A potential antibody repertoire diversification mechanism through tyrosine sulfation for biotherapeutics engineering and production

The diversity of three hypervariable loops in antibody heavy chain and light chain, termed the complementarity-determining regions (CDRs), defines antibody’s binding affinity and specificity owing to the direct contact between the CDRs and antigens. These CDR regions typically contain tyrosine (Tyr) residues that are known to engage in both nonpolar and pi stacking interaction with antigens through their complementary aromatic ring side chains. Nearly two decades ago, sulfotyrosine residue (sTyr), a negatively charged Tyr formed by Golgi-localized membrane-bound tyrosylprotein sulfotransferases during protein trafficking, were also found in the CDR regions and shown to play an important role in modulating antibody-antigen interaction. This breakthrough finding demonstrated that antibody repertoire could be further diversified through post-translational modifications, in addition to the conventional genetic recombination. This review article summarizes the current advances in the understanding of the Tyr-sulfation modification mechanism and its application in potentiating protein-protein interaction for antibody engineering and production. Challenges and opportunities are also discussed

A Quantitative LC-MS/MS Method for Determination of a Small Molecule Agonist of EphA2 in Mouse Plasma and Brain Tissue

Compound 27 {1, 12‐bis[4‐(4‐amino‐6,7‐dimethoxyquinazolin‐2‐yl)piperazin‐1‐yl]dodecane‐1,12‐dione} is a novel small molecule agonist of EphA2 receptor tyrosine kinase. It showed much improved activity for the activation of EphA2 receptor compared with the parental compound doxazosin. To support further pharmacological and toxicological studies of the compound, a method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC–MS/MS) has been developed for the quantification of this compound. Liquid–liquid extraction was used to extract the compound from mouse plasma and brain tissue homogenate. Reverse‐phase chromatography with gradient elution was performed to separate compound 27 from the endogenous molecules in the matrix, followed by MS detection using positive ion multiple reaction monitoring mode. Multiple reaction monitoring transitions m/z 387.3 → 290.1 and m/z 384.1 → 247.1 were selected for monitoring compound 27 and internal standard prazosin, respectively. The linear calibration range was 2–200 ng/mL with the intra‐ and inter‐day precision and accuracy within the acceptable range. This method was successfully applied to the quantitative analysis of compound 27 in mouse plasma and brain tissue with different drug administration routes

Differences in transcription between free-living and CO_2-activated third-stage larvae of Haemonchus contortus

Background: The disease caused by Haemonchus contortus, a blood-feeding nematode of small ruminants, is of major economic importance worldwide. The infective third-stage larva (L3) of this gastric nematode is enclosed in a cuticle (sheath) and, once ingested with herbage by the host, undergoes an exsheathment process that marks the transition from the free-living (L3) to the parasitic (xL3) stage. This study explored changes in gene transcription associated with this transition and predicted, based on comparative analysis, functional roles for key transcripts in the metabolic pathways linked to larval development. Results: Totals of 101,305 (L3) and 105,553 (xL3) expressed sequence tags (ESTs) were determined using 454 sequencing technology, and then assembled and annotated; the most abundant transcripts encoded transthyretin-like, calcium-binding EF-hand, NAD(P)-binding and nucleotide-binding proteins as well as homologues of Ancylostoma-secreted proteins (ASPs). Using an in silico-subtractive analysis, 560 and 685 sequences were shown to be uniquely represented in the L3 and xL3 stages, respectively; the transcripts encoded ribosomal proteins, collagens and elongation factors (in L3), and mainly peptidases and other enzymes of amino acid catabolism (in xL3). Caenorhabditis elegans orthologues of transcripts that were uniquely transcribed in each L3 and xL3 were predicted to interact with a total of 535 other genes, all of which were involved in embryonic development. Conclusion: The present study indicated that some key transcriptional alterations taking place during the transition from the L3 to the xL3 stage of H. contortus involve genes predicted to be linked to the development of neuronal tissue (L3 and xL3), formation of the cuticle (L3) and digestion of host haemoglobin (xL3). Future efforts using next-generation sequencing and bioinformatic technologies should provide the efficiency and depth of coverage required for the determination of the complete transcriptomes of different developmental stages and/or tissues of H. contortus as well as the genome of this important parasitic nematode. Such advances should lead to a significantly improved understanding of the molecular biology of H. contortus and, from an applied perspective, to novel methods of intervention

Alzheimer\u27s Disease Drug Development Pipeline: 2019

Introduction Alzheimer\u27s disease (AD) has few available treatments, and there is a high rate of failure in AD drug development programs. Study of the AD drug development pipeline can provide insight into the evolution of drug development and how best to optimize development practices. Methods We reviewed clinicaltrials.gov and identified all pharmacologic AD trials of all agents currently being developed for treatment of AD. Results There are 132 agents in clinical trials for the treatment of AD. Twenty-eight agents are in 42 phase 3 trials; 74 agents are in 83 phase 2 trials; and 30 agents are in 31 phase 1 trials. There is an increase in the number of agents in each phase compared with that in the 2018 pipeline. Nineteen agents in trials target cognitive enhancement, and 14 are intended to treat neuropsychiatric and behavioral symptoms. There are 96 agents in disease modification trials; of these, 38 (40%) have amyloid as the primary target or as one of several effects. Eighteen of the antiamyloid agents are small molecules, and 20 are monoclonal antibodies or biological therapies. Seven small molecules and ten biologics have tau as a primary or combination target (18%). Amyloid is the most common specific target in phase 3 and phase 2 disease modification trials. Novel biomarkers (e.g., neurofilament light), new outcomes (e.g., AD Composite Score [ADCOMS]), enrollment of earlier populations, and innovative trial designs (e.g., Bayesian adaptive designs) are new features in recent clinical trials. Discussion Drug development continues robustly at all phases despite setbacks in several programs in the recent past. Continuing unmet needs require a commitment to growing and accelerating the pipeline

Impact of Eastern Redcedar Proliferation on Water Resources in the Great Plains USA—Current State of Knowledge

In the Great Plains of the central United States, water resources for human and aquatic life rely primarily on surface runoff and local recharge from rangelands that are under rapid transformation to woodland by the encroachment of Eastern redcedar (redcedar; Juniperus virginiana) trees. In this synthesis, the current understanding and impact of redcedar encroachment on the water budget and water resources available for non-ecosystem use are reviewed. Existing studies concluded that the conversion from herbaceous-dominated rangeland to redcedar woodland increases precipitation loss to canopy interception and vegetation transpiration. The decrease of soil moisture, particularly for the subsurface soil layer, is widely documented. The depletion of soil moisture is directly related to the observed decrease in surface runoff, and the potential of deep recharge for redcedar encroached watersheds. Model simulations suggest that complete conversion of the rangelands to redcedar woodland at the watershed and basin scale in the South-central Great Plains would lead to reduced streamflow throughout the year, with the reductions of streamflow between 20 to 40% depending on the aridity of the climate of the watershed. Recommended topics for future studies include: (i) The spatial dynamics of redcedar proliferation and its impact on water budget across a regional hydrologic network; (ii) the temporal dynamics of precipitation interception by the herbaceous canopy; (iii) the impact of redcedar infilling into deciduous forests such as the Cross Timbers and its impact on water budget and water availability for non-ecosystem use; (iv) land surface and climate interaction and cross-scale hydrological modeling and forecasting; (v) impact of redcedar encroachment on sediment production and water quality; and (vi) assessment and efficacy of different redcedar control measures in restoring hydrological functions of watershed

Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins

PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication

MLPerf Inference Benchmark

Machine-learning (ML) hardware and software system demand is burgeoning. Driven by ML applications, the number of different ML inference systems has exploded. Over 100 organizations are building ML inference chips, and the systems that incorporate existing models span at least three orders of magnitude in power consumption and five orders of magnitude in performance; they range from embedded devices to data-center solutions. Fueling the hardware are a dozen or more software frameworks and libraries. The myriad combinations of ML hardware and ML software make assessing ML-system performance in an architecture-neutral, representative, and reproducible manner challenging. There is a clear need for industry-wide standard ML benchmarking and evaluation criteria. MLPerf Inference answers that call. In this paper, we present our benchmarking method for evaluating ML inference systems. Driven by more than 30 organizations as well as more than 200 ML engineers and practitioners, MLPerf prescribes a set of rules and best practices to ensure comparability across systems with wildly differing architectures. The first call for submissions garnered more than 600 reproducible inference-performance measurements from 14 organizations, representing over 30 systems that showcase a wide range of capabilities. The submissions attest to the benchmark's flexibility and adaptability.Comment: ISCA 202

Rasagiline Effects on Glucose Metabolism, Cognition, and Tau in Alzheimer’s Dementia

Background: A Phase II proof of concept (POC) randomized clinical trial was conducted to evaluate the effects of rasagiline, a monoamine oxidase B (MAO-B) inhibitor approved for Parkinson disease, in mild to moderate Alzheimer\u27s disease (AD). The primary objective was to determine if 1 mg of rasagiline daily for 24 weeks is associated with improved regional brain metabolism (fluorodeoxyglucose–positron emission tomography [FDG-PET]) compared to placebo. Secondary objectives included measurement of effects on tau PET and evaluation of directional consistency of clinical end points. Methods: This was a double-blind, parallel group, placebo-controlled, community-based, three-site trial of 50 participants randomized 1:1 to receive oral rasagiline or placebo (NCT02359552). FDG-PET was analyzed for the presence of an AD-like pattern as an inclusion criterion and as a longitudinal outcome using prespecified regions of interest and voxel-based analyses. Tau PET was evaluated at baseline and longitudinally. Clinical outcomes were analyzed using an intention-to-treat (ITT) model. Results: Fifty patients were randomized and 43 completed treatment. The study met its primary end point, demonstrating favorable change in FDG-PET differences in rasagiline versus placebo in middle frontal (P \u3c 0.025), anterior cingulate (P \u3c 0.041), and striatal (P \u3c 0.023) regions. Clinical measures showed benefit in quality of life (P \u3c 0.04). Digit Span, verbal fluency, and Neuropsychiatric Inventory (NPI) showed non-significant directional favoring of rasagiline; no effects were observed in Alzheimer\u27s Disease Assessment Scale-Cognitive Subscale (ADAS-cog) or activities of daily living. Rasagiline was generally well tolerated with low rates of adverse events and notably fewer neuropsychiatric symptoms in the active treatment group. Discussion: These outcomes illustrate the potential benefits of rasagiline on clinical and neuroimaging measures in patients with mild to moderate AD. Rasagiline appears to affect neuronal activity in frontostriatal pathways, with associated clinical benefit potential warranting a more fully powered trial. This study illustrated the potential benefit of therapeutic repurposing and an experimental medicine proof-of-concept design with biomarkers to characterize patient and detect treatment response