Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex

Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named “PGDM1400–1412,” show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC₅₀ of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.Multiple methods have been developed to isolate HIV broadly neutralizing antibodies (bnAbs) (1–12). Hybridoma and phage display techniques were used to isolate the first generation of bnAbs including b12, 2F5, 2G12, 4E10, and Z13 (13–20). These antibodies exhibit a range of neutralization breadth against primary isolates from 30 to 90% but have moderate neutralization potency (median IC₅₀ of ∼2–4 µg/mL). Access to infected donors who have high serum titers of bnAbs (21, 22) and the availability of newer approaches for isolating human mAbs have recently enabled the discovery of a new generation of more potent bnAbs (1–4, 6–8).One of the newer approaches involves the sorting and activation of large numbers of memory B cells using cytokine-secreting feeder cells and the subsequent high-throughput screening of supernatants for neutralization. This method led to the identification and characterization of the first of the new generation of bnAbs, PG9 and PG16 (1), and since then has revealed several sites of vulnerability to bnAb recognition on HIV envelope (Env) (1–4, 6, 7). An alternative method of bnAb isolation involves the use of soluble Env molecules or scaffold proteins as baits to select single IgG⁺ memory B cells of interest by cell sorting (6, 8, 9, 23, 24). However, soluble baits have not been successful in isolating antibody responses targeting quaternary epitopes, including the trimer-apex site surrounding the N160 glycan, because the protein constructs used to date have not properly mimicked native Env trimers. To address this problem, GFP-labeled 293T cells that express cell-surface Env, called “GFP-293T^BaL cells,” were used recently to isolate antibodies 3BC176 and 3BC315 (10, 25). These antibodies do not bind soluble monomeric gp120 but do bind Env trimer, demonstrating the utility of the approach, but the method was reported to be less efficient than the use of soluble protein baits (10, 25).The favorable antigenic profile of the soluble BG505 SOSIP.664 gp140 trimer opens the possibility of its use for isolating quaternary-specific antibodies by single-cell sorting (26). To this end, we used BG505 SOSIP.664 gp140 to select for memory B cells from a donor from whom we previously had isolated the trimer-specific bnAbs PGT141–145 (3, 21). (For naming of PGT and PGDM bnAbs, please see SI Materials and Methods, Antibody Nomenclature.) We describe the isolation of previously unidentified somatic variants that are highly divergent from PGT145 and display a range of neutralization breadth and potency, with some being broader and more potent than the previously described PGT145 family members. Overall, the results reveal a mosaic of antibody responses against the trimer-apex site of vulnerability that have important implications for immunogen design in general and for the future optimization of BG505 SOSIP.664 and related native-like trimers as vaccine candidates.     

Spatiotemporal control of epithelial remodeling by regulated myosin phosphorylation

