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SUMMARY: Dinoflagellates have a rich history of characterization of their membrane‐reinforcing sterols because of a structural diversity and chemotaxonomic utility uncommon to other classes of algae. The Kareniaceae are no exception in that they produce sterols, often containing a Δ8(14) nuclear unsaturation, which are rare in most other dinoflagellate groups. A continuing goal of our laboratory is to examine the sterols of previously uncharacterized dinoflagellates to assess their sterol‐based chemotaxonomy compared to other members of the Dinophyceae. Asterodinium gracile, a member of the Kareniaceae, has not been commercially available for study until recently. To this end, our objective was to characterize the sterols of A. gracile to determine whether they match the 4α‐methyl‐substituted, Δ8(14)‐nuclear‐unsaturated sterols, such as (24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (gymnodinosterol; C29:2) and 27‐nor‐(24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (brevesterol; C28:2), of most species within the canonical genera Karenia, Karlodinium and Takayama, or instead the 4‐desmethyl sterols, such as 27‐nor‐(24R)‐23‐methyl‐ergosta‐8(14),22‐dien‐3β‐ol (C28:2), of its chemotaxonomically atypical, yet closest phylogenetic relative, Karenia papilionacea. We have observed A. gracile to produce two 4α‐methyl‐substituted sterols with the suggested structures of 4α‐methyl‐5α‐ergosta‐8(14)‐en‐3β‐ol (C29:1) and 4α‐methyl‐5α‐ergosta‐8(14),24(28)‐dien‐3β‐ol (amphisterol, C29:2), as major sterols amongst five other minor sterols, which included the common dinoflagellate sterol cholest‐5‐en‐3β‐ol (cholesterol, C27:1), but did not include gymnodinosterol or brevesterol (as in most canonical Kareniaceae) or 27‐nor‐(24R)‐23‐methyl‐ergosta‐8(14),22‐dien‐3β‐ol (as found in K. papilionacea). Detection of amphisterol and 4α‐methyl‐5α‐ergosta‐8(14)‐en‐3β‐ol is notable because they are sterols associated with some species of Amphidinium, such as Amphidinium carterae, and, to our knowledge, have not been observed in a member of the Kareniaceae before. Discovery of these major sterols in A. gracile expands our knowledge of the range of sterols produced by the Kareniaceae and indicates a shared chemotaxonomy with some species of Amphidinium. [ABSTRACT FROM AUTHOR]

Small, low-power, and inexpensive marine depth sensors are of interest for a myriad of applications from maritime security to environmental monitoring. Recently, laser-induced graphene (LIG) piezoresistive pressure sensors have been proposed given their rapid fabrication and large dynamic range. In this work, the practicality of LIG integration into fieldable deep ocean (1 km) depth sensors in bulk is explored. Initially, a design of experiments (DOEs) approach evaluated laser engraver fabrication parameters such as line length, line width, laser speed, and laser power on resultant resistances of LIG traces. Next, uniaxial compression and thermal testing at relevant ocean pressures up to 10.3 MPa and temperatures between 0 and 25 °C evaluated the piezoresistive response of replicate sensors and determined the individual characterization of each, which is necessary. Additionally, bare LIG sensors showed larger resistance changes with temperature (ΔR ≈ 30 kΩ) than pressure (ΔR ≈ 1–15 kΩ), indicating that conformal coatings are required to both thermally insulate and electrically isolate traces from surrounding seawater. Sensors encapsulated with two dip-coated layers of 5 wt% polydimethylsiloxane (PDMS) silicone and submerged in water baths from 0 to 25 °C showed significant thermal dampening (ΔR ≈ 0.3 kΩ), indicating a path forward for the continued development of LIG/PDMS composite structures. This work presents both the promises and limitations of LIG piezoresistive depth sensors and recommends further research to validate this platform for global deployment. [ABSTRACT FROM AUTHOR]

