Lack of avoidance of the fungal entomopathogen, Metarhizium brunneum, by male Agriotes obscurus beetles.

Fungal entomopathogens can greatly reduce the fitness of their hosts, and it is therefore expected that susceptible insects will be selected to avoid exposure to pathogens. Metarhizium brunneum is a fungal pathogen that can infect Agriotes obscurus, which in its larval form is a destructive agricultural pest and is repelled by the presence of M. brunneum conidia. Due to the subterranean nature of larval A. obscurus, recent research has focused on targeting adult A. obscurus with M. brunneum. No-choice and choice behavioural assays were conducted to determine if male adult A. obscurus avoid M. brunneum mycosed cadavers, or conidia applied to either food or soil. To further investigate the response of A. obscurus beetles to conspecific cadavers, the movement and behaviour of beetles placed at the centre of a semi-circular arrangement of mycosed or control cadavers was examined using motion tracking software. We found little evidence to suggest that A. obscurus male beetles avoid M. brunneum conidia or mycosed conspecific cadavers or alter their behaviour in their presence.

Burkitt lymphoma expresses oncofetal chondroitin sulfate without being a reservoir for placental malaria sequestration.

Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria-infected erythrocytes and facilitates a highly specific binding to the placenta. ofCS is absent in other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor site for VAR2CSA-expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA-specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non-malignant tissue encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL-derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL-derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy.

Recommendations for HLA-B*15:02 and HLA-A*31:01 genetic testing to reduce the risk of carbamazepine-induced hypersensitivity reactions.

OBJECTIVE: To systematically review evidence on genetic risk factors for carbamazepine (CBZ)-induced hypersensitivity reactions (HSRs) and provide practice recommendations addressing the key questions: (1) Should genetic testing for HLA-B*15:02 and HLA-A*31:01 be performed in patients with an indication for CBZ therapy to reduce the occurrence of CBZ-induced HSRs? (2) Are there subgroups of patients who may benefit more from genetic testing for HLA-B*15:02 or HLA-A*31:01 compared to others? (3) How should patients with an indication for CBZ therapy be managed based on their genetic test results? METHODS: A systematic literature search was performed for HLA-B*15:02 and HLA-A*31:01 and their association with CBZ-induced HSRs. Evidence was critically appraised and clinical practice recommendations were developed based on expert group consensus. RESULTS: Patients carrying HLA-B*15:02 are at strongly increased risk for CBZ-induced Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) in populations where HLA-B*15:02 is common, but not CBZ-induced hypersensitivity syndrome (HSS) or maculopapular exanthema (MPE). HLA-B*15:02-positive patients with CBZ-SJS/TEN have been reported from Asian countries only, including China, Thailand, Malaysia, and India. HLA-B*15:02 is rare among Caucasians or Japanese; no HLA-B*15:02-positive patients with CBZ-SJS/TEN have been reported so far in these groups. HLA-A*31:01-positive patients are at increased risk for CBZ-induced HSS and MPE, and possibly SJS/TEN and acute generalized exanthematous pustulosis (AGEP). This association has been shown in Caucasian, Japanese, Korean, Chinese, and patients of mixed origin; however, HLA-A*31:01 is common in most ethnic groups. Not all patients carrying either risk variant develop an HSR, resulting in a relatively low positive predictive value of the genetic tests. SIGNIFICANCE: This review provides the latest update on genetic markers for CBZ HSRs, clinical practice recommendations as a basis for informed decision making regarding the use of HLA-B*15:02 and HLA-A*31:01 genetic testing in patients with an indication for CBZ therapy, and identifies knowledge gaps to guide future research. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

A retrospective comparative study with historical control to determine the effectiveness of platelet-rich plasma as part of nonoperative treatment of acute achilles tendon rupture.

PURPOSE: The aim of this study was to evaluate the effectiveness of platelet-rich plasma (PRP) in the nonoperative treatment of acute Achilles tendon rupture. METHODS: This was a comparative study that included a prospective cohort and a historical control group. The control group was formed from a randomized trial in which one arm of the trial underwent nonoperative treatment, including accelerated functional rehabilitation after acute Achilles tendon rupture identical to that performed in the prospective treatment group. Patients in the prospective group were recruited consecutively and were administered 2 injections of PRP during the first 2 weeks after the injury. The primary outcome was isokinetic plantar flexion strength at 1 and 2 years after injury. Secondary outcomes included range of motion (ROM), calf circumference, and Leppilahti score. The ankle-hindfoot scale (American Orthopedic Functional Ankle Scale [AOFAS]) was administered to patients who received the PRP injection in the prospective group but was not measured for the historical group. RESULTS: A total of 73 patients participated in the prospective PRP study group and were compared with a retrospective control group of 72 patients from a previous randomized controlled trial (RCT). The mean difference between groups in isokinetic plantar flexion strength (injured/uninjured) at 1 year after injury was -4.3% (95% confidence interval [CI], -15.9 to 7.3; P = .5) and 2.4% (95% CI, -8.6 to 13.5; P = .7) at 30°/s and 60°/s, respectively. Results at 2 years after injury were -3.1% (95% CI, -13.5 to 7.2; P = .6) and 4.8% (95% CI, -3.5 to 13.1; P = .3) at 30°/s and 60°/s, respectively. All secondary outcomes were also not statistically different. CONCLUSIONS: The results of this study suggest that there is no measurable clinical benefit to the addition of PRP to the treatment regimen for nonoperatively treated acute Achilles tendon rupture. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Limitation of radiological T3 subclassification of rectal cancer due to paucity of mesorectal fat in Chinese patients.

OBJECTIVES: To describe the thickness of mesorectal fat in local Chinese population and its impact on rectal cancer staging. DESIGN: Case series. SETTING: Two local regional hospitals in Hong Kong. PATIENTS: Consecutive patients referred for multidisciplinary board meetings from January to October 2012 were selected. MAIN OUTCOME MEASURES: Reports of cases that had undergone staging magnetic resonance imaging for histologically proven rectal cancer were retrospectively retrieved and reviewed by two radiologists. All magnetic resonance imaging examinations were acquired with 1.5T magnetic resonance imaging. Measurements were made by agreement between the two radiologists. The distance in mm was obtained in the axial plane at levels of 5 cm, 7.5 cm, and 10 cm from the anal verge. Four readings were obtained at each level, namely, anterior, left lateral, posterior, and right lateral positions. RESULTS: A total of 25 patients (16 males, 9 females) with a median age of 69 (range, 38-84) years were included in the study. Mean thickness of the mesorectal fat at 5 cm, 7.5 cm, and 10 cm from the anal verge was 3.1 mm (standard deviation, 3.0 mm), 9.8 mm (5.3 mm), and 11.8 mm (4.2 mm), respectively. The proportions of patients with mean mesorectal fat thickness of <15 mm were 100%, 84%, and 75% at 5 cm, 7.5 cm, and 10 cm from the anal verge, respectively. The thickness of mesorectal fat was the least anteriorly, and <15 mm at all three arbitrary levels (P<0.001). CONCLUSION: The thickness of mesorectal fat was <15 mm in the majority of patients and in most positions. Tumours invading 10 mm beyond the serosa on magnetic resonance imaging may paradoxically threaten the circumferential resection margin in Chinese patients. Use of T3 subclassification of rectal cancer in Chinese patients may be limited.

Design and Evaluation of ZD06519, a Novel Camptothecin Payload for Antibody Drug Conjugates.

In recent years, the field of antibody drug conjugates (ADC) has seen a resurgence, largely driven by the clinical benefit observed in patients treated with ADCs incorporating camptothecin-based topoisomerase I inhibitor payloads. Herein, we present the development of a novel camptothecin ZD06519 (FD1), which has been specifically designed for its application as an ADC payload. A panel of camptothecin analogs with different substituents at the C-7 and C-10 positions of the camptothecin core was prepared and tested in vitro. Selected compounds spanning a range of potency and hydrophilicity were elaborated into drug-linkers, conjugated to trastuzumab, and evaluated in vitro and in vivo. ZD06519 was selected on the basis of its favorable properties as a free molecule and as an antibody conjugate, which include moderate free payload potency (∼1 nmol/L), low hydrophobicity, strong bystander activity, robust plasma stability, and high-monomeric ADC content. When conjugated to different antibodies using a clinically validated MC-GGFG-based linker, ZD06519 demonstrated impressive efficacy in multiple cell line-derived xenograft models and noteworthy tolerability in healthy mice, rats, and non-human primates.

Developing a Nanopore Sequencing Workflow for Protein Engineering Applications.

Sequencing plays a critical role in protein engineering, where the genetic information encoding for a desired mutation can be identified. We evaluated the performance of two commercially available NGS technologies (Illumina NGS and nanopore sequencing) on the available mutant libraries that were either previously constructed for other protein engineering projects or constructed in-house for this study. The sequencing results from Illumina sequencing indicated that a substantial proportion of the reads exhibited strand exchange, which mixed information of different mutants. When nanopore sequencing was used, the occurrence of strand exchange was substantially reduced compared with that of Illumina sequencing. We then developed a new library preparation workflow for nanopore sequencing and were successful in further reducing the incidence of strand exchange. The optimized workflow was successfully used to aid selection of improved alcohol dehydrogenase mutants in cells where their activities were coupled with the cell growth rate. The workflow quantified the enrichment fold change of most mutants in the library (size = 1728) in the growth-based selection passaging. A mutant that was >500% more active than its parent variant was identified based on the fold change data but not with the absolute abundance data (random sampling of the passaged cells), highlighting the usefulness of this rapid and affordable sequencing workflow in protein engineering.