Spatiotemporally regulated actomyosin contractility generates the forces that drive epithelial cell rearrangements and tissue remodeling. Phosphorylation of the myosin II regulatory light chain (RLC) promotes the assembly of myosin monomers into active contractile filaments and is an essential mechanism regulating the level of myosin activity. However, the effects of phosphorylation on myosin localization, dynamics, and function during epithelial remodeling are not well understood. In Drosophila, planar polarized myosin contractility is required for oriented cell rearrangements during elongation of the body axis. We show that regulated myosin phosphorylation influences spatial and temporal properties of contractile behavior at molecular, cellular, and tissue length scales. Expression of myosin RLC variants that prevent or mimic phosphorylation both disrupt axis elongation, but have distinct effects at the molecular and cellular levels. Unphosphorylatable RLC produces fewer, slower cell rearrangements, whereas phosphomimetic RLC accelerates rearrangement and promotes higher-order cell interactions. Quantitative live imaging and biophysical approaches reveal that both phosphovariants reduce myosin planar polarity and mechanical anisotropy, altering the orientation of cell rearrangements during axis elongation. Moreover, the localized myosin activator Rho-kinase is required for spatially regulated myosin activity, even when the requirement for phosphorylation is bypassed by the expression of phosphomimetic myosin RLC. These results indicate that myosin phosphorylation influences both the level and the spatiotemporal regulation of myosin activity, linking molecular properties of myosin activity to tissue morphogenesis.Contractile assemblies of actin filaments and the nonmuscle myosin II motor protein produce mechanical forces that generate changes in cell shape during cytokinesis, cell movements during cell migration, and the dynamic remodeling of multicellular tissues (1–3). During development, spatiotemporal patterns of actomyosin contractility remodel simple epithelia into functional tissues with complex form and structure. Contractile force generation requires the assembly of inactive myosin monomers into active bipolar filaments (4). Phosphorylation of the myosin II regulatory light chain promotes myosin filament assembly and the movement of the myosin motor along actin filaments in vitro (5, 6) and is necessary for cytokinesis (7–9), oogenesis (7, 10, 11), and tissue morphogenesis (12–15). However, the in vivo effects of regulatory light chain phosphorylation on myosin localization, dynamics, and force generation, and how these properties are integrated to achieve complex changes in the shapes of tissues, are not well understood.Epithelial elongation in the Drosophila embryo is driven by cell rearrangements that are coordinated by spatiotemporal patterns of actomyosin contractility. Myosin contractility is spatially regulated in the plane of the epithelium (termed planar polarity), driving the planar polarized contraction of cell interfaces oriented close to perpendicular to the anterior–posterior axis (AP edges) (16–20). New interfaces preferentially form between dorsal and ventral cells (DV edges), resulting in oriented cell rearrangements that rapidly elongate the body axis of the animal from head to tail (16–18, 21). The myosin II activator Rho-associated protein kinase (Rho-kinase) localizes to regions of cells that display strong actomyosin activity during development (14, 15, 22) and promotes myosin contractility by phosphorylating the myosin regulatory light chain (RLC) and inactivating myosin phosphatase (23). Rho-kinase is required for localized myosin contractility during Drosophila axis elongation (14), neural tube closure in chick (22), and in other epithelia that undergo planar polarized cell behaviors (24, 25). Localized phosphorylation by Rho-kinase could therefore serve as an instructive cue that determines where and when myosin is activated within the cell. However, constitutively active myosin variants that evade this upstream regulation are sufficient to support normal myosin function during cytokinesis (26, 27), oogenesis (7), and wing hair formation (12), indicating that in some contexts regulated myosin phosphorylation is dispensable for morphogenesis. This raises the alternative possibility that the primary requirement for phosphorylation is to activate myosin, and additional mechanisms regulate the spatiotemporal patterns of myosin localization within epithelia.Here we use time-lapse imaging and biophysical approaches to analyze the role of myosin phosphorylation in polarized cell rearrangements during Drosophila axis elongation. Myosin II regulatory light chain variants that prevent or mimic phosphorylation both disrupt axis elongation, but have distinct effects on myosin localization, dynamics, and cell behavior. These results indicate that regulated myosin phosphorylation provides an instructive cue that directs both the magnitude and spatiotemporal pattern of myosin activity during epithelial remodeling, linking molecular-scale myosin activity to cell behavior and tissue morphogenesis.     

Dysfunctional breathing treated with continuous positive airway pressure in newly diagnosed obstructive sleep apnoea: a prospective cohort study

A prospective cohort study investigating patients with obstructive sleep apnoea (OSA) was conducted to determine the prevalence of dysfunctional breathing and if continuous positive airway pressure (CPAP) therapy improves associated symptoms. Almost half of newly diagnosed patients with OSA had dysfunctional breathing and CPAP was not an effective treatment. Dysfunctional breathing is common in patients with OSA.     

Frequency of laboratory testing and associated abnormalities in patients with hypertension

Clinical practice guidelines recommend several routine laboratory tests in patients diagnosed with hypertension. However, the rates of clinically relevant laboratory abnormalities are unknown. Therefore, we conducted a retrospective cohort study using administrative and laboratory data of patients diagnosed with hypertension between April 2010 and March 2015 in Alberta, Canada. Laboratory investigations for renal function, serum electrolytes (sodium and potassium), low‐density lipoprotein (LDL) cholesterol, and diabetes (fasting blood glucose and hemoglobin A1c), measured within 1 year of diagnosis, were examined, and the frequency of abnormalities determined. A total of 225 296 cases of incident hypertension were identified. Of these, 74.3% received at least one of the four guideline‐recommended laboratory tests, but only 42.3% received all four tests. Patients who received any testing, compared to subjects who did not, were on average older (median age 55.9 vs 51.2 years, P < .001) and had more comorbidity (14.5% vs 2.8% with a Charlson comorbidity index ≥ 3, P < .001). Laboratory abnormalities with the potential to affect clinical decision‐making were more common among multi‐comorbid patients. Patients with renal dysfunction (6.7% vs 11.6%, 26.3%, P < .001), electrolyte abnormalities (9.8% vs 12.6%, 20.5%, P < .001), and diabetes (13.4% vs 25.1% vs 38.8%, P < .001) were found in patients with Charlson scores of 0 vs 1‐2 vs ≥3, respectively. Our study found most patients diagnosed with hypertension received some laboratory testing, but rates of laboratory testing and frequency of abnormalities varied by clinical context. Testing and abnormalities detected were both more common among older patients and patients with comorbidities.     

Insight into the 24‐hour ambulatory central blood pressure in adolescents and young adults

This study attempted to investigate the behavior of 24‐hour central ambulatory blood pressure (ABP) in adolescents and young adults. Adolescents and young adults (age 10‐25 years) referred for elevated blood pressure (BP) and healthy volunteers had simultaneous 24‐hour peripheral (brachial) and central (aortic) ABP monitoring using the same automated upper‐arm cuff device (Mobil‐O‐Graph 24h PWA). Central BP was calculated by the device using two different calibration methods (C1SBP using peripheral systolic (pSBP)/diastolic BP and C2SBP using mean arterial/diastolic BP). A total of 136 participants (age 17.9 ± 4.7 years, 54% adolescents, 77% males, 25% volunteers, 34% with elevated peripheral ABP) were analyzed. Twenty‐four‐hour pSBP was higher than C1SBP, with this difference being more pronounced during daytime than nighttime (16.3 ± 4.5 and 10.5 ± 3.2 mm Hg, respectively, P < .001). Younger age, higher body height, and male gender were associated with greater systolic ABP amplification (pSBP‐C1SBP difference). C1SBP followed the variation pattern of pSBP, yet with smaller nighttime dip (8.4 ± 6.0% vs 11.9 ± 4.6%, P < .001), whereas C2SBP increased (2.4 ± 7.2%) during nighttime sleep (P < .001 for comparison with pSBP change). Older age remained independent determinant of larger nighttime BP fall for pSBP and C1SBP, whereas male gender predicted a larger nighttime C2SBP rise. These data suggest that the calibration method of the BP monitor considerably influences the diurnal variation in central BP, showing a lesser nocturnal dip than pSBP or even nocturnal BP rise, which are determined by the individual''s age and gender.     

Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress

The occurrence of repeat-associated non-ATG (RAN) translation, an atypical form of translation of expanded repeats that results in the synthesis of homopolymeric expansion proteins, is becoming more widely appreciated among microsatellite expansion disorders. Such disorders include amyotrophic lateral sclerosis and frontotemporal dementia caused by a hexanucleotide repeat expansion in the C9ORF72 gene (c9FTD/ALS). We and others have recently shown that this bidirectionally transcribed repeat is RAN translated, and the “c9RAN proteins” thusly produced form neuronal inclusions throughout the central nervous system of c9FTD/ALS patients. Nonetheless, the potential contribution of c9RAN proteins to disease pathogenesis remains poorly understood. In the present study, we demonstrate that poly(GA) c9RAN proteins are neurotoxic and may be implicated in the neurodegenerative processes of c9FTD/ALS. Specifically, we show that expression of poly(GA) proteins in cultured cells and primary neurons leads to the formation of soluble and insoluble high molecular weight species, as well as inclusions composed of filaments similar to those observed in c9FTD/ALS brain tissues. The expression of poly(GA) proteins is accompanied by caspase-3 activation, impaired neurite outgrowth, inhibition of proteasome activity, and evidence of endoplasmic reticulum (ER) stress. Of importance, ER stress inhibitors, salubrinal and TUDCA, provide protection against poly(GA)-induced toxicity. Taken together, our data provide compelling evidence towards establishing RAN translation as a pathogenic mechanism of c9FTD/ALS, and suggest that targeting the ER using small molecules may be a promising therapeutic approach for these devastating diseases.     

Pathways to Assignment of Payees

How clients come to be assigned representative payees and/or conservators to manage their funds is not well understood. We compared clients assigned a payee during a clinical trial of a money management-based intervention to those not assigned payees and examined antecedents to payee assignment. One year after randomization, significantly more clients assigned to the advisor teller money manager (ATM) money management intervention were assigned payees than participants in the control condition (10 of 47 vs. 2 of 43; p = .02); those assigned payees had lower baseline GAF scores and participated more in study therapies. Several ATM clients were assigned payees after third parties paid more attention to clients’ finances, and others after having negotiated storage of their funds with the ATM money manager during the study. Assignment of payees appears to be influenced by whether third parties critically attend to how clients’ manage funds and by clients’ receptiveness to having a payee.