Targeting human CALR‐mutated MPN progenitors with a neoepitope‐directed monoclonal antibody

Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co‐expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK‐STAT signalling, TPO‐independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR‐dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.     

Remarkably Low KIR and HLA Diversity in Amerindians Reveals Signatures of Strong Purifying Selection Shaping the Centromeric KIR Region

The killer-cell immunoglobulin-like receptors (KIR) recognize human leukocyte antigen (HLA) molecules to regulate the cytotoxic and inflammatory responses of natural killer cells. KIR genes are encoded by a rapidly evolving gene family on chromosome 19 and present an unusual variation of presence and absence of genes and high allelic diversity. Although many studies have associated KIR polymorphism with susceptibility to several diseases over the last decades, the high-resolution allele-level haplotypes have only recently started to be described in populations. Here, we use a highly innovative custom next-generation sequencing method that provides a state-of-art characterization of KIR and HLA diversity in 706 individuals from eight unique South American populations: five Amerindian populations from Brazil (three Guarani and two Kaingang); one Amerindian population from Paraguay (Aché); and two urban populations from Southern Brazil (European and Japanese descendants from Curitiba). For the first time, we describe complete high-resolution KIR haplotypes in South American populations, exploring copy number, linkage disequilibrium, and KIR–HLA interactions. We show that all Amerindians analyzed to date exhibit the lowest numbers of KIR–HLA interactions among all described worldwide populations, and that 83–97% of their KIR–HLA interactions rely on a few HLA-C molecules. Using multiple approaches, we found signatures of strong purifying selection on the KIR centromeric region, which codes for the strongest NK cell educator receptors, possibly driven by the limited HLA diversity in these populations. Our study expands the current knowledge of KIR genetic diversity in populations to understand KIR–HLA coevolution and its impact on human health and survival.     

The Pseudomonas aeruginosa homeostasis enzyme AlgL clears the periplasmic space of accumulated alginate during polymer biosynthesis

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of chronic infection in the lungs of individuals with cystic fibrosis. After colonization, P. aeruginosa often undergoes a phenotypic conversion to mucoidy, characterized by overproduction of the alginate exopolysaccharide. This conversion is correlated with poorer patient prognoses. The majority of genes required for alginate synthesis, including the alginate lyase, algL, are located in a single operon. Previous investigations of AlgL have resulted in several divergent hypotheses regarding the protein’s role in alginate production. To address these discrepancies, we determined the structure of AlgL and, using multiple sequence alignments, identified key active site residues involved in alginate binding and catalysis. In vitro enzymatic analysis of active site mutants highlights R249 and Y256 as key residues required for alginate lyase activity. In a genetically engineered P. aeruginosa strain where alginate biosynthesis is under arabinose control, we found that AlgL is required for cell viability and maintaining membrane integrity during alginate production. We demonstrate that AlgL functions as a homeostasis enzyme to clear the periplasmic space of accumulated polymer. Constitutive expression of the AlgU/T sigma factor mitigates the effects of an algL deletion during alginate production, suggesting that an AlgU/T-regulated protein or proteins can compensate for an algL deletion. Together, our study demonstrates the role of AlgL in alginate biosynthesis, explains the discrepancies observed previously across other P. aeruginosa ΔalgL genetic backgrounds, and clarifies the existing divergent data regarding the function of AlgL as an alginate degrading enzyme.     

Evaluation of an adapted version of the Diabetes Prevention Program for low- and middle-income countries: A cluster randomized trial to evaluate “Lifestyle Africa” in South Africa

