Artificial (beta / alpha)8 barrel enzymes by in vitro evolution

Natural evolution has yielded countless enzymes of the (β/α)8 barrel fold (TIM barrel). This versatile fold is highly favored by natural enzymes to catalyze a wide array of reactions and appears in five of the six enzymatic classes. Therefore, the (β/α)8 barrel fold is an attractive starting point for enzyme engineering. Several examples of modified TIM barrel enzymes have been reported that accept different substrates or even catalyze a different reaction. However, engineering new activities into the (β/α)8 barrel fold is apparently still far more challenging than its ubiquitous role in nature suggests. Please click Additional Files below to see the full abstract

Mapping charge to function relationships of the DNA mimic protein Ocr

This thesis investigates the functional consequences of neutralising the negative charges on the bacteriophage T7 antirestriction protein ocr. The ocr molecule is a small highly negatively charged, protein homodimer that mimics a short DNA duplex upon binding to the Type I Restriction Modification (RM) system. Thus, ocr facilitates phage infection by binding to and inactivating the host RM system. The aim of this study was to analyse the effect of reducing the negative charge on the ocr molecule by mutating the acidic residues of the protein. The ocr molecule (117 residues) is replete with Asp and Glu residues; each monomer of the homodimer contains 34 acidic residues. Our strategy was to begin with a synthetic gene in which all the acidic residues of ocr had been neutralised. This so called ‘positive ocr’ (or pocr) was used as a template to gradually reintroduce codons for acidic residues by adapting the ISOR strategy proposed by D.S.Tawfik. After each round of mutagenesis an average of 4-6 acidic residues were incorporated into pocr. In this fashion a series of mutant libraries in which acidic residues were progressively introduced into pocr was generated. A high-throughput in vivo selection assay was developed and validated by assessing the antirestriction behaviour of a number of mutants of the DNA mimic proteins wtOcr and Orf18 ArdA. Further to this, selective screening of the libraries allowed us to select clones that displayed antirestriction activity. These mutants were purified and in vitro characterisation confirmed these mutants as displaying the minimum number of acidic residues deemed critical for the activity of ocr. This in vitro process effectively simulated the evolution of the charge mimicry of ocr. Moreover, we were able to tune the high-throughput assay to different selection criteria in order to elucidate various levels of functionality and unexpected changes in phenotype. This approach enables us to map the “in vitro” evolution of ocr to identify acidic residues that are required for protein expression, solubility and function proceeding to a fully functional antirestriction protein

Laparoscopic cholecystectomy in a patient with pneumonectomy

Post pneumonectomy patients pose a challenge to the anaesthetist owing to the altered respiratory mechanics and decreased respiratory reserve. Performing laparoscopic surgery in such patients further deteriorates the already compromised lung functions. Authors report a case of laparoscopic cholecystectomy performed in post pneumonectomy patient. A clear understanding of respiratory mechanics and post pneumonectomy physiological changes helped us to administer a safe anaesthesia and safe perioperative outcome

Nature-inspired engineering of an artificial RNA ligase created by in vitro selection

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Contraceptive efficacy and antioxidant potential of Leptadenia reticulata bark extracts in male albino rats

Birth control measures available are primarily for women which are hormonal supplements that are increasing cancer risks and reproductive health issues. Male contraceptive options are effective and available, i.e. barrier methods and vasectomy. Condoms are failure-prone and single-use, while a vasectomy is a permanent sterilization method done surgically, and reversion is not always successful and expensive. A promising oral male contraceptive drug candidate is yet to be discovered. This study investigated the contraceptive efficacy and antioxidant potential of various extracts of Leptadenia reticulata bark in male rats. To study the effects of various extracts (ethanolic and petroleum ether) of L. reticulata bark in male rats, oral administration at the dose level of 250 mg/kg body weight/ day was done for 60 days. Observations were made for body and organ weight, hematology, serum biochemical chemistry, testosterone and antioxidants, lipid profile, sperm parameters (density and motility) and histological changes (reproductive organs). As compared to control in treated groups (TP and bark petroleum ether extract), a significant reduction (P ≤ 0.001) was perceived in sperm motility and density, as well as reproductive organ weight, serum testosterone, and serum antioxidant parameters like SOD. Histological observations revealed arrest in spermatogenesis and reduced seminiferous tubule diameter, mature Leydig cells, secondary spermatogonia, and spermatids which caused a substantial increase in LPO and GSH. From the research findings, it can be concluded that bark petroleum ether extract of L. reticulata possesses contraceptive potential in male albino rats and can serve as a safe and reversible oral contraceptive for males

The evolutionary pathway from a biologically inactive polypeptide sequence to a folded, active structural mimic of DNA

The protein Ocr (overcome classical restriction) from bacteriophage T7 acts as a mimic of DNA and inhibits all Type I restriction/modification (RM) enzymes. Ocr is a homodimer of 116 amino acids and adopts an elongated structure that resembles the shape of a bent 24 bp DNA molecule. Each monomer includes 34 acidic residues and only six basic residues. We have delineated the mimicry of Ocr by focusing on the electrostatic contribution of its negatively charged amino acids using directed evolution of a synthetic form of Ocr, termed pocr, in which all of the 34 acidic residues were substituted for a neutral amino acid. In vivo analyses confirmed that pocr did not display any antirestriction activity. Here, we have subjected the gene encoding pocr to several rounds of directed evolution in which codons for the corresponding acidic residues found in Ocr were specifically re-introduced. An in vivo selection assay was used to detect antirestriction activity after each round of mutation. Our results demonstrate the variation in importance of the acidic residues in regions of Ocr corresponding to different parts of the DNA target which it is mimicking and for the avoidance of deleterious effects on the growth of the host

Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions and validated through next generation sequencing

We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous codons are described. Utility of the method is demonstrated via fabrication of complementarity determining regions (CDR) in antibody fragment libraries and next generation sequencing (NGS) analysis of their quality and diversity

Mapping genomic and transcriptomic alterations spatially in epithelial cells adjacent to human breast carcinoma.

Almost all genomic studies of breast cancer have focused on well-established tumours because it is technically challenging to study the earliest mutational events occurring in human breast epithelial cells. To address this we created a unique dataset of epithelial samples ductoscopically obtained from ducts leading to breast carcinomas and matched samples from ducts on the opposite side of the nipple. Here, we demonstrate that perturbations in mRNA abundance, with increasing proximity to tumour, cannot be explained by copy number aberrations. Rather, we find a possibility of field cancerization surrounding the primary tumour by constructing a classifier that evaluates where epithelial samples were obtained relative to a tumour (cross-validated micro-averaged AUC = 0.74). We implement a spectral co-clustering algorithm to define biclusters. Relating to over-represented bicluster pathways, we further validate two genes with tissue microarrays and in vitro experiments. We highlight evidence suggesting that bicluster perturbation occurs early in tumour development

Heterogeneity of circulating tumour cell-associated genomic gains in breast cancer and its association with the host immune response.

Tumor cells that preferentially enter circulation include the precursors of metastatic cancer. Previously, we characterized circulating tumor cells (CTC) from patients with breast cancer and identified a signature of genomic regions with recurrent copy-number gains. Through FISH, we now show that these CTC-associated regions are detected within the matched untreated primary tumors of these patients (21% to 69%, median 55.5%, n = 19). Furthermore, they are more prevalent in the metastases of patients who died from breast cancer after multiple rounds of treatment (70% to 100%, median 93%, samples n = 41). Diversity indices revealed that higher spatial heterogeneity for these regions within primary tumors is associated with increased dissemination and metastasis. An identified subclone with multiple regions gained (MRG clone) was enriched in a posttreatment primary breast carcinoma as well as multiple metastatic tumors and local breast recurrences obtained at autopsy, indicative of a distinct early subclone with the capability to resist multiple lines of treatment and eventually cause death. In addition, multiplex immunofluorescence revealed that tumor heterogeneity is significantly associated with the degree of infiltration of B lymphocytes in triple-negative breast cancer, a subtype with a large immune component. Collectively, these data reveal the functional potential of genetic subclones that comprise heterogeneous primary breast carcinomas and are selected for in CTCs and posttreatment breast cancer metastases. In addition, they uncover a relationship between tumor heterogeneity and host immune response in the tumor microenvironment. SIGNIFICANCE: As breast cancers progress, they become more heterogeneous for multiple regions amplified in circulating tumor cells, and intratumoral spatial heterogeneity is associated with the immune landscape