Suicide gene therapy with an adenovirus expressing the fusion gene CD::UPRT in human glioblastomas: different sensitivities correlate with p53 status

BACKGROUND: Several gene therapy strategies have been designed for cancer treatment. Intra-tumoral injection of adenoviruses expressing pro-drug-converting enzymes is one such strategy. Although the efficacy of these therapies was tested in animal models, little work has been devoted to the determination of critical variables for success. In this work, we aimed at better understanding variables that affect the cytosine deaminase::uracil phosphoribosyl transferase (CD::UPRT)-based strategy in two human glioblastomas. METHODS: We have constructed two adenoviruses expressing either CD or the fusion protein CD::UPRT. We have tested their anti-tumor activity in combination with 5-fluorocytosine (5-FC) in the glioblastoma cell lines U87 and U251, which are p53-wt and p53-deficient, respectively. Anti-tumor activity has also been investigated in spheroid structures. RESULTS: The superiority of CD::UPRT over CD was confirmed in both glioblastoma cells. We found that the pro-drug concentration required for anti-tumor activity was 9-fold higher in U251 than in U87, while multiplicity of infection (MOI) as low as 6 was sufficient to achieve 50% killing. Bystander activity was observed with as few as 2 and 6% transduced cells for U87 and U251, respectively. Differences in sensitivity between U87 and U251 were not due to differences in transduction, transgene expression, or intercellular transport, but were related to 5-FU sensitivity and p53 status. Also, it is noteworthy that, in contrast to U87, U251 spheroids barely responded to the treatment, whereas their monolayer counterparts were very sensitive. CONCLUSIