Growth stage and ensiling: impact on chemical composition, conservation quality and in situ ruminal degradability of whole-crop oat.

BACKGROUND: The effects of growth stage (GS) and ensiling were assessed on whole-crop oat's (Avena sativa L. cv. Cantara) chemical composition, silage fermentation quality and in situ ruminal degradability. Oat was harvested and ensiled at six GS: boot, heading, water ripe, early milk, early dough and grain ripe (144, 151, 178, 234, 362 and 512 g kg-1 of dry matter (DM) of whole-crop forage, respectively). RESULTS: GS influenced chemical composition, silage fermentation quality and ruminal degradability of whole-crop oat. Lower DM and higher water-soluble carbohydrates (WSC) contents lead to adequate forage compaction and fermentation at early GS; however, effluent was produced until the dough stage (L and Q; P ≤ 0.003). Advancing in maturity increased (P < 0.001) crop yield (4.5 to 9.4 t DM ha-1 ), DM (144 to 512 g kg-1 ), neutral detergent fibre (NDF; 537 to 571 g kg-1 DM), lignin (44.6 to 71.3 g kg-1 DM) and starch contents (26.4 to 201 g kg-1 DM), and reduced (P < 0.001) crude protein (107 to 60 g kg-1 DM) and WSC (115 to 17.5 g kg-1 DM). DM and NDF ruminal degradability declined with maturity for fresh and ensiled forages (L and Q; P < 0.05). Density and buffering capacity decreased with GS (L and Q; P < 0.001), whereas pH and soluble protein increased (L and Q; P ≤ 0.004). CONCLUSION: The growth stage of oat influenced the nutritive value and ruminal degradation to a greater extent than ensiling, and thus it can play a paramount role in whole-crop oat silage quality. © 2021 Society of Chemical Industry.

A robust heterodimeric Fc platform engineered for efficient development of bispecific antibodies of multiple formats.

Bispecific monoclonal antibodies can bind two protein targets simultaneously and enable therapeutic modalities inaccessible by traditional mAbs. Bispecific formats containing a heterodimeric Fc region are of particular interest, as a heterodimeric Fc empowers both bispecificity and altered valencies while retaining the developability and druggability of a monoclonal antibody. We present a robust heterodimeric Fc platform, called the XmAb® bispecific platform, engineered for efficient development of bispecific antibodies and Fc fusions of multiple formats. First, we engineer a purification solution for proteins containing a heterodimeric Fc using engineered isoelectric point differences in the Fc region that enable straightforward purification of the heterodimeric species. Then, we combine this purification solution with a novel set of Fc substitutions capable of achieving heterodimer yields over 95% with little change in thermostability. Next, we illustrate the flexibility of our heterodimeric Fc with a case study in which a wide range of tumor-associated antigen × CD3 bispecifics are generated, differing in choice of tumor antigen, affinities for both tumor antigen and CD3, and tumor antigen valency. Finally, we present manufacturing data reinforcing the robustness of the heterodimeric Fc platform at scale.

Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling.

Cellular demolition during apoptosis is completed by executioner caspases, that selectively cleave more than 1,500 proteins but whose individual roles are challenging to assess. Here, we used an optimized site-specific and inducible protease to examine the role of a classic apoptotic node, the caspase-activated DNase (CAD). CAD is activated when caspases cleave its endogenous inhibitor ICAD, resulting in the characteristic DNA laddering of apoptosis. We describe a posttranscriptional gene replacement (PTGR) approach where endogenous biallelic ICAD is knocked down and simultaneously replaced with an engineered allele that is susceptible to inducible cleavage by tobacco etch virus protease. Remarkably, selective activation of CAD alone does not induce cell death, although hallmarks of DNA damage are detected in human cancer cell lines. Our data strongly support that the highly cooperative action of CAD and inhibition of DNA repair systems are critical for the DNA laddering phenotype in apoptosis. Furthermore, the PTGR approach provides a general means for replacing wild-type protein function with a precisely engineered mutant at the transcriptional level that should be useful for cell engineering studies.

Virus-polymer hybrid nanowires tailored to detect prostate-specific membrane antigen.

We demonstrate the de novo fabrication of a biosensor, based upon virus-containing poly(3,4-ethylene-dioxythiophene) (PEDOT) nanowires, that detects prostate-specific membrane antigen (PSMA). This development process occurs in three phases: (1) isolation of a M13 virus with a displayed polypeptide receptor, from a library of ≈10(11) phage-displayed peptides, which binds PSMA with high affinity and selectivity, (2) microfabrication of PEDOT nanowires that entrain these virus particles using the lithographically patterned nanowire electrodeposition (LPNE) method, and (3) electrical detection of the PSMA in high ionic strength (150 mM salt) media, including synthetic urine, using an array of virus-PEDOT nanowires with the electrical resistance of these nanowires for transduction. The electrical resistance of an array of these nanowires increases linearly with the PSMA concentration from 20 to 120 nM in high ionic strength phosphate-buffered fluoride (PBF) buffer, yielding a limit of detection (LOD) for PSMA of 56 nM.

Synthesis of a virus electrode for measurement of prostate specific membrane antigen.

Though relatively unexploited in biosensor applications, phage display technology can provide versatile recognition scaffolds for detection of cancer markers and other analytes. This chapter details protocols for covalent attachment of viruses directly to electrodes for reagent-free detection of analytes in real-time. Customization of binding specificity leverages selections with large phage display libraries prior to covalent attachment of the selected virus to the electrode. The methods described here utilize electrochemical impedance spectroscopy (EIS) to detect molecular recognition between M13 phage bound to a Au electrode and the following analytes: prostate specific membrane antigen (PSMA), positive and negative control antibodies (p-Ab and n-Ab, respectively). Because of a thick layer built on the Au electrode, the real impedance (Zre) increases reliably with S/N ratios upon noncovalent binding to PSMA (approximately 14) and p-Ab (approximately 20).

Direct electrical transduction of antibody binding to a covalent virus layer using electrochemical impedance.

Electrochemical impedance spectroscopy is used to detect the binding of a 148.2 kDa antibody to a "covalent virus layer" (CVL) immobilized on a gold electrode. The CVL consisted of M13 phage particles covalently anchored to a 3 mm diameter gold disk electrode. The ability of the CVL to distinguish this antibody ("p-Ab") from a second, nonbinding antibody ("n-Ab") was evaluated as a function of the frequency and phase of the measured current relative to the applied voltage. The binding of p-Ab to the CVL was correlated with a change in the resistance, reducing it at low frequency (1-40 Hz) while increasing it at high frequency (2-140 kHz). The capacitance of the CVL was virtually uncorrelated with p-Ab binding. At both low and high frequency, the electrode resistance was linearly dependent on the p-Ab concentration from 20 to 266 nM but noise compromised the reproducibility of the p-Ab measurement at frequencies below 40 Hz. A "signal-to-noise" ratio for antibody detection was computed based upon the ratio between the measured resistance change upon p-Ab binding and the standard deviation of this change obtained from multiple measurements. In spite of the fact that the impedance change upon p-Ab binding in the low frequency domain was more than 100 times larger than that measured at high frequency, the S/N ratio at high frequency was higher and virtually independent of frequency from 4 to 140 kHz. Attempts to release p-Ab from the CVL using 0.05 M HCl, as previously described for mass-based detection, caused a loss of sensitivity that may be associated with a transition of these phage particles within the CVL from a linear to a coiled conformation at low pH.

Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins.

While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRASG12V, and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi screen identified integrin and Wnt signaling proteins as critical to RAS-transformed cells. We show that antibodies targeting CDCP1, a protein common to our proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and immunotherapeutic payloads to RAS-transformed cancer cells and report for RAS signaling status in vivo. Taken together, this work presents a technological platform for attacking RAS from outside the cell.

A Split-Abl Kinase for Direct Activation in Cells.

To dissect the cellular roles of individual kinases, it is useful to design tools for their selective activation. We describe the engineering of a split-cAbl kinase (sKin-Abl) that is rapidly activated in cells with rapamycin and allows temporal, dose, and compartmentalization control. Our design strategy involves an empirical screen in mammalian cells and identification of split site in the N lobe. This split site leads to complete loss of activity, which can be restored upon small-molecule-induced dimerization in cells. Remarkably, the split site is transportable to the related Src Tyr kinase and the distantly related Ser/Thr kinase, AKT, suggesting broader applications to kinases. To quantify the fold induction of phosphotyrosine (pTyr) modification, we employed quantitative proteomics, NeuCode SILAC. We identified a number of known Abl substrates, including autophosphorylation sites and novel pTyr targets, 432 pTyr sites in total. We believe that this split-kinase technology will be useful for direct activation of protein kinases in cells.

Computational design and selections for an engineered, thermostable terpene synthase.

Terpenoids include structurally diverse antibiotics, flavorings, and fragrances. Engineering terpene synthases for control over the synthesis of such compounds represents a long sought goal. We report computational design, selections, and assays of a thermostable mutant of tobacco 5-epi-aristolochene synthase (TEAS) for the catalysis of carbocation cyclization reactions at elevated temperatures. Selection for thermostability included proteolytic digestion followed by capture of intact proteins. Unlike the wild-type enzyme, the mutant TEAS retains enzymatic activity at 65°C. The thermostable terpene synthase variant denatures above 80°C, approximately twice the temperature of the wild-type enzyme.

Covalent virus layer for mass-based biosensing.

M13 virus particles were covalently attached to a planar gold-coated quartz crystal microbalance (QCM) through reaction with a self-assembled monolayer of N-hydroxysuccinimide thioctic ester, followed by incorporation of the blocking agent bovine serum albumin. This immobilization chemistry produced a phage multilayer having a coverage equivalent to approximately 6.5 close-packed monolayers of the virus. The properties of this "covalent virus surface" or CVS for the mass-based detection of a 148.2 kDa antibody were then evaluated in a phosphate buffer using a flow injection analysis system. The mass of the CVS increased with exposure to an antibody (p-Ab) known to bind the phage particles with high affinity. Bound p-Ab was removed by washing with 0.5 M HCl thereby regenerating the sensor surface. A calibration plot for p-Ab binding was constructed by repetitively exposing the surface to p-Ab at concentrations between 6.6 and 200 nM and HCl rinsing after each exposure. The mass-concentration relationship was linear with a sensitivity of 0.018 microg/(cm2 nM) and a limit of detection of 7 nM or 1.3 pmol. The CVS could be saturated with high doses of p-Ab enabling the determination that an average of approximately 140 binding sites are available per M13 phage particle. Exposure of the CVS to a second, nonbinding antibody (n-Ab) did not cause a measurable mass change. These results demonstrate that the covalent virus layer is a rugged, selective, and sensitive means for carrying out mass-based biodetection.

Exploring biochemistry and cellular biology with protein libraries.

Polypeptide libraries cast a broad net for defining enzyme and binding protein specificities. In addition to uncovering rules for molecular recognition, the binding preferences and functional group tolerances from such libraries can reveal mechanisms underlying biochemical and cellular processes. Ligands obtained from protein libraries can also provide pharmaceutical lead compounds and even reagents to further explore cell biology. Here, we review selected recent examples of protein libraries demonstrating these principles. In particular, we focus on combinatorial libraries composed of randomized peptides or variations of a single protein. The characteristics of various techniques for library constructions and screening are also briefly surveyed.