Structure-Activity Relationships of Bis-Intercalating Peptides and Their Application as Antibody-Drug Conjugate Payloads.

Synthetic analogs based on the DNA bis-intercalating natural product peptides sandramycin and quinaldopeptin were investigated as antibody drug conjugate (ADC) payloads. Synthesis, biophysical characterization, and in vitro potency of 34 new analogs are described. Conjugation of an initial drug-linker derived from a novel bis-intercalating peptide produced an ADC that was hydrophobic and prone to aggregation. Two strategies were employed to improve ADC physiochemical properties: addition of a solubilizing group in the linker and the use of an enzymatically cleavable hydrophilic mask on the payload itself. All ADCs showed potent in vitro cytotoxicity in high antigen expressing cells; however, masked ADCs were less potent than payload matched unmasked ADCs in lower antigen expressing cell lines. Two pilot in vivo studies were conducted using stochastically conjugated DAR4 anti-FRα ADCs, which showed toxicity even at low doses, and site-specific conjugated (THIOMAB) DAR2 anti-cMet ADCs that were well tolerated and highly efficacious.

An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

C2 feedstock-based biomanufacturing of value-added chemicals.

Engineering microbes to produce value-added chemicals from C6/C5 sugars sometimes requires long biosynthetic pathways, which causes carbon loss due to involving multiple metabolic branch nodes, leading to a lower product yield. Using C2 feedstocks derived from gaseous, cellulosic, and plastic wastes could establish shorter biosynthetic pathways to produce some target chemicals, for example, acetyl-CoA-derived natural products. Utilizing these waste-derived feedstocks would also contribute to reducing the carbon footprint of the chemical industry. In this review, we highlighted the promising waste-processing technologies that could provide C2 feedstocks that are compatible with microbial fermentation. We also analyzed the recent metabolic engineering works in which the microorganisms/fermentation processes were modified/optimized to utilize acetate, ethanol, or ethylene glycol more efficiently.

Producing aromatic amino acid from corn husk by using polyols as intermediates.

Agricultural biomass remains as one of the commonly found waste on Earth. Although valorisation of these wastes has been studied in detail, the fermentation-based processes still need improvement due to the high cost of hydrolysing enzymes, and the presence of growth inhibitors which constrains the fermentation to produce high-value products. To address these challenges, we developed an integrated process in this study combining abiotic- and bio-catalysis to produce l-tyrosine from corn husk. The first step involved a one-pot hydrolytic hydrogenation tandem reaction without the use of the expensive enzymes, which yielded a mixture of polyols and sugars. Without any purification, these crude hydrolysates can be almost completely utilized by an engineered Escherichia coli strain, which did not exhibit any growth inhibition. The strain produced 0.44 g/L l-tyrosine from 10 g/L crude corn husk hydrolysates, demonstrating the feasibility of converting agricultural biomass into a valuable aromatic amino acid via an integrated process.

Application of purging biotinylated liposomes from plasma to elucidate influx and efflux processes associated with accumulation of liposomes in solid tumors.

Although small, 100-nm liposomes are known to selectively accumulate in solid tumors, the individual contributions of liposome influx and egress rates are not well understood. The aim of this work was to determine influx and efflux kinetics for 100-nm, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol) liposomes by inducing aggregate formation of biotinylated liposomes upon administering avidin. Injecting 50 microg of neutravidin intravenously to mice that had previously been administered 100 mg/kg DPSC/Chol liposomes containing 0.5 mol% biotin-conjugated lipid resulted in >90% elimination of the liposomes from plasma within 1 h. This rapid removal by the reticuloendothelial system (RES) permitted the determination of the tumor efflux kinetics due to negligible tumor influx after neutravidin injection. The tumor efflux rate constant (k(-1)) was determined to be 0.041 h(-1) when neutravidin was injected 4 h after liposome injection. This allowed the determination of the tumor influx rate constant (k(1)), which under these conditions was 0.022 h(-1). Therefore, DSPC/Chol liposomal accumulation, in LS180 solid tumors, is dictated primarily by plasma liposome concentrations and liposome egress is comparable or slightly faster than influx into the tumors. This method is applicable for a wide range of lipid doses, and can be used to characterize influx and efflux parameters at different time points after accumulation. The application, therefore, has the potential to be used to fully characterize the impact of different liposome parameters such as lipid composition, steric stabilization, size and dose on tumor accumulation kinetics.

Clinical practice guideline: CYP2D6 genotyping for safe and efficacious codeine therapy.

This guideline is intended to provide a basis for informed decision-making regarding genetic testing to identify those individuals who will not benefit from codeine therapy, as well as those who are at an increased risk for codeine-induced toxicity. This guideline addresses the following key questions: 1) Should genetic testing for CYP2D6 be performed in patients prior to the initiation of codeine therapy? 2) How should patients with an indication for codeine therapy be managed based on their genotyping results for CYP2D6?