Sebastian O. Wendel

Cell Based Drug Delivery: Micrococcus luteus Loaded Neutrophils as Chlorhexidine Delivery Vehicles in a Mouse Model of Liver Abscesses in Cattle.

The recent WHO report on antibiotic resistances shows a dramatic increase of microbial resistance against antibiotics. With only a few new antibiotics in the pipeline, a different drug delivery approach is urgently needed. We have obtained evidence demonstrating the effectiveness of a cell based drug delivery system that utilizes the innate immune system as targeting carrier for antibacterial drugs. In this study we show the efficient loading of neutrophil granulocytes with chlorhexidine and the complete killing of E. coli as well as Fusobacterium necrophorum in in-vitro studies. Fusobacterium necrophorum causes hepatic abscesses in cattle fed high grain diets. We also show in a mouse model that this delivery system targets infections of F. necrophorum in the liver and reduces the bacterial burden by an order of magnitude from approximately 2•106 to 1•105.

High-Risk Alphapapillomavirus Oncogenes Impair the Homologous Recombination Pathway

ABSTRACT Persistent high-risk genus human Alphapapillomavirus (HPV) infections cause nearly every cervical carcinoma and a subset of tumors in the oropharyngeal tract. During the decades required for HPV-associated tumorigenesis, the cellular genome becomes significantly destabilized. Our analysis of cervical tumors from four separate data sets found a significant upregulation of the homologous-recombination (HR) pathway genes. The increased abundance of HR proteins can be replicated in primary cells by expression of the two HPV oncogenes (E6 and E7) required for HPV-associated transformation. HPV E6 and E7 also enhanced the ability of HR proteins to form repair foci, and yet both E6 and E7 reduce the ability of the HR pathway to complete double-strand break (DSB) repair by about 50%. The HPV oncogenes hinder HR by allowing the process to begin at points in the cell cycle when the lack of a sister chromatid to serve as a homologous template prevents completion of the repair. Further, HPV E6 attenuates repair by causing RAD51 to be mislocalized away from both transient and persistent DSBs, whereas HPV E7 is only capable of impairing RAD51 localization to transient lesions. Finally, we show that the inability to robustly repair DSBs causes some of these lesions to be more persistent, a phenotype that correlates with increased integration of episomal DNA. Together, these data support our hypothesis that HPV oncogenes contribute to the genomic instability observed in HPV-associated malignancies by attenuating the repair of damaged DNA. IMPORTANCE This study expands the understanding of HPV biology, establishing a direct role for both HPV E6 and E7 in the destabilization of the host genome by blocking the homologous repair of DSBs. To our knowledge, this is the first time that both viral oncogenes were shown to disrupt this DSB repair pathway. We show that HPV E6 and E7 allow HR to initiate at an inappropriate part of the cell cycle. The mislocalization of RAD51 away from DSBs in cells expressing HPV E6 and E7 hinders HR through a distinct mechanism. These observations have broad implications. The impairment of HR by HPV oncogenes may be targeted for treatment of HPV+ malignancies. Further, this attenuation of repair suggests HPV oncogenes may contribute to tumorigenesis by promoting the integration of the HPV genome, a common feature of HPV-transformed cells. Our data support this idea since HPV E6 stimulates the integration of episomes.

Channel blocking of MspA revisited.

Porin A from Mycobacterium smegmatis (MspA) is a highly stable, octameric channel protein, which acts as the main transporter of electrolytes across the cell membrane. MspA features a narrow, negatively charged constriction zone, allowing stable binding of various analytes thereby blocking the channel. Investigation of channel blocking of mycobacterial porins is of significance in developing alternate treatment methods for tuberculosis. The concept that ruthenium(II)quaterpyridinium complexes have the capability to act as efficient channel blockers for MspA and related porins, emerged after very high binding constants were measured by high-performance liquid chromatography and steady-state luminescence studies. Consequently, the interactions between the ruthenium(II) complex RuC2 molecules and MspA, leading to RuC2@MspA assemblies, have been studied utilizing time-resolved absorption/emission, atomic force microscopy, dynamic light scattering, ζ potential measurements, and isothermal titration calorimetry. The results obtained provide evidence for the formation of clusters/large aggregates of RuC2 and MspA. The results are of interest with respect to utilizing prospective channel blockers in porins. The combination of results from conceptually different techniques shed some light onto the chemical nature of MspA-channel blocker interactions thus contributing to the development of a paradigm for channel blocking.

Early breast cancer screening using iron/iron oxide-based nanoplatforms with sub-femtomolar limits of detection.

Summary Proteases, including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins (CTS) exhibit numerous functions in tumor biology. Solid tumors are characterized by changes in protease expression levels by tumor and surrounding tissue. Therefore, monitoring protease levels in tissue samples and liquid biopsies is a vital strategy for early cancer detection. Water-dispersable Fe/Fe3O4-core/shell based nanoplatforms for protease detection are capable of detecting protease activity down to sub-femtomolar limits of detection. They feature one dye (tetrakis(carboxyphenyl)porphyrin (TCPP)) that is tethered to the central nanoparticle by means of a protease-cleavable consensus sequence and a second dye (Cy 5.5) that is directly linked. Based on the protease activities of urokinase plasminogen activator (uPA), MMPs 1, 2, 3, 7, 9, and 13, as well as CTS B and L, human breast cancer can be detected at stage I by means of a simple serum test. By monitoring CTS B and L stage 0 detection may be achieved. This initial study, comprised of 46 breast cancer patients and 20 apparently healthy human subjects, demonstrates the feasibility of protease-activity-based liquid biopsies for early cancer diagnosis.

Loss of Genome Fidelity: Beta HPVs and the DNA Damage Response

While the role of genus alpha human papillomaviruses in the tumorigenesis and tumor maintenance of anogenital and oropharyngeal cancers is well-established, the role of genus beta human papilloviruses (β-HPVs) in non-melanoma skin cancers (NMSCs) is less certain. Persistent β-HPV infections cause NMSCs in sun-exposed skin of people with a rare genetic disorder, epidermodysplasia verruciformis. However, β-HPV infections in people without epidermodysplasia verruciformis are typically transient. Further, β-HPV gene expression is not necessary for tumor maintenance in the general population as on average there is fewer than one copy of the β-HPV genome per cell in NMSC tumor biopsies. Cell culture, epidemiological, and mouse model experiments support a role for β-HPV infections in the initiation of NMSCs through a “hit and run” mechanism. The virus is hypothesized to act as a cofactor, augmenting the genome destabilizing effects of UV. Supporting this idea, two β-HPV proteins (β-HPV E6 and E7) disrupt the cellular response to UV exposure and other genome destabilizing events by abrogating DNA repair and deregulating cell cycle progression. The aberrant damage response increases the likelihood of oncogenic mutations capable of driving tumorigenesis independent of a sustained β-HPV infection or continued viral protein expression. This review summarizes what is currently known about the deleterious effects of β-HPV on genome maintenance in the context of the viruss putative role in NMSC initiation.

Adaptation of Mycobacterium smegmatis to an industrial scale medium and isolation of the mycobacterial porinMspA

The adaptation of the organism to a simple and cost-effective growth medium is mandatory in developing a process for large scale production of the octamericporinMspA, which is isolated from Mycobacterium smegmatis. A fermentation optimization with the minimal nutrients required for growth has been performed. During the fermentation, the iron- and ammonium chloride concentrations in the medium were varied to determine their impact on the observed growth rates and cell mass yields. Common antibiotics to control contamination were eliminated in favor of copper sulfate to reduce costs. MspA has been successfully isolated from the harvested M. smegmatisusing aqueous nOPOE (n-octyloligooxyethylene) at 65°C. Because of the extraordinary stability of MspA, it is possible to denature and precipitate virtually all other proteins and contaminants by following this approach. To further purify the product, acetone is used for precipitation. Gel electrophoresis confirmed the presence and purity of MspA. A maximum of 840µg (via Bradford assay) of pure MspA per liter of the optimized simple growth medium has been obtained. This is a 40% increase with respect to the previously reported culture medium for MspA.

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzymes recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Fermentation optimization of Mycobacterium smegmatis using experimental design

The exceptionally stable mycobacterial protein porin A (MspA) from Mycobacterium smegmatis has potential applications in protein-based solar cells, and as a biotemplate for nano-wires and nano-dots. These applications would be enabled by an efficient and cost effective method to grow the host organism at high cell mass yields, and recover purified MspA. In this work, the cell mass yield was maximized and costs lowered by applying experimental design (varying nitrogen and iron contents according to a Doehlert matrix) based on a minimal fermentation medium that was reported earlier. Glucose use was minimized by adjusting glucose feed based on analyzing residual glucose after fermentation. The costs for extracted and purified MspA were lowered by 67% for the Original Research Article British Journal of Applied Science & Technology, 4(10): 1472-1484, 2014 1473 minimum medium and the optimized composition derived here compared to commercial medium (7H9 Middlebrook).