SUMMARY: Asterodinium gracile is a morphologically distinct, star‐shaped member of the Kareniaceae with, like canonical Kareniaceae, a tertiary plastid of haptophyte origin. However, A. gracile's complement of carotenoid photosynthetic pigments has been shown to be chemotaxonomically atypical in that it possesses much less fucoxanthin when compared to that of other, canonical Kareniaceae in the genera Karenia, Karlodinium, and Takayama, also with a tertiary plastid of haptophyte origin. To date, Karenia mikimotoi, Karenia papilionacea, and Karenia selliformis are the only canonical Kareniaceae that have been shown to have a chemotaxonomically atypical carotenoid pigment composition in that they possess a gyroxanthin diester‐like carotenoid not observed in other species of Karenia, Karlodinium, or Takayama (recognizing that Karenia, in general, produces fucoxanthin derivatives not observed in Karlodinium or Takayama). As a photosynthetic organism, K. mikimotoi has been shown to resemble Karenia brevis such that both species possess the chloroplast‐associated galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively) enriched with octadecapentaenoic acid (18:5(n‐3)) in the sn‐1 position, and hexadecenoic acid (16:0) and tetradecanoic acid (14:0) at the sn‐2 position. However, K. mikimotoi is chemotaxonomically atypical beyond its carotenoid composition in that it possesses MGDG and DGDG with hexadecatetraenoic acid (16:4(n‐3)), which has not been observed in any other members of the Kareniaceae, in the sn‐2 position as major galactolipids. The goal of this study was to characterize the galactolipids of A. gracile with the hypothesis that they would also be atypical when compared to other canonical Kareniaceae because of A. gracile's atypical carotenoid pigment composition. To this end, we report that like K. brevis and K. mikimotoi, A. gracile produces MGDG and DGDG enriched in 18:5(n‐3) at the sn‐1 position and C14 fatty acids, such as 14:0, at the sn‐2 position, and like K. mikimotoi, it produces 18:5(n‐3)/16:4(n‐3) MGDG, yet here as its most abundant galactolipid. [ABSTRACT FROM AUTHOR]

Various pathophysiological situations of negative energy balance involve the intense depletion of the body's energy reserves. White adipose tissue is a central place to store energy and a major endocrine organ. As a model of choice to better understand how the white adipose tissue dynamically responds to changes in substrate availability, we used the prolonged fasting paradigm, which is characterized by successive periods of stimulated (phase 2) and then reduced (phase 3) lipid mobilization/utilization. Using omics analyses, we report a regulatory transcriptional program in rat epididymal (EPI) adipose tissue favoring lipolysis during phase 2 and repressing it during phase 3. Changes in gene expression levels of lipases, lipid droplet-associated factors, and the proteins involved in cAMP-dependent and cAMP-independent regulation of lipolysis are highlighted. The mRNA and circulating levels of adipose-secreted factors were consistent with the repression of insulin signaling during prolonged fasting. Other molecular responses are discussed, including the regulation of leptin and adiponectin levels, the specific changes reflecting an increased fibrinolysis and a possible protein catabolism-related energy saving mechanism in late fasting. Finally, some differences between internal and subcutaneous (SC) adipose tissues are also reported. These data provide a comprehensive molecular basis of adipose tissue responses when facing a major energetic challenge. [ABSTRACT FROM AUTHOR]

We report on arrayed germanium-on-silicon waveguide photodetectors for high-power analog applications utilizing the AIM Photonics silicon foundry. Photodetector arrays with two, four, and eight elements reach output powers of −0.4 dBm, 10 dBm, and 14.3 dBm at 18 GHz, 12 GHz, and 5 GHz, respectively. The 4-photodiode array has an output third order intercept point (OIP3) above 20 dBm up to 12 GHz and shows a 5-dB improvement over a single photodiode. An integrated 20 GHz receiver based on a pair of balanced photodiodes and a Mach–Zehnder delay line interferometer is successfully demonstrated in an optical phase-modulated link. [ABSTRACT FROM AUTHOR]

The bioproduction of recombinant monoclonal antibodies results in complex mixtures of a main isoform and numerous macro- and microvariants. Monoclonal antibodies therefore present different levels of heterogeneities ranging from primary sequence variants to post-translational modifications. Among these heterogeneities, the truncation and fragmentation of the primary amino-acid sequence result in shorter or cleaved polypeptide chains while the incomplete processing of the signal peptide produces N-terminal elongated polypeptide chains. Here, we present an in-gel protein N-terminal chemical derivatization method using (N-succinimidyloxycarbonylmethyl)-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP). This chemical tag enhances the detection by mass spectrometry of the N-terminal positions of proteins and allows their unambiguous assignment without altering the identification of internal digestion peptides. This method adds just one step to the classical peptide mapping workflow. Using this in-gel N-TOP (N-terminal oriented proteomics) strategy, the N-terminal sequence heterogeneities of several monoclonal antibodies, among which are minor unexpected proteoforms, were successfully detected and characterized. [ABSTRACT FROM AUTHOR]

