Evaluation of LC-MS data for the absolute quantitative analysis of marker proteins

The serum complexity makes the absolute quantitative analysis of medium to low-abundant proteins very challenging. Tens of thousands proteins are present in human serum and dispersed over an extremely wide dynamic range. The reliable identification and quantitation of proteins, which are potential biomarkers of disease, in serum or plasma as matrix still represents one of the most difficult analytical challenges. The difficulties arise from the presence of a few, but highly abundant proteins in serum and from the non-availability of isotope-labeled proteins, which serve to calibrate the method and to account for losses during sample preparation. For the absolute quantitation of serum proteins, we have developed an analytical scheme based on first-dimension separation of the intact proteins by anion-exchange high-performance liquid chromatography (HPLC), followed by proteolytic digestion and second-dimension separation of the tryptic peptides by reversed-phase HPLC in combination with electrospray ionization mass spectrometry (ESI-MS). The potential of mass spectrometric peptide identification in complex mixtures by means of peptide mass fingerprinting (PMF) and peptide fragment fingerprinting (PFF) was evaluated and compared utilizing synthetic mixtures of commercially available proteins and electrospray-ion trap- or electrospray time-of-flight mass spectrometers. While identification of peptides by PFF is fully supported by automated spectrum interpretation and database search routines, reliable identification by PMF still requires substantial efforts of manual calibration and careful data evaluation in order to avoid false positives. Quantitation of the identified peptides, however, is preferentially performed utilizing full-scan mass spectral data typical of PMF. Algorithmic solutions for PMF that incorporate both recalibration and automated feature finding on the basis of peak elution profiles and isotopic patterns are therefore highly desirable in order to speed up the process of data evaluation and calculation of quantitative results. Calibration for quantitative analysis of serum proteins was performed upon addition of known amounts of authentic protein to the serum sample. This was essential for the analysis of human serum samples, for which isotope-labeled protein standards are usually not available. We present the application of multidimensional HPLC-ESI-MS to the absolute quantitative analysis of myoglobin in human serum, a very sensitive biomarker for myocardial infarction. It was possible to determine myoglobin concentrations in human serum down to 100-500 ng/mL. Calibration graphs were linear over at least one order of magnitude and the relative standard deviation of the method ranged from 7-15%

Belowground carbon transfer across mycorrhizal networks among trees: Facts, not fantasy

The mycorrhizal symbiosis between fungi and plants is among the oldest, ubiquitous and most important interactions in terrestrial life on Earth. Carbon (C) transfer across a common mycorrhizal network (CMN) was demonstrated over half a century ago in the lab (Reid and Woods 1969), and later in the field (Simard et al. 1997). Recent years have seen ample progress in this research direction, including evidence for ecological significance of carbon transfer (Klein et al. 2016). Furthermore, specific cases where the architecture of mycorrhizal networks have been mapped (Beiler et al. 2015) and CMNC transfer from mature trees to seedlings has been demonstrated (Orrego 2018) have suggested that trees in forests are more connected than once thought (Simard 2021). In a recent Perspective, Karst et al. (2023) offered a valuable critical review warning of overinterpretation and positive citation bias in CMN research. It concluded that while there is evidence for C movement among plants, the importance of CMNs remains unclear, as noted by others too (Henriksson et al. 2023). Here we argue that while some of these claims are justified, factual evidence about belowground C transfer across CMNs is solid and accumulating

Solution conformations of a linked construct of the Zika virus NS2B-NS3 protease

The Zika virus presents a serious risk for global health. Crystal structures of different constructs of the Zika virus NS2B-NS3 protease (NS2B-NS3pro) have been determined with the aim to provide a basis for rational drug discovery. In these structures, the C-terminal β-hairpin of NS2B, NS2Bc, was observed to be either disordered (open conformation) or bound to NS3pro complementing the substrate binding site (closed conformation). Enzymatically active constructs of flaviviral NS2B-NS3 proteases commonly used for inhibitor testing contain a covalent peptide linker between NS2B and NS3pro. Using a linked construct of Zika virus NS2B-NS3pro, we studied the location of NS2Bc relative to NS3pro in solution by pseudocontact shifts generated by a paramagnetic lanthanide tag attached to NS3pro. Both closed and open conformations were observed with different inhibitors. As the NS2B co-factor is involved in substrate binding of flaviviral NS2B-NS3 proteases, the destabilization of the closed conformation in the linked construct makes it an attractive tool to search for inhibitors that interfere with the formation of the enzymatically active, closed conformation.C.N. thanks the Alexander vonHumbolt Foundation for a Feodor-Lynen fellowship. Financialsupport by the Australian Research Council is gratefully acknowl-edged. C.K. acknowledges support by the Deutsche For-schungsgemeinschaft, grant KL 1356/3

Measurement of the dynamical dipolar coupling in a pair of magnetic nano-disks using a Ferromagnetic Resonance Force Microscope

