Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational,structure-guided Fv engineering

Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies.     

Reduced genetic diversity and increased reproductive isolation follow population‐level loss of larval dispersal in a marine gastropod

Population‐level consequences of dispersal ability remain poorly understood, especially for marine animals in which dispersal is typically considered a species‐level trait governed by oceanographic transport of microscopic larvae. Transitions from dispersive (planktotrophic) to nondispersive, aplanktonic larvae are predicted to reduce connectivity, genetic diversity within populations, and the spatial scale at which reproductive isolation evolves. However, larval dimorphism within a species is rare, precluding population‐level tests. We show the sea slug Costasiella ocellifera expresses both larval morphs in Florida and the Caribbean, regions with divergent mitochondrial lineages. Planktotrophy predominated at 11 sites, 10 of which formed a highly connected and genetically diverse Caribbean metapopulation. Four populations expressed mainly aplanktonic development and had markedly reduced connectivity, and lower genetic diversity at one mitochondrial and six nuclear loci. Aplanktonic dams showed partial postzygotic isolation in most interpopulation crosses, regardless of genetic or geographic distance to the sire's source, suggesting that outbreeding depression affects fragmented populations. Dams from genetically isolated and neighboring populations also exhibited premating isolation, consistent with reinforcement contingent on historical interaction. By increasing self‐recruitment and genetic drift, the loss of dispersal may thus initiate a feedback loop resulting in the evolution of reproductive isolation over small spatial scales in the sea.     

Stroke Damage Is Exacerbated by Nano-Size Particulate Matter in a Mouse Model

This study examines the effects of nano-size particulate matter (nPM) exposure in the setting of murine reperfused stroke. Particulate matter is a potent source of inflammation and oxidative stress. These processes are known to influence stroke progression through recruitment of marginally viable penumbral tissue into the ischemic core. nPM was collected in an urban area in central Los Angeles, impacted primarily by traffic emissions. Re-aerosolized nPM or filtered air was then administered to mice through whole body exposure chambers for forty-five cumulative hours. Exposed mice then underwent middle cerebral artery occlusion/ reperfusion. Following cerebral ischemia/ reperfusion, mice exposed to nPM exhibited significantly larger infarct volumes and less favorable neurological deficit scores when compared to mice exposed to filtered air. Mice exposed to nPM also demonstrated increases in markers of inflammation and oxidative stress in the region of the ischemic core. The findings suggest a detrimental effect of urban airborne particulate matter exposure in the setting of acute ischemic stroke.     

A plague of waterfleas (<Emphasis Type="Italic">Bythotrephes</Emphasis>): impacts on microcrustacean community structure,seasonal biomass,and secondary production in a large inland-lake complex

The spiny cladoceran (Bythotrephes longimanus) is an invasive, predaceous zooplankter that is expanding from Great Lakes coastal waters into inland lakes within a northern latitudinal band. In a large, Boundary Water lake complex (largely within Voyageurs National Park), we use two comparisons, a 2-year spatial and a 12-year temporal, to quantify seasonal impacts on food webs and biomass, plus a preliminary calculation of secondary production decline. Bythotrephes alters the seasonal biomass pattern by severely depressing microcrustaceans during summer and early fall, when the predator is most abundant. Cladoceran and cyclopoid copepods suffer the most serious population declines, although the resistant cladoceran Holopedium is favored in spatial comparisons. Microcrustacean biomass is reduced 40–60 % and secondary production declines by about 67 %. The microcrustacean community shifts towards calanoid copepods. The decline in secondary production is due both to summer biomass loss and to the longer generation times of calanoid copepods (slower turnover). The Bythotrephes “top-down” perturbation appears to hold across small, intermediate, and large-sized lakes (i.e. appears scale-independent), and is pronounced when Bythotrephes densities reach 20–40 individuals L^?1. Induction tests with small cladocerans (Bosmina) suggest that certain native prey populations do not sense the exotic predator and are “blind-sided”. Failure of prey to deploy defenses could explain the disproportionate community impacts in New World versus Old World lakes.     

Control of cerebral blood velocity with furosemide-induced hypovolemia and upright tilt

The purpose of this study was to test the hypothesis that exacerbated reductions of cerebral blood velocity (CBV) during upright tilt with dehydration are associated with impaired cerebrovascular control. Nine healthy men were tilted head-up (HUT) to 70° for 10 min on two occasions separated by 7 days under euhydration (EUH) and dehydration (DEH; 40 mg of furosemide and water restriction) conditions. Beat-by-beat arterial pressures and CBV were measured during a 5-min supine baseline and during the first (T1) and last (T2) 5 min of HUT. Cerebral autoregulation and arterial baroreflex sensitivity were assessed in the frequency domain with cross-spectral techniques. DEH reduced plasma volume by 10% (P = 0.008) and supine mean CBV (CBV(mean)) by 11% (P = 0.002). Mean arterial pressure (MAP), stroke volume, and baroreflex sensitivity decreased during HUT (P ≤ 0.002), but absolute reductions were similar between hydration conditions, with the exception of stroke volume, which was lower at T1 during DEH than EUH (P = 0.04). CBV(mean) during DEH was lower (7 cm/s) over the course of the entire 10 min of HUT (P ≤ 0.004) than during EUH. Low-frequency oscillations (0.07-0.2 Hz) of MAP and CBV(mean) and MAP-CBV(mean) coherence were higher during DEH than EUH at T1 (P ≤ 0.02), but not at T2. Our results suggest that increased coherence between arterial pressure and CBV with the combination of DEH and HUT are indicative of altered cerebrovascular control. Increased CBV oscillations with DEH may reflect acute protective mechanisms to ensure adequate cerebral perfusion under conditions of reduced central blood volume.     

