shown to be biocompatible in vitro, and to reduce αSN neurotoxicity and reactive oxygen species (ROS) levels with no influence on intracellular calcium in neuronal cells, emphasizing a prospective role for NLPs in reducing αSN pathogenicity in vivo as well as utility as a vehicle for drug delivery.