International audienceWe perform an extensive experimental spectroscopic study of the collective spin-wave dynamics occurring in a pair of magnetic nano-disks coupled by the magneto-dipolar interaction. For this, we take advantage of the stray field gradient produced by the magnetic tip of a ferromagnetic resonance force microscope (f-MRFM) to continuously tune and detune the relative resonance frequencies between two adjacent nano-objects. This reveals the anti-crossing and hybridization of the spin-wave modes in the pair of disks. At the exact tuning, the measured frequency splitting between the binding and anti-binding modes precisely corresponds to the strength of the dynamical dipolar coupling $\Omega$. This accurate f-MRFM determination of $\Omega$ is measured as a function of the separation between the nano-disks. It agrees quantitatively with calculations of the expected dynamical magneto-dipolar interaction in our sample

Ibrutinib-based therapy reinvigorates CD8 T cells compared to chemoimmunotherapy: immune-monitoring from the E1912 trial

Bruton's tyrosine kinase Inhibitors (BTKis) that target B cell receptor signaling have led to a paradigm shift in CLL treatment. BTKis have been shown to reduce abnormally high CLL-associated T cell counts and the expression of immune checkpoint receptors concomitantly with tumor reduction. However, the impact of BTKi therapy on T cell function has not been fully characterized. Here, we performed longitudinal immunophenotypic and functional analysis of pre- and on-treatment (6- and 12-months) peripheral blood samples from patients in the phase 3 E1912 trial comparing ibrutinib-rituximab to fludarabine, cyclophosphamide and rituximab (FCR). Intriguingly, we report that despite reduced overall T cell counts, higher numbers of T cells including effector CD8+ subsets at baseline and at the 6-month time-point associated with no infections and favorable progression-free survival (PFS) in the ibrutinib-rituximab arm. Assays demonstrated enhanced anti-CLL T cell killing function during ibrutinib-rituximab, including a switch from predominantly CD4+ T-cell:CLL immune synapses at baseline to increased CD8+ lytic synapses on-therapy. Conversely, in the FCR arm, higher T cell numbers correlated with adverse clinical responses and showed no functional improvement. We further demonstrate the potential of exploiting rejuvenated T cell cytotoxicity during ibrutinib-rituximab using the bispecific antibody glofitamab - supporting combination immunotherapy approaches

Novel mutations expand the clinical spectrum of DYNC1H1-associated spinal muscular atrophy

OBJECTIVE To expand the clinical phenotype of autosomal dominant congenital spinal muscular atrophy with lower extremity predominance (SMA-LED) due to mutations in the dynein, cytoplasmic 1, heavy chain 1 (DYNC1H1) gene. METHODS Patients with a phenotype suggestive of a motor, non-length-dependent neuronopathy predominantly affecting the lower limbs were identified at participating neuromuscular centers and referred for targeted sequencing of DYNC1H1. RESULTS We report a cohort of 30 cases of SMA-LED from 16 families, carrying mutations in the tail and motor domains of DYNC1H1, including 10 novel mutations. These patients are characterized by congenital or childhood-onset lower limb wasting and weakness frequently associated with cognitive impairment. The clinical severity is variable, ranging from generalized arthrogryposis and inability to ambulate to exclusive and mild lower limb weakness. In many individuals with cognitive impairment (9/30 had cognitive impairment) who underwent brain MRI, there was an underlying structural malformation resulting in polymicrogyric appearance. The lower limb muscle MRI shows a distinctive pattern suggestive of denervation characterized by sparing and relative hypertrophy of the adductor longus and semitendinosus muscles at the thigh level, and diffuse involvement with relative sparing of the anterior-medial muscles at the calf level. Proximal muscle histopathology did not always show classic neurogenic features. CONCLUSION Our report expands the clinical spectrum of DYNC1H1-related SMA-LED to include generalized arthrogryposis. In addition, we report that the neurogenic peripheral pathology and the CNS neuronal migration defects are often associated, reinforcing the importance of DYNC1H1 in both central and peripheral neuronal functions

Solar Energetic Particles Produced by a Slow Coronal Mass Ejection at ∼0.25 au

We present an analysis of Parker Solar Probe (PSP) IS⊙IS observations of ~30–300 keV n⁻¹ ions on 2018 November 11 when PSP was about 0.25 au from the Sun. Five hours before the onset of a solar energetic particle (SEP) event, a coronal mass ejection (CME) was observed by STEREO-A/COR2, which crossed PSP about a day later. No shock was observed locally at PSP, but the CME may have driven a weak shock earlier. The SEP event was dispersive, with higher energy ions arriving before the lower energy ones. Timing suggests the particles originated at the CME when it was at ~7.4R_⊙. SEP intensities increased gradually from their onset over a few hours, reaching a peak, and then decreased gradually before the CME arrived at PSP. The event was weak, having a very soft energy spectrum (−4 to −5 spectral index). The earliest arriving particles were anisotropic, moving outward from the Sun, but later, the distribution was observed to be more isotropic. We present numerical solutions of the Parker transport equation for the transport of 30–300 keV n⁻¹ ions assuming a source comoving with the CME. Our model agrees well with the observations. The SEP event is consistent with ion acceleration at a weak shock driven briefly by the CME close to the Sun, which later dissipated before arriving at PSP, followed by the transport of ions in the interplanetary magnetic field