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- ItemDetection of Protein Glycosylation Using Tip-Enhanced Raman Scattering(Columbus, Ohio : American Chemical Society, 2016) Cowcher, David P.; Deckert-Gaudig, Tanja; Brewster, Victoria L.; Ashton, Lorna; Deckert, Volker; Goodacre, RoystonThe correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.
- ItemHuman spermbots for patient-representative 3D ovarian cancer cell treatment(Cambridge : RSC Publ., 2020) Xu, Haifeng; Medina-Sánchez, Mariana; Zhang, Wunan; Seaton, Melanie P. H.; Brison, Daniel R.; Edmondson, Richard J.; Taylor, Stephen S.; Nelson, Louisa; Zeng, Kang; Bagley, Steven; Ribeiro, Carla; Restrepo, Lina P.; Lucena, Elkin; Schmidt, Christine K.; Schmidt, Oliver G.Cellular micromotors are attractive for locally delivering high concentrations of drug, and targeting hard-to-reach disease sites such as cervical cancer and early ovarian cancer lesions by non-invasive means. Spermatozoa are highly efficient micromotors perfectly adapted to traveling up the female reproductive system. Indeed, bovine sperm-based micromotors have shown potential to carry drugs toward gynecological cancers. However, due to major differences in the molecular make-up of bovine and human sperm, a key translational bottleneck for bringing this technology closer to the clinic is to transfer this concept to human material. Here, we successfully load human sperm with Doxorubicin (DOX) and perform treatment of 3D cervical cancer and patient-representative ovarian cancer cell cultures, resulting in strong anticancer cell effects. Additionally, we define the subcellular localization of the chemotherapeutic drug within human sperm, using high-resolution optical microscopy. We also assess drug effects on sperm motility and viability over time, employing sperm samples from healthy donors as well as assisted reproduction patients. Finally, we demonstrate guidance and release of human drug-loaded sperm onto cancer tissues using magnetic microcaps, and show the sperm microcap loaded with a second anticancer drug, camptothecin (CPT), which unlike DOX is not suitable for directly loading into sperm due to its hydrophobic nature. This co-drug delivery approach opens up novel targeted combinatorial drug therapies for future applications. © 2020 The Royal Society of Chemistry.
- ItemMolecular characterisation of extended-spectrum ß-lactamase producing Escherichia coli in wild birds and cattle, Ibadan, Nigeria(London : BioMed Central, 2021) Fashae, Kayode; Engelmann, Ines; Monecke, Stefan; Braun, Sascha D.; Ehricht, RalfBackground: Antimicrobial resistance (AMR) is an increasing global health concern reducing options for therapy of infections and also for perioperative prophylaxis. Many Enterobacteriaceae cannot be treated anymore with third generation cephalosporins (3GC) due to the production of certain 3GC hydrolysing enzymes (extended spectrum beta-lactamases, ESBLs). The role of animals as carriers and vectors of multi-resistant bacteria in different geographical regions is poorly understood. Therefore, we investigated the occurrence and molecular characteristics of ESBL-producing Escherichia coli (E. coli) in wild birds and slaughtered cattle in Ibadan, Nigeria. Cattle faecal samples (n = 250) and wild bird pooled faecal samples (cattle egrets, Bubulcus ibis, n = 28; white-faced whistling duck, Dendrocygna viduata, n = 24) were collected and cultured on cefotaxime-eosin methylene blue agar. Antimicrobial susceptibility was determined by agar diffusion assays and all 3GC resistant isolates were genotypically characterised for AMR genes, virulence associated genes (VAGs) and serotypes using DNA microarray-based assays. Results: All 3GC resistant isolates were E. coli: cattle (n = 53), egrets (n = 87) and whistling duck (n = 4); cultured from 32/250 (12.8%), 26/28 (92.9%), 2/24(8.3%), cattle, egrets and whistling duck faecal samples, respectively. blaCTX-M gene family was prevalent; blaCTX-M15 (83.3%) predominated over blaCTX-M9 (11.8%). All were susceptible to carbapenems. The majority of isolates were resistant to at least one of the other tested antimicrobials; multidrug resistance was highest in the isolates recovered from egrets. The isolates harboured diverse repositories of other AMR genes (including strB and sul2), integrons (predominantly class 1) and VAGs. The isolates recovered from egrets harboured more AMR genes; eight were unique to these isolates including tetG, gepA, and floR. The prevalent VAGs included hemL and iss; while 14 (including sepA) were unique to certain animal isolates. E. coli serotypes O9:H9, O9:H30 and O9:H4 predominated. An identical phenotypic microarray profile was detected in three isolates from egrets and cattle, indicative of a clonal relationship amongst these isolates. Conclusion: Wild birds and cattle harbour diverse ESBL-producing E. coli populations with potential of inter-species dissemination and virulence. Recommended guidelines to balance public health and habitat conservation should be implemented with continuous surveillance. © 2021, The Author(s).
