Varietal differences in protein polymer built-up of wheat at different temperature and nitrogen regimes during grain filling

Both radionuclide imaging and near-infrared fluorescent (NIRF) imaging have a high sensitivity to detect tumors in vivo. The combination of these modalities using dual-labeled antibodies may allow both preoperative and intraoperative tumor localization and may be used in image-guided surgery to ensure complete resection of tumor tissue. Here, we evaluated the potential of dual-modality imaging of prostate cancer with the monoclonal antibody D2B, directed against an extracellular domain of prostate-specific membrane antigen (PSMA). For these studies, D2B was labeled both with (111)In and with the NIRF dye IRDye800CW.D2B was conjugated with N-hydroxysuccinimide-IRDye800CW and p-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid (ITC-DTPA) and subsequently radiolabeled with (111)In. For biodistribution and NIRF imaging, (111)In-DTPA-D2B-IRDye800CW (2 μg, 0.55 MBq/mouse) was injected intravenously into BALB/c nude mice with subcutaneous PSMA-expressing LNCaP tumors (right flank) and PSMA-negative PC3 tumors (left flank). The biodistribution was determined at 1, 2, 3, and 7 d after injection. In addition, micro-SPECT/CT and NIRF imaging with (111)In-DTPA-D2B-IRDye800CW (3 μg, 8.5 MBq/mouse) was performed on mice with intraperitoneally growing LS174T-PSMA tumors.(111)In-DTPA-D2B-IRDye800CW specifically accumulated in subcutaneous PSMA-positive LNCaP tumors (45.8 ± 8.0 percentage injected dose per gram at 168 h after injection), whereas uptake in subcutaneous PSMA-negative PC3 tumors was significantly lower (6.6 ± 1.3 percentage injected dose per gram at 168 h after injection). Intraperitoneal LS174T-PSMA tumors could be visualized specifically with both micro-SPECT/CT and NIRF imaging at 2 d after injection, and the feasibility of image-guided resection of intraperitoneal tumors was demonstrated in this model.Dual-labeled (111)In-DTPA-D2B-IRDye800CW enables specific and sensitive detection of prostate cancer lesions in vivo with micro-SPECT/CT and NIRF imaging. In addition to preoperative micro-SPECT/CT imaging to detect tumors, NIRF imaging enables image-guided surgical resection. These preclinical findings warrant clinical studies with (111)In-DTPA-D2B-IRDye800CW to improve tumor detection and resection in prostate cancer patients

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10

Identification of sVSG117 as an immunodiagnostic antigen and evaluation of a dual-antigen lateral flow test for the diagnosis of human african trypanosomiasis

The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT.Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank.In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use

Tuberculosis treatment intervention trials in Africa: A cross-sectional bibliographic study and spatial analysis

Background Mycobacterium Tuberculosis (TB) poses a substantial burden in sub-Saharan Africa and is the leading cause of death amongst infectious diseases. Randomised controlled trials (RCTs) are regarded as the gold standard for evaluating the effectiveness of interventions. We aimed to describe published TB treatment trials conducted in Africa. Methods This is a cross-sectional study of published TB trials conducted in at least one African country. In November 2019, we searched three databases using the validated Africa search filter and Cochrane’s sensitive trial string. Published RCTs conducted in at least one African country were included for analysis. Records were screened for eligibility. Co-reviewers assisted with duplicate data extraction. Extracted data included: the country where studies were conducted, publication dates, ethics statement, trial registration number, participant’s age range. We used Cochrane’s Risk of Bias criteria to assess methodological quality. Results We identified 10,495 records; 175 trials were eligible for inclusion. RCTs were published between 1952 and 2019. The median sample size was 206 participants (interquartile range: 73–657). Most trials were conducted in South Africa (n = 83) and were drug therapy trials (n = 130). First authors were from 30 countries globally. South Africa had the most first authors (n = 55); followed by the United States of America (USA) (n = 28) and Great Britain (n = 14) with fewer other African countries contributing to the first author tally. Children under 13 years of age eligible to participate in the trials made up 17/175 trials (9.71%). International governments (n = 29) were the most prevalent funders. Ninety-four trials provided CONSORT flow diagrams. Methodological quality such as allocation concealment and blinding were poorly reported or unclear in most trials. Conclusions By mapping African TB trials, we were able to identify potential research gaps. Many of the global north’s researchers were found to be the lead authors in these African trials. Few trials tested behavioural interventions compared to drugs, and far fewer tested interventions on children compared to adults to improve TB outcomes. Lastly, funders and researchers should ensure better methodological quality reporting of trials

Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 A° resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for Cclade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool

Randomized controlled trials of malaria intervention trials in Africa, 1948 to 2007: a descriptive analysis

<p>Abstract</p> <p>Background</p> <p>Nine out of ten deaths from malaria occur in sub-Saharan Africa. Various control measures have achieved some progress in the control of the disease, but malaria is still a major public health problem in Africa. Randomized controlled trials (RCTs) are universally considered the best study type to rigorously assess whether an intervention is effective. The study reported here provides a descriptive analysis of RCTs reporting interventions for the prevention and treatment of malaria conducted in Africa, with the aim of providing detailed information on their main clinical and methodological characteristics, that could be used by researchers and policy makers to help plan future research.</p> <p>Methods</p> <p>Systematic searches for malaria RCTs were conducted using electronic databases (Medline, Embase, the Cochrane Library), and an African geographic search filter to identify RCTs conducted in Africa was applied. Results were exported to the statistical package STATA 8 to obtain a random sample from the overall data set. Final analysis of trial characteristics was done in a double blinded fashion by two authors using a standardized data extraction form.</p> <p>Results</p> <p>A random sample of 92 confirmed RCTs (from a total of 943 reports obtained between 1948 and 2007) was prepared. Most trials investigated drug treatment in children with uncomplicated malaria. Few trials reported on treatment of severe malaria or on interventions in pregnant women. Most trials were of medium size (100-500 participants), individually randomized and based in a single centre. Reporting of trial quality was variable. Although three-quarter of trials provided information on participants' informed consent and ethics approval, more details are needed.</p> <p>Conclusions</p> <p>The majority of malaria RCT conducted in Africa report on drug treatment and prevention in children; there is need for more research done in pregnant women. Sources of funding, informed consent and trial quality were often poorly reported. Overall, clearer reporting of trials is needed.</p

Llama Antibody Fragments Recognizing Various Epitopes of the CD4bs Neutralize a Broad Range of HIV-1 Subtypes A, B and C

Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120Ds2), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B′/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides

Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols

Drug Resistance in Eukaryotic Microorganisms

Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies

Calpain 3 Is a Rapid-Action, Unidirectional Proteolytic Switch Central to Muscle Remodeling

Calpain 3 (CAPN3) is a cysteine protease that when mutated causes Limb Girdle Muscular Dystrophy 2A. It is thereby the only described Calpain family member that genetically causes a disease. Due to its inherent instability little is known of its substrates or its mechanism of activity and pathogenicity. In this investigation we define a primary sequence motif underlying CAPN3 substrate cleavage. This motif can transform non-related proteins into substrates, and identifies >300 new putative CAPN3 targets. Bioinformatic analyses of these targets demonstrate a critical role in muscle cytoskeletal remodeling and identify novel CAPN3 functions. Among the new CAPN3 substrates are three E3 SUMO ligases of the Protein Inhibitor of Activated Stats (PIAS) family. CAPN3 can cleave PIAS proteins and negatively regulates PIAS3 sumoylase activity. Consequently, SUMO2 is deregulated in patient muscle tissue. Our study thus uncovers unexpected crosstalk between CAPN3 proteolysis and protein sumoylation, with strong implications for muscle remodeling