Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins

Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased k_cat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.     

An extended combinatorial 15N, 13Cα, and $$ ^{13} {\text{C}}^{\prime } $$ labeling approach to protein backbone resonance assignment

Solution NMR studies of α-helical membrane proteins are often complicated by severe spectral crowding. In addition, hydrophobic environments like detergent micelles, isotropic bicelles or nanodiscs lead to considerably reduced molecular tumbling rates which translates into line-broadening and low sensitivity. Both difficulties can be addressed by selective isotope labeling methods. In this publication, we propose a combinatorial protocol that utilizes four different classes of labeled amino acids, in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in ¹⁵N or ¹³C isotopes individually as well as simultaneously. This results in eight different combinations of dipeptides giving rise to cross peaks in ¹H–¹⁵N correlated spectra. Their differentiation is achieved by recording a series of HN-detected 2D triple-resonance spectra. The utility of this new scheme is demonstrated with a homodimeric 142-residue membrane protein in DHPC micelles. Restricting the number of selectively labeled samples to three allowed the identification of the amino-acid type for 77 % and provided sequential information for 47 % of its residues. This enabled us to complete the backbone resonance assignment of the uniformly labeled protein merely with the help of a 3D HNCA spectrum, which can be collected with reasonable sensitivity even for relatively large, non-deuterated proteins.     

Phylogenetic systematics of leaffishes (Teleostei: Polycentridae,Nandidae)

The Asian (nandid) and Afro‐Neotropical (polycentrid) leaffishes represent two superficially similar, but historically poorly diagnosed families – a situation resulting in a convoluted systematic history. Here, and including for the first time in a molecular study all leaffish genera, we generate a hypothesis of the phylogenetic history of both groups. We analyse a multilocus molecular data set encompassing 257 acanthomorph taxa, carry out a survey and assessment of selected osteological characters for the polycentrid leaffishes and also provide a reanalysis of previously published morphological data. Our results confirm: (1) that the Polycentridae and Nandidae are only remotely related, and hence, the classic leaffishes are diphyletic; (2) that the Polycentridae is monophyletic, with new skeletal synapomorphies being congruent with molecular data in placing the enigmatic Afronandus – a taxon that thus far has never been included in any molecular study – as sistergroup to the remaining genera; (3) the monophyly of the Nandidae + Badidae and their inclusion into a larger monophyletic group – along with the Pristolepididae, Anabantoidei and Channoidei – comprising the Labyrinthici sensu Rosen & Patterson. We also review the morphological and molecular evidence for both the conflicting placement of Pristolepis and the putative sistergroup relationship between the labyrinth fishes (Anabantoidei) and snakeheads (Channoidei).     

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea.A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS™ clones with insert sizes ∼20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs.Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter β-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue.This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.     

Discovery of new molecular subtypes in oesophageal adenocarcinoma

A large number of patients suffering from oesophageal adenocarcinomas do not respond to conventional chemotherapy; therefore, it is necessary to identify new predictive biomarkers and patient signatures to improve patient outcomes and therapy selections. We analysed 87 formalin-fixed and paraffin-embedded (FFPE) oesophageal adenocarcinoma tissue samples with a reverse phase protein array (RPPA) to examine the expression of 17 cancer-related signalling molecules. Protein expression levels were analysed by unsupervised hierarchical clustering and correlated with clinicopathological parameters and overall patient survival. Proteomic analyses revealed a new, very promising molecular subtype of oesophageal adenocarcinoma patients characterised by low levels of the HSP27 family proteins and high expression of those of the HER family with positive lymph nodes, distant metastases and short overall survival. After confirmation in other independent studies, our results could be the foundation for the development of a Her2-targeted treatment option for this new patient subgroup of oesophageal adenocarcinoma.     

An evaluation of the clinical assessments of under-five febrile children presenting to primary health facilities in rural Ghana

Background

The shift to test-based management of malaria represents an important departure from established practice under the Integrated Management of Childhood Illnesses (IMCI). The possibility of false results of tests for malaria and co-morbidity, however, make it important that guidelines in IMCI case assessment are still followed.

Methods and Findings

We conducted a cross-sectional observational study to evaluate current practices in IMCI-based assessment of febrile children in 10 health centres and 5 district hospitals, with follow up of a subset of children to determine day 7–10 post-treatment clinical outcome. Clinical consultation, examination and prescribing practices were recorded using a checklist by trained non-medical observers. The facility case management of 1,983 under-five years old febrile children was observed and 593 followed up at home on days 5–10. The mean number of tasks performed from the 11 tasks expected to be done by the IMCI guidelines was 6 (SD 1.6). More than 6 tasks were performed in only 35% of children and this varied substantially between health facilities (range 3–85%). All 11 tasks were performed in only 1% of children. The most commonly performed tasks were temperature measurement (91%) and weighing (88%). Respiratory rate was checked in only 4% of children presenting with cough or difficulty in breathing. The likelihood of performing “better than average number of tasks” (>6) was higher when the consultation was done by medical assistants than doctors (O.R. = 3.16, 1.02–9.20). The number of tasks performed during assessment did not, however, influence clinical outcome (O.R. = 1.02, 0.83–1.24).

Conclusion

Facility-tailored interventions are needed to improve adherence to IMCI guidelines incorporating test-based management of malaria. Studies are needed to re-evaluate the continued validity of tasks defined in IMCI case assessment guidelines.