The rapid growth of approved biotherapeutics, e.g., monoclonal antibodies or immunoglobulins G (IgGs), demands improved techniques for their quality control. Traditionally, proteolysis-based bottom-up mass spectrometry (MS) has been employed. However, the long, multistep sample preparation protocols required for bottom-up MS are known to potentially introduce artifacts in the original sample. For this reason, a top-down MS approach would be preferable. The current performance of top-down MS of intact monoclonal IgGs, though, enables reaching only up to 30% sequence coverage, with incomplete sequencing of the complementarity determining regions which are fundamental for IgG's antigen binding. Here, we describe a middle-down MS protocol based on the use of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS), which is capable of digesting IgGs in only ~30 min. After chemical reduction, the obtained ~25 kDa proteolytic fragments were analyzed by reversed phase liquid chromatography (LC) coupled online with an electron transfer dissociation (ETD)-enabled hybrid Orbitrap Fourier transform mass spectrometer (Orbitrap Elite FTMS). Upon optimization of ETD and product ion transfer parameters, results show that up to ~50% sequence coverage for selected IgG fragments is reached in a single LC run and up to ~70% when data obtained by distinct LC-MS runs are averaged. Importantly, we demonstrate the potential of this middle-down approach in the identification of oxidized methionine residues. The described approach shows a particular potential for the analysis of IgG mixtures. [ABSTRACT FROM AUTHOR]

The protein ReP1-NCXSQ was isolated from the cytosol of squid nerves and has been shown to be required for MgATP stimulation of the squid nerve Na+/Ca2+ exchanger NCXSQ1. In order to determine its mode of action and the corresponding biologically active ligand, sequence analysis, crystal structures and mass-spectrometric studies of this protein and its Tyr128Phe mutant are reported. Sequence analysis suggests that it belongs to the CRABP family in the FABP superfamily. The X-ray structure at 1.28 Å resolution shows the FABP β-barrel fold, with a fatty acid inside the barrel that makes a relatively short hydrogen bond to Tyr128 and shows a double bond between C9 and C10 but that is disordered beyond C12. Mass-spectrometric studies identified this fatty acid as palmitoleic acid, confirming the double bond between C9 and C10 and establishing a length of 16 C atoms in the aliphatic chain. This acid was caught inside during the culture in Escherichia coli and therefore is not necessarily linked to the biological activity. The Tyr128Phe mutant was unable to activate the Na+/Ca2+ exchanger and the corresponding crystal structure showed that without the hydrogen bond to Tyr128 the palmitoleic acid inside the barrel becomes disordered. Native mass-spectrometric analysis confirmed a lower occupancy of the fatty acid in the Tyr128Phe mutant. The correlation between (i) the lack of activity of the Tyr128Phe mutant, (ii) the lower occupancy/disorder of the bound palmitoleic acid and (iii) the mass-spectrometric studies of ReP1-NCXSQ suggests that the transport of a fatty acid is involved in regulation of the NCXSQ1 exchanger, providing a novel insight into the mechanism of its regulation. In order to identify the biologically active ligand, additional high-resolution mass-spectrometric studies of the ligands bound to ReP1-NCXSQ were performed after incubation with squid nerve vesicles both with and without MgATP. These studies clearly identified palmitic acid as the fatty acid involved in regulation of the Na+/Ca2+ exchanger from squid nerve. [ABSTRACT FROM AUTHOR]

Highlights: [•] Extensive biosimilar/reference mAb comparison is required in biosimilar development. [•] In EU and USA, biosimilar/reference mAb primary amino-acid sequences must be the same. [•] MS, chromatography and electrophoresis are central in biosimilar characterization. [•] MS approaches allow detection of small differences in biosimilar/originator mAbs. [•] HDX/native/quantitative MS will become pivotal techniques for comparability studies. [ABSTRACT FROM AUTHOR]

The chloroquine resistance transporter PfCRT of the human malaria parasite Plasmodium falciparum confers resistance to the former first-line antimalarial drug chloroquine, and it modulates the responsiveness to a wide range of quinoline and quinoline-like compounds. PfCRT is post-translationally modified by phosphorylation, palmitoylation, and, possibly, ubiquitination. However, the impact of these post-translational modifications on P. falciparum biology and, in particular, the drug resistance-conferring activity of PfCRT has remained elusive. Here, we confirm phosphorylation at Ser-33 and Ser-411 of PfCRT of the chloroquine-resistant P. falciparum strain Dd2 and show that kinase inhibitors can sensitize drug responsiveness. Using CRISPR/Cas9 genome editing to generate genetically engineered PfCRT variants in the parasite, we further show that substituting Ser-33 with alanine reduced chloroquine and quinine resistance by ~50% compared with the parental P. falciparum strain Dd2, whereas the phosphomimetic amino acid aspartic acid could fully and glutamic acid could partially reconstitute the level of chloroquine/quinine resistance. Transport studies conducted in the parasite and in PfCRT-expressing Xenopus laevis oocytes linked phosphomimetic substitution at Ser-33 to increased transport velocity. Our data are consistent with phosphorylation of Ser-33 relieving an autoinhibitory intramolecular interaction within PfCRT, leading to a stimulated drug transport activity. Our findings shed additional light on the function of PfCRT and suggest that chloroquine could be reevaluated as an antimalarial drug by targeting the kinase in P. falciparum that phosphorylates Ser-33 of PfCRT. [ABSTRACT FROM AUTHOR]