- ItemEvaluating the potential of dietary crude protein manipulation in reducing ammonia emissions from cattle and pig manure: A meta-analysis(Dordrecht [u.a.] : Springer Science + Business Media B.V, 2017-11-22) Sajeev, Erangu Purath Mohankumar; Amon, Barbara; Ammon, Christian; Zollitsch, Werner; Winiwarter, WilfriedDietary manipulation of animal diets by reducing crude protein (CP) intake is a strategic NH3 abatement option as it reduces the overall nitrogen input at the very beginning of the manure management chain. This study presents a comprehensive meta-analysis of scientific literature on NH3 reductions following a reduction of CP in cattle and pig diets. Results indicate higher mean NH3 reductions of 17 ± 6% per %-point CP reduction for cattle as compared to 11 ± 6% for pigs. Variability in NH3 emission reduction estimates reported for different manure management stages and pig categories did not indicate a significant influence. Statistically significant relationships exist between CP reduction, NH3 emissions and total ammoniacal nitrogen content in manure for both pigs and cattle, with cattle revealing higher NH3 reductions and a clearer trend in relationships. This is attributed to the greater attention given to feed optimization in pigs relative to cattle and also due to the specific physiology of ruminants to efficiently recycle nitrogen in situations of low protein intake. The higher NH3 reductions in cattle highlights the opportunity to extend concepts of feed optimization from pigs and poultry to cattle production systems to further reduce NH3 emissions from livestock manure. The results presented help to accurately quantify the effects of NH3 abatement following reduced CP levels in animal diets distinguishing between animal types and other physiological factors. This is useful in the development of emission factors associated with reduced CP as an NH3 abatement option. © 2017, The Author(s).
- ItemCharacterisation of S. aureus/MRSA CC1153 and review of mobile genetic elements carrying the fusidic acid resistance gene fusC([London] : Macmillan Publishers Limited, part of Springer Nature, 2021) Monecke, Stefan; Müller, Elke; Braun, Sascha D.; Armengol-Porta, Marc; Bes, Michèle; Boswihi, Samar; El-Ashker, Maged; Engelmann, Ines; Gawlik, Darius; Gwida, Mayada; Hotzel, Helmut; Nassar, Rania; Reissig, Annett; Ruppelt-Lorz, Antje; Senok, Abiola; Somily, Ali M.; Udo, Edet E.; Ehricht, RalfWhile many data on molecular epidemiology of MRSA are available for North America, Western Europe and Australia, much less is known on the distribution of MRSA clones elsewhere. Here, we describe a poorly known lineage from the Middle East, CC1153, to which several strains from humans and livestock belong. Isolates were characterised using DNA microarrays and one isolate from the United Arab Emirates was sequenced using Nanopore technology. CC1153 carries agr II and capsule type 5 genes. Enterotoxin genes are rarely present, but PVL is common. Associated spa types include t504, t903 and t13507. PVL-positive CC1153-MSSA were found in Egyptian cattle suffering from mastitis. It was also identified among humans with skin and soft tissue infections in Saudi Arabia, France and Germany. CC1153-MRSA were mainly observed in Arabian Gulf countries. Some isolates presented with a previously unknown SCCmec/SCCfus chimeric element in which a mec B complex was found together with the fusidic acid resistance gene fusC and accompanying genes including ccrA/B-1 recombinase genes. Other isolates carried SCCmec V elements that usually also included fusC. Distribution and emergence of CC1153-MRSA show the necessity of molecular characterization of MRSA that are resistant to fusidic acid. These strains pose a public health threat as they combine resistance to beta-lactams used in hospitals as well as to fusidic acid used in the community. Because of the high prevalence of fusC-positive MRSA in the Middle East, sequences and descriptions of SCC elements harbouring fusC and/or mecA are reviewed. When comparing fusC and its surrounding regions from the CC1153 strain to available published sequences, it became obvious that there are four fusC alleles and five distinct types of fusC gene complexes reminiscent to the mec complexes in SCCmec elements. Likewise, they are associated with different sets of ccrA/B recombinase genes and additional payload that might include entire mec complexes or SCCmec elements.
- ItemSensor-based detection of the severity of hyperkeratosis in the teats of dairy cows(Basel : MDPI AG, 2018) Demba, S.; Hoffmann, G.; Ammon, C.; Rose-Meierhöfer, S.The aim of this study was to evaluate whether the severity of hyperkeratosis (HK) in the teats of dairy cows can be assessed by a dielectric measurement. The study focused on surveying the occurrence of hyperkeratosis in a total of 241 teats of lactating dairy cows. A scoring system consisting of four categories was used to macroscopically assess the severity of HK. Additionally, the dielectric constant (DC) of all teats with milkability was measured in a double iteration with the MoistureMeterD (Delfin Technologies, Kuopio, Finland) on four different days. The Spearman rank correlation coefficient revealed a negative correlation between the DC and HK score (rs = −0.55 to −0.36). The results of the regression analysis showed that the DC values differed significantly between healthy teat ends (≤2) and teat ends with HK (≥3). Thus, the non-invasive measurement of DC provides a promising method of objectively assessing the occurrence and severity of HK.
- ItemHigh resolution spectroscopy reveals fibrillation inhibition pathways of insulin([London] : Macmillan Publishers Limited, part of Springer Nature, 2016) Deckert-Gaudig, Tanja; Deckert, VolkerFibril formation implies the conversion of a protein’s native secondary structure and is associated with several neurodegenerative diseases. A better understanding of fibrillation inhibition and fibril dissection requires nanoscale molecular characterization of amyloid structures involved. Tip-enhanced Raman scattering (TERS) has already been used to chemically analyze amyloid fibrils on a sub-protein unit basis. Here, TERS in combination with atomic force microscopy (AFM), and conventional Raman spectroscopy characterizes insulin assemblies generated during inhibition and dissection experiments in the presence of benzonitrile, dimethylsulfoxide, quercetin, and β-carotene. The AFM topography indicates formation of filamentous or bead-like insulin self-assemblies. Information on the secondary structure of bulk samples and of single aggregates is obtained from standard Raman and TERS measurements. In particular the high spatial resolution of TERS reveals the surface conformations associated with the specific agents. The insulin aggregates formed under different inhibition and dissection conditions can show a similar morphology but differ in their β-sheet structure content. This suggests different aggregation pathways where the prevention of the β-sheet stacking of the peptide chains plays a major role. The presented approach is not limited to amyloid-related reasearch but can be readily applied to systems requiring extremely surface-sensitive characterization without the need of labels.