Targeted activation of toll-like receptors: conjugation of a toll-like receptor 7 agonist to a monoclonal antibody maintains antigen binding and specificity

Therapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of anti-tumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to non-specific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an anti-tumour mAb (the anti-hCD 20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods and yield, molecular substitution ratio, retention of TLR7 activity and specificity of antigen binding were compared. Both conjugation methods produced rituximab-UC-1V150 conjugates with UC-1V150 : rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method, and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional crosslinker was firstly reacted with rituximab, and secondly to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65-78%. Rituximab-UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28-53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab-UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells whilst no non-specific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible

High potency of lipid conjugated TLR7 agonist requires nanoparticulate or liposomal formulation

Conjugation of small molecule agonists of Toll-like receptor 7 (TLR7) to proteins, lipids, or polymers is known to modulate potency, and the physical form or formulation of these conjugates is likely to have a major effect on their immunostimulatory activity. Here, we studied the effect of formulation on potency of a 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) conjugated TLR7 agonist (DOPE-TLR7a) alongside assessing physical form using Dynamic Light Scattering (DLS), Nanosight Particle Tracking (NTA) analysis and Small Angle X-ray Scattering (SAXS). A very high potency of DOPE-TLR7a conjugate (EC50 around 9 nM) was observed either when prepared by direct dilution from DMSO or when formulated into 400-700 nm large multilamella liposomes containing dimethyldioctadecylammonium bromide salt (DDA) and DOPE. When prepared by dissolution in DMSO followed by dilution in aqueous culture medium, 93 ± 5 nm nanoparticles were formed. Without dilution from solution in DMSO, no nanoparticles were observed, and no immunostimulatory activity could be detected without this formulation step. SAXS analysis of the conjugate after DMSO dissolution/water dilution revealed a lamellar order with a layer spacing of 68.7 Å, which correlates with arrangement in groups of 3 bilayers. The addition of another immunostimulatory glycolipid, trehalose-6,6-dibehenate (TDB), to DOPE:DDA liposomes gave no further increase in immunostimulatory activity beyond that provided by incorporating DOPE-TLR7a. Given the importance of nanoparticle or liposomal formulation for activity, we conclude a major mechanism for increase in potency when TLR7 agonists are conjugated to macromolecules is through alteration of physical form

Teachers' classroom feedback: still trying to get it right

This article examines feedback traditionally given by teachers in schools. Such feedback tends to focus on children's acquisition and retrieval of externally prescribed knowledge which is then assessed against mandated tests. It suggests that, from a sociocultural learning perspective, feedback directed towards such objectives may limit children's social development. In this article, I draw on observation and interview data gathered from a group of 27 9- to 10-year olds in a UK primary school. These data illustrate the children's perceived need to conform to, rather than negotiate, the teacher's feedback comments. They highlight the children's sense that the teacher's feedback relates to school learning but not to their own interests. The article also includes alternative examples of feedback which draw on children's own inquiries and which relate to the social contexts within which, and for whom, they act. It concludes by suggesting that instead of looking for the right answer to the question of what makes teachers' feedback effective in our current classrooms, a more productive question might be how a negotiation can be opened up among teachers and learners themselves, about how teachers' feedback could support children's learning most appropriately

How temporal patterns in rainfall determine the geomorphology and carbon fluxes of tropical peatlands

Tropical peatlands now emit hundreds of megatons of carbon dioxide per year because of human disruption of the feedbacks that link peat accumulation and groundwater hydrology. However, no quantitative theory has existed for how patterns of carbon storage and release accompanying growth and subsidence of tropical peatlands are affected by climate and disturbance. Using comprehensive data from a pristine peatland in Brunei Darussalam, we show how rainfall and groundwater flow determine a shape parameter (the Laplacian of the peat surface elevation) that specifies, under a given rainfall regime, the ultimate, stable morphology, and hence carbon storage, of a tropical peatland within a network of rivers or canals. We find that peatlands reach their ultimate shape first at the edges of peat domes where they are bounded by rivers, so that the rate of carbon uptake accompanying their growth is proportional to the area of the still-growing dome interior. We use this model to study how tropical peatland carbon storage and fluxes are controlled by changes in climate, sea level, and drainage networks. We find that fluctuations in net precipitation on timescales from hours to years can reduce long-term peat accumulation. Our mathematical and numerical models can be used to predict long-term effects of changes in temporal rainfall patterns and drainage networks on tropical peatland geomorphology and carbon storage

Discovery of the Optical Afterglow and Host Galaxy of Short GRB 181123B at z = 1.754: Implications for Delay Time Distributions

We present the discovery of the optical afterglow and host galaxy of the Swift short-duration gamma-ray burst (SGRB) GRB 181123B. Observations with Gemini-North starting ≈9.1 hr after the burst reveal a faint optical afterglow with i ≈ 25.1 mag at an angular offset of 0.”59 ± 0.”16 from its host galaxy. Using grizYJHK observations, we measure a photometric redshift of the host galaxy of z = 1.77^(+0.30)_(−0.17). From a combination of Gemini and Keck spectroscopy of the host galaxy spanning 4500–18000 Å, we detect a single emission line at 13390 Å, inferred as Hβ at z = 1.754 ± 0.001 and corroborating the photometric redshift. The host galaxy properties of GRB 181123B are typical of those of other SGRB hosts, with an inferred stellar mass of ≈9.1 × 10⁹ M⊙, a mass-weighted age of ≈0.9 Gyr, and an optical luminosity of ≈0.9L*. At z = 1.754, GRB 181123B is the most distant secure SGRB with an optical afterglow detection and one of only three at z > 1.5. Motivated by a growing number of high-z SGRBs, we explore the effects of a missing z > 1.5 SGRB population among the current Swift sample on delay time distribution (DTD) models. We find that lognormal models with mean delay times of ≈4–6 Gyr are consistent with the observed distribution but can be ruled out to 95% confidence, with an additional ≈one to five Swift SGRBs recovered at z > 1.5. In contrast, power-law models with ∝t⁻¹ are consistent with the redshift distribution and can accommodate up to ≈30 SGRBs at these redshifts. Under this model, we predict that ≈1/3 of the current Swift population of SGRBs is at z > 1. The future discovery or recovery of existing high-z SGRBs will provide significant discriminating power on their DTDs and thus their formation channels

Discovery of the Optical Afterglow and Host Galaxy of Short GRB 181123B at z = 1.754: Implications for Delay Time Distributions

We present the discovery of the optical afterglow and host galaxy of the Swift short-duration gamma-ray burst (SGRB) GRB 181123B. Observations with Gemini-North starting ≈9.1 hr after the burst reveal a faint optical afterglow with i ≈ 25.1 mag at an angular offset of 0farcs59 ± 0farcs16 from its host galaxy. Using grizYJHK observations, we measure a photometric redshift of the host galaxy of $z={1.77}_{-0.17}^{+0.30}$. From a combination of Gemini and Keck spectroscopy of the host galaxy spanning 4500–18000 Å, we detect a single emission line at 13390 Å, inferred as Hβ at z = 1.754 ± 0.001 and corroborating the photometric redshift. The host galaxy properties of GRB 181123B are typical of those of other SGRB hosts, with an inferred stellar mass of ≈9.1 × 109 M ⊙, a mass-weighted age of ≈0.9 Gyr, and an optical luminosity of ≈0.9L*. At z = 1.754, GRB 181123B is the most distant secure SGRB with an optical afterglow detection and one of only three at z > 1.5. Motivated by a growing number of high-z SGRBs, we explore the effects of a missing z > 1.5 SGRB population among the current Swift sample on delay time distribution (DTD) models. We find that lognormal models with mean delay times of ≈4–6 Gyr are consistent with the observed distribution but can be ruled out to 95% confidence, with an additional ≈one to five Swift SGRBs recovered at z > 1.5. In contrast, power-law models with ∝t −1 are consistent with the redshift distribution and can accommodate up to ≈30 SGRBs at these redshifts. Under this model, we predict that ≈1/3 of the current Swift population of SGRBs is at z > 1. The future discovery or recovery of existing high-z SGRBs will provide significant discriminating power on their DTDs and thus their formation channels

Net greenhouse gas balance of fibre wood plantation on peat in Indonesia

Tropical peatlands cycle and store large amounts of carbon in their soil and biomass1,2,3,4,5. Climate and land-use change alters greenhouse gas (GHG) fluxes of tropical peatlands, but the magnitude of these changes remains highly uncertain6,7,8,9,10,11,12,13,14,15,16,17,18,19. Here we measure net ecosystem exchanges of carbon dioxide, methane and soil nitrous oxide fluxes between October 2016 and May 2022 from Acacia crassicarpa plantation, degraded forest and intact forest within the same peat landscape, representing land-cover-change trajectories in Sumatra, Indonesia. This allows us to present a full plantation rotation GHG flux balance in a fibre wood plantation on peatland. We find that the Acacia plantation has lower GHG emissions than the degraded site with a similar average groundwater level (GWL), despite more intensive land use. The GHG emissions from the Acacia plantation over a full plantation rotation (35.2 ± 4.7 tCO2-eq ha−1 year−1, average ± standard deviation) were around two times higher than those from the intact forest (20.3 ± 3.7 tCO2-eq ha−1 year−1), but only half of the current Intergovernmental Panel on Climate Change (IPCC) Tier 1 emission factor (EF)20 for this land use. Our results can help to reduce the uncertainty in GHG emissions estimates, provide an estimate of the impact of land-use change on tropical peat and develop science-based peatland management practices as nature-based climate solutions