BackgroundLow- and middle-income countries (LMICs) are experiencing major increases in diabetes and cardiovascular conditions linked to overweight and obesity. Lifestyle interventions such as the United States National Diabetes Prevention Program (DPP) developed in high-income countries require adaptation and cultural tailoring for LMICs. The objective of this study was to evaluate the efficacy of “Lifestyle Africa,” an adapted version of the DPP tailored for an underresourced community in South Africa compared to usual care.Methods and findingsParticipants were residents of a predominantly Xhosa-speaking urban township of Cape Town, South Africa characterized by high rates of poverty. Participants with body mass index (BMI) ≥ 25 kg/m² who were members of existing social support groups or “clubs” receiving health services from local nongovernmental organizations (NGOs) were enrolled in a cluster randomized controlled trial that compared Lifestyle Africa (the intervention condition) to usual care (the control condition). The Lifestyle Africa intervention consisted of 17 video-based group sessions delivered by trained community health workers (CHWs). Clusters were randomized using a numbered list of the CHWs and their assigned clubs based on a computer-based random allocation scheme. CHWs, participants, and research team members could not be blinded to condition. Percentage weight loss (primary outcome), hemoglobin A1c (HbA1c), blood pressure, triglycerides, and low-density lipoprotein (LDL) cholesterol were assessed 7 to 9 months after enrollment. An individual-level intention-to-treat analysis was conducted adjusting for clustering within clubs and baseline values. Trial registration is at ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT03342274","term_id":"NCT03342274"}}NCT03342274). Between February 2018 and May 2019, 782 individuals were screened, and 494 were enrolled. Participants were predominantly retired (57% were receiving a pension) and female (89%) with a mean age of 68 years. Participants from 28 clusters were allocated to Lifestyle Africa (15, n = 240) or usual care (13, n = 254). Fidelity assessments indicated that the intervention was generally delivered as intended. The modal number of sessions held across all clubs was 17, and the mean attendance of participants across all sessions was 61%. Outcome assessment was completed by 215 (90%) intervention and 223 (88%) control participants. Intent-to-treat analyses utilizing multilevel modeling included all randomized participants. Mean weight change (primary outcome) was −0.61% (95% confidence interval (CI) = −1.22, −0.01) in Lifestyle Africa and −0.44% (95% CI = −1.06, 0.18) in control with no significant difference (group difference = −0.17%; 95% CI = −1.04, 0.71; p = 0.71). However, HbA1c was significantly lower at follow-up in Lifestyle Africa compared to the usual care group (mean difference = −0.24, 95% CI = −0.39, −0.09, p = 0.001). None of the other secondary outcomes differed at follow-up: systolic blood pressure (group difference = −1.36; 95% CI = −6.92, 4.21; p = 0.63), diastolic blood pressure (group difference = −0.39; 95% CI = −3.25, 2.30; p = 0.78), LDL (group difference = −0.07; 95% CI = −0.19, 0.05; p = 0.26), triglycerides (group difference = −0.02; 95% CI = −0.20, 0.16; p = 0.80). There were no unanticipated problems and serious adverse events were rare, unrelated to the intervention, and similar across groups (11 in Lifestyle Africa versus 13 in usual care). Limitations of the study include the lack of a rigorous dietary intake measure and the high representation of older women.ConclusionsIn this study, we found that Lifestyle Africa was feasible for CHWs to deliver and, although it had no effect on the primary outcome of weight loss or secondary outcomes of blood pressure or triglycerides, it had an apparent small significant effect on HbA1c. The study demonstrates the potential feasibility of CHWs to deliver a program without expert involvement by utilizing video-based sessions. The intervention may hold promise for addressing cardiovascular disease (CVD) and diabetes at scale in LMICs.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03342274","term_id":"NCT03342274"}}NCT03342274.

In a cluster randomized trial, Delwyn Catley and colleagues evaluate an adapted version of the Diabetes Prevention Program in South Africa.     

SHP-2 positively regulates myogenesis by coupling to the Rho GTPase signaling pathway

Myogenesis is an intricate process that coordinately engages multiple intracellular signaling cascades. The Rho family GTPase RhoA is known to promote myogenesis, however, the mechanisms controlling its regulation in myoblasts have yet to be fully elucidated. We show here that the SH2-containing protein tyrosine phosphatase, SHP-2, functions as an early modulator of myogenesis by regulating RhoA. When MyoD was expressed in fibroblasts lacking functional SHP-2, muscle-specific gene activity was impaired and abolition of SHP-2 expression by RNA interference inhibited muscle differentiation. By using SHP-2 substrate-trapping mutants, we identified p190-B RhoGAP as a SHP-2 substrate. When dephosphorylated, p190-B RhoGAP has been shown to stimulate the activation of RhoA. During myogenesis, p190-B RhoGAP was tyrosyl dephosphorylated concomitant with the stimulation of SHP-2's phosphatase activity. Moreover, overexpression of a catalytically inactive mutant of SHP-2 inhibited p190-B RhoGAP tyrosyl dephosphorylation, RhoA activity, and myogenesis. These observations strongly suggest that SHP-2 dephosphorylates p190-B RhoGAP, leading to the activation of RhoA. Collectively, these data provide a mechanistic basis for RhoA activation in myoblasts and demonstrate that myogenesis is critically regulated by the actions of SHP-2 on the p190-B Rho GAP/RhoA pathway.     

Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations

A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters. Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization. Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate. Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized. FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C. For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids. The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis. As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated.     

Antimicrobial activity of fractions and compounds from Calophyllumbrasiliense (Clusiaceae/Guttiferae)

Calophyllum brasiliense (Clusiaceae/Guttiferae) is a native Brazilian medicinal plant traditionally used against several diseases, including infectious pathologies. Crude methanolic extracts (CME) and two fractions, denoted non-polar (soluble in chloroform) and polar (nonsoluble in chloroform), were prepared from different parts of the plant (roots, stems, leaves, flowers and fruits) and studied. The following compounds were isolated and tested against pathogenic bacteria and yeasts by determination of the minimal inhibitory concentration (MIC): brasiliensic acid (1), gallic acid (2), epicatechin (3), protocatechuic acid (4), friedelin (5) and 1,5-dihydroxyxanthone (6). The results indicated that all the parts of the plant exhibited antimicrobial activity against Gram-positive bacteria, which are selectively inhibited by components of C. brasiliense. No activity was observed against Gram-negative bacteria and yeasts tested. Regarding the isolated compounds, substance 4 showed antimicrobial activity against all the tested microorganisms, whereas compound 6 exhibited antimicrobial activity only against Gram-positive bacteria. The results from the current study confirm and justify the popular use of this plant to treat infectious processes.