A general approach to antibody thermostabilization

Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.     

Cash transfers for HIV prevention: A systematic review

BackgroundGiven the success of cash programs in improving health outcomes and addressing upstream drivers of HIV risk such as poverty and education, there has been an increasing interest in their potential to improve HIV prevention and care outcomes. Recent reviews have documented the impacts of structural interventions on HIV prevention, but evidence about the effects of cash transfer programs on HIV prevention has not been systematically reviewed for several years.Methods and findingsWe did a systematic review of published and unpublished literature to update and summarize the evidence around cash programs for HIV prevention from January 2000 to December 17, 2020. We included studies with either a cash transfer intervention, savings program, or program to reduce school costs. Included studies measured the program’s impact on HIV infection, other sexually transmitted infections (STIs), or sexual behaviors. We screened 1,565 studies and examined 78 in full-text review to identify a total of 45 peer-reviewed publications and reports from 27 different interventions or populations. We did not do a meta-analysis given the range of outcomes and types of cash transfer interventions assessed. Most studies were conducted in sub-Saharan Africa (N = 23; South Africa, Tanzania, Malawi, Lesotho, Kenya, Uganda, Zimbabwe, Zambia, and eSwatini) followed by Mexico (N = 2), the United States (N = 1), and Mongolia (N = 1)). Of the 27 studies, 20 (72%) were randomized trials, 5 (20%) were observational studies, 1 (4%) was a case–control study, and 1 (4%) was quasi-experimental. Most studies did not identify a strong association between the program and sexual behaviors, except sexual debut (10/18 finding an association; 56%). Eight of the 27 studies included HIV biomarkers, but only 3 found a large reduction in HIV incidence or prevalence, and the rest found no statistically significant association. Of the studies that identified a statistically significant association with other STIs (N = 4/8), 2 involved incentives for staying free of the STI, and the other 2 were cash transfer programs for adolescent girls that had conditionalities related to secondary schooling. Study limitations include the small number of studies in key populations and examining interventions to reduce school costs and matched saving programs.ConclusionsThe evidence base for large-scale impacts of cash transfers reducing HIV risk is limited; however, government social protection cash transfer programs and programs that incentivize school attendance among adolescent girls and young women show the greatest promise for HIV prevention.

Marie Stoner and co-workers assess the evidence on potential benefits of cash transfer interventions for HIV prevention.     

Lysophosphatidic acid induces endothelial cell death by modulating the redox environment

Oxidant stress plays a significant role in hypoxic-ischemic injury to the susceptible microvascular endothelial cells. During oxidant stress, lysophosphatidic acid (LPA) concentrations increase. We explored whether LPA caused cytotoxicity to neuromicrovascular cells and the potential mechanisms thereof. LPA caused a dose-dependent death of porcine cerebral microvascular as well as human umbilical vein endothelial cells; cell death appeared oncotic rather than apoptotic. LPA-induced cell death was mediated via LPA(1) receptor, because the specific LPA(1) receptor antagonist THG1603 fully abrogated LPA's effects. LPA decreased intracellular GSH levels and induced a p38 MAPK/JNK-dependent inducible nitric oxide synthase (NOS) expression. Pretreatment with the antioxidant GSH precursor N-acetyl-cysteine (NAC), as well as with inhibitors of NOS [N(omega)-nitro-l-arginine (l-NNA); 1400W], significantly prevented LPA-induced endothelial cell death (in vitro) to comparable extents; as expected, p38 MAPK (SB203580) and JNK (SP-600125) inhibitors also diminished cell death. LPA did not increase indexes of oxidation (isoprostanes, hydroperoxides, and protein nitration) but did augment protein nitrosylation. Endothelial cytotoxicity by LPA in vitro was reproduced ex vivo in brain and in vivo in retina; THG1603, NAC, l-NNA, and combined SB-203580 and SP600125 prevented the microvascular rarefaction. Data implicate novel properties for LPA as a modulator of the cell redox environment, which partakes in endothelial cell death and ensued neuromicrovascular rarefaction.     

Analysis of SRC Oncogenic Signaling in Colorectal Cancer by Stable Isotope Labeling with Heavy Amino Acids in Mouse Xenografts

The non-receptor tyrosine kinase SRC is frequently deregulated in human colorectal cancer (CRC), and SRC increased activity has been associated with poor clinical outcomes. In nude mice engrafted with human CRC cells, SRC over-expression favors tumor growth and is accompanied by a robust increase in tyrosine phosphorylation in tumor cells. How SRC contributes to this tumorigenic process is largely unknown. We analyzed SRC oncogenic signaling in these tumors by means of a novel quantitative proteomic analysis. This method is based on stable isotope labeling with amino acids of xenograft tumors by the addition of [¹³C₆]-lysine into mouse food. An incorporation level greater than 88% was obtained in xenograft tumors after 30 days of the heavy lysine diet. Quantitative phosphoproteomic analysis of these tumors allowed the identification of 61 proteins that exhibited a significant increase in tyrosine phosphorylation and/or association with tyrosine phosphorylated proteins upon SRC expression. These mainly included molecules implicated in vesicular trafficking and signaling and RNA binding proteins. Most of these proteins were specific targets of SRC signaling in vivo, as they were not identified by analysis via stable isotope labeling by amino acids in cell culture (SILAC) of the same CRC cells in culture. This suggests that oncogenic signaling induced by SRC in tumors significantly differs from that induced by SRC in cell culture. We next confirmed this notion experimentally with the example of the vesicular trafficking protein and SRC substrate TOM1L1. We found that whereas TOM1L1 depletion only slightly affected SRC-induced proliferation of CRC cells in vitro, it drastically decreased tumor growth in xenografted nude mice. We thus concluded that this vesicular trafficking protein plays an important role in SRC-induced tumor growth. Overall, these data show that SILAC analysis in mouse xenografts is a valuable approach for deciphering tyrosine kinase oncogenic signaling in vivo.The non-receptor tyrosine kinase (TK)¹ SRC mediates cellular signaling induced by growth factors and integrins (1) leading to cell growth, survival, and migration. It also has oncogenic activity when deregulated, a role originally described for the constitutively active v-SRC (2) that has since been observed in a large variety of human cancers (3). Remarkably, elevated SRC kinase activity has been found in more than 80% of colorectal cancers (CRCs) to levels (5- to 10-fold) consistent with oncogenic properties (4). Moreover, SRC deregulation has been associated with poor clinical outcomes (3), suggesting an additional function of SRC during late tumorigenesis. SRC deregulation largely occurs in the absence of mutations in the SRC gene. Instead, it primarily involves protein over-expression (2) and inhibition of SRC negative regulators, such as the transmembrane protein Cbp/PAG (5, 6). A large body of evidence indicates that SRC deregulation is an important event in colon tumorigenesis (3, 6). Indeed, SRC controls growth, survival, and invasion of some CRC cell lines in vitro (4). Moreover, it contributes to tumor growth, angiogenesis, and metastasis formation in mouse colon tumor xenograft models (7–11). However, our knowledge of the SRC-dependent oncogenic signaling pathway in CRC is largely incomplete, mostly because the majority of data have been obtained in two-dimensional cell culture models. Moreover, the standard culture conditions of CRC cells do not allow the recapitulation of all the SRC-dependent signaling cascades that are activated during tumorigenesis to promote tumor growth, angiogenesis, and interactions with the microenvironment.MS-based quantitative phosphoproteomic technology has been a valuable tool for deciphering signaling pathways initiated by a given TK (12). Particularly, the method of stable isotope labeling with amino acids in cell culture (SILAC) has been employed for the characterization of oncogenic TK signaling pathways in cell culture, including HER2 (13) and BCR-ABL (14). We recently used this powerful approach to investigate SRC-dependent oncogenic signaling in CRC cells (15) and identified the first SRC-dependent tyrosine “phosphoproteome” in these cells. Additionally, we found that SRC phosphorylated a small cluster of TKs that mediate its oncogenic activity, thus uncovering a TK network that is important for the induction of CRC cell growth (15). Whether these signaling processes also operate in vivo is, however, currently unknown.SRC oncogenic signaling could be investigated in vivo using similar MS-based quantitative phosphoproteomic approaches in animal models or tumor biopsies. However, the application of the SILAC method in vivo has been challenging until recently because it requires efficient protein labeling in different tissues, which is conditioned by the rate of de novo protein synthesis. Recently, Mann et al. described the successful development of a SILAC approach for labeling mice that is based on the addition of [¹³C₆]-lysine to their food (16). They reported complete labeling from the F2 generation. Similar SILAC approaches were then described for additional multicellular organisms, such as worms (17), flies (18), and zebrafish (19). Here, we report a similar SILAC approach in which we labeled tumors in nude mice xenografted with human CRC cells. We reasoned that the high rate of de novo protein synthesis occurring in tumors should allow efficient tumor labeling in a short period of time. Indeed, we obtained consistent (>88%) labeling of the tumor proteome by feeding xenografted mice with the SILAC mouse diet for only 30 days. We then used this approach to compare the tyrosine phosphoproteome of SRC over-expressing tumors (labeled with heavy amino acids) and of control tumors (labeled with light amino acids) and report the first SRC-dependent tyrosine phosphoproteome of CRC in vivo. Finally, comparison of the in vivo and in vitro SRC-dependent tyrosine phosphoproteomes showed that some of the SRC substrates were specifically activated only in CRC xenograft tumors, and not in cultured CRC cells.     