Free Fatty Acid Effects on the Atrial Myocardium: Membrane Ionic Currents Are Remodeled by the Disruption of T-Tubular Architecture

Background

Epicardial adiposity and plasma levels of free fatty acids (FFAs) are elevated in atrial fibrillation, heart failure and obesity, with potentially detrimental effects on myocardial function. As major components of epicardial fat, FFAs may be abnormally regulated, with a potential to detrimentally modulate electro-mechanical function. The cellular mechanisms underlying such effects of FFAs are unknown.

Objective

To determine the mechanisms underlying electrophysiological effects of palmitic (PA), stearic (SA) and oleic (OA) FFAs on sheep atrial myocytes.

Methods

We used electrophysiological techniques, numerical simulations, biochemistry and optical imaging to examine the effects of acutely (≤ 15 min), short-term (4–6 hour) or 24-hour application of individual FFAs (10 μM) on isolated ovine left atrial myocytes (LAMs).

Results

Acute and short-term incubation in FFAs resulted in no differences in passive or active properties of isolated left atrial myocytes (LAMs). 24-hour application had differential effects depending on the FFA. PA did not affect cellular passive properties but shortened (p<0.05) action potential duration at 30% repolarization (APD₃₀). APD₅₀ and APD₈₀ were unchanged. SA had no effect on resting membrane potential but reduced membrane capacitance by 15% (p<0.05), and abbreviated APD at all values measured (p≤0.001). OA did not significantly affect passive or active properties of LAMs. Measurement of the major voltage-gated ion channels in SA treated LAMs showed a ~60% reduction (p<0.01) of the L-type calcium current (I_Ca-L) and ~30% reduction (p<0.05) in the transient outward potassium current (I_TO). A human atrial cell model recapitulated SA effects on APD. Optical imaging showed that SA incubated for 24 hours altered t-tubular structure in isolated cells (p<0.0001).

Conclusions

SA disrupts t-tubular architecture and remodels properties of membrane ionic currents in sheep atrial myocytes, with potential implications in arrhythmogenesis.     

Dynamic model of CHO cell metabolism

Fed-batch cultures are extensively used for the production of therapeutic proteins. However, process optimization is hampered by lack of quantitative models of mammalian cellular metabolism in these cultures. This paper presents a new kinetic model of CHO cell metabolism and a novel framework for simulating the dynamics of metabolic and biosynthetic pathways of these cells grown in fed-batch culture. The model defines a subset of the intracellular reactions with kinetic rate expressions based on extracellular metabolite concentrations and temperature- and redox-dependent regulatory variables. The simulation uses the rate expressions to calculate pseudo-steady state flux distributions and extracellular metabolite concentrations at discrete time points. Experimental data collected in this study for several different CHO cell fed-batch cultures are used to derive the rate expressions, fit the parameters, and validate the model. The simulations accurately predicted the effects of process variables, including temperature shift, seed density, specific productivity, and nutrient concentrations.     

Revision of the genus Menevia Schaus, 1928 (Lepidoptera,Mimallonoidea, Mimallonidae) with the description of 11 new species

The Neotropical genus Menevia Schaus, 1928 is revised to include 18 species, 11 of which are new. Two species, Menevia ostia comb. n. and Menevia parostia comb. n. are transferred from Pamea Walker, 1855 to Menevia. Four species-groups are diagnosed for the first time based on external characters and male genitalia morphology. The following new species are described: Menevia rosea sp. n., Menevia torvamessoria sp. n., Menevia magna sp. n., Menevia menapia sp. n., Menevia mielkei sp. n., Menevia australis sp. n., Menevia vulgaris sp. n., Menevia franclemonti sp. n., Menevia vulgaricula sp. n., Menevia cordillera sp. n., and Menevia delphinus sp. n.. A neotype is designated for Mimallo plagiata Walker, 1855, which has since been placed in Menevia. Mimallo saturata Walker, 1855 is interpreted to be a nomen dubium.     

Expression of Mirlet7 family microRNAs in response to retinoic acid-induced spermatogonial differentiation in mice

Spermatogonial differentiation is orchestrated by the precise control of gene expression involving retinoic acid signaling. MicroRNAs have emerged as important regulators of spermatogenesis, and here we show that the Mirlet7 family miRNAs are expressed in mouse spermatogonia and spermatocytes. Retinoic acid significantly leads to the induction of Mirlet7 miRNAs through suppression of Lin28. We further confirmed both in vitro and in vivo that expressions of Mycn, Ccnd1, and Col1a2, which are targets of Mirlet7, were downregulated during spermatogonial differentiation. These results suggest that Mirlet7 family miRNAs play a role in retinoic acid-induced spermatogonial differentiation.     

Pristine Early Eocene Wood Buried Deeply in Kimberlite from Northern Canada

We report exceptional preservation of fossil wood buried deeply in a kimberlite pipe that intruded northwestern Canada’s Slave Province 53.3±0.6 million years ago (Ma), revealed during excavation of diamond source rock. The wood originated from forest surrounding the eruption zone and collapsed into the diatreme before resettling in volcaniclastic kimberlite to depths >300 m, where it was mummified in a sterile environment. Anatomy of the unpermineralized wood permits conclusive identification to the genus Metasequoia (Cupressaceae). The wood yields genuine cellulose and occluded amber, both of which have been characterized spectroscopically and isotopically. From cellulose δ¹⁸O and δ²H measurements, we infer that Early Eocene paleoclimates in the western Canadian subarctic were 12–17°C warmer and four times wetter than present. Canadian kimberlites offer Lagerstätte-quality preservation of wood from a region with limited alternate sources of paleobotanical information.