Photosynthesis of temperate Eucalyptus globulus trees outside their native range has limited adjustment to elevated CO2 and climate warming

Eucalyptus species are grown widely outside of their native ranges in plantations on all vegetated continents of the world. We predicted that such a plantation species would show high potential for acclimation of photosynthetic traits across a wide range of growth conditions, including elevated [CO₂] and climate warming. To test this prediction, we planted temperate Eucalyptus globulus Labill. seedlings in climate‐controlled chambers in the field located >700 km closer to the equator than the nearest natural occurrence of this species. Trees were grown in a complete factorial combination of elevated CO₂ concentration (eC; ambient [CO₂] +240 ppm) and air warming treatments (eT; ambient +3 °C) for 15 months until they reached ca. 10 m height. There was little acclimation of photosynthetic capacity to eC and hence the CO₂‐induced photosynthetic enhancement was large (ca. 50%) in this treatment during summer. The warming treatment significantly increased rates of both carboxylation capacity (V_cmax) and electron transport (J_max) (measured at a common temperature of 25 °C) during winter, but decreased them significantly by 20–30% in summer. The photosynthetic CO₂ compensation point in the absence of dark respiration (Γ*) was relatively less sensitive to temperature in this temperate eucalypt species than for warm‐season tobacco. The temperature optima for photosynthesis and J_max significantly changed by about 6 °C between winter and summer, but without further adjustment from early to late summer. These results suggest that there is an upper limit for the photosynthetic capacity of E. globulus ssp. globulus outside its native range to acclimate to growth temperatures above 25 °C. Limitations to temperature acclimation of photosynthesis in summer may be one factor that defines climate zones where E. globulus plantation productivity can be sustained under anticipated global environmental change.     

Obesity and body fat classification in the metabolic syndrome: Impact on cardiometabolic risk metabotype

Objective:

Obesity is a key factor in the development of the metabolic syndrome (MetS), which is associated with increased cardiometabolic risk. We investigated whether obesity classification by BMI and body fat percentage (BF%) influences cardiometabolic profile and dietary responsiveness in 486 MetS subjects (LIPGENE dietary intervention study).

Design and Methods:

Anthropometric measures, markers of inflammation and glucose metabolism, lipid profiles, adhesion molecules, and hemostatic factors were determined at baseline and after 12 weeks of four dietary interventions (high saturated fat (SFA), high monounsaturated fat (MUFA), and two low fat high complex carbohydrate (LFHCC) diets, one supplemented with long chain n‐3 polyunsaturated fatty acids (LC n‐3 PUFAs)).

Results:

About 39 and 87% of subjects classified as normal and overweight by BMI were obese according to their BF%. Individuals classified as obese by BMI (≥30 kg/m²) and BF% (≥25% (men) and ≥35% (women)) (OO, n = 284) had larger waist and hip measurements, higher BMI and were heavier (P < 0.001) than those classified as nonobese by BMI but obese by BF% (NOO, n = 92). OO individuals displayed a more proinflammatory (higher C reactive protein (CRP) and leptin), prothrombotic (higher plasminogen activator inhibitor‐1 (PAI‐1)), proatherogenic (higher leptin/adiponectin ratio) and more insulin resistant (higher HOMA‐IR) metabolic profile relative to the NOO group (P < 0.001). Interestingly, tumor necrosis factor‐α (TNF‐α) concentrations were lower post‐intervention in NOO individuals compared with OO subjects (P < 0.001).

Conclusions:

In conclusion, assessing BF% and BMI as part of a metabotype may help to identify individuals at greater cardiometabolic risk than BMI alone.     

Deciphering structure-activity relationships in a series of Tat/TAR inhibitors

A series of pentameric “Polyamide Amino Acids” (PAAs) compounds derived from the same trimeric precursor have been synthesized and investigated as HIV TAR RNA ligands, in the absence and in the presence of a Tat fragment. All PAAs bind TAR with similar sub-micromolar affinities but their ability to compete efficiently with the Tat fragment strongly differs, IC₅₀ ranging from 35 nM to >2 μM. While NMR and CD studies reveal that all PAA interact with TAR at the same site and induce globally the same RNA conformational change upon binding, a comparative thermodynamic study of PAA/TAR equilibria highlights distinct TAR binding modes for Tat competitor and non-competitor PAAs. This led us to suggest two distinct interaction modes that have been further validated by molecular modeling studies. While the binding of Tat competitor PAAs induces a contraction at the TAR bulge region, the binding of non-competitor ones widens it. This could account for the distinct PAA ability to compete with Tat fragment. Our work illustrates how comparative thermodynamic studies of a series of RNA ligands of same chemical family are of value for understanding their binding modes and for rationalizing structure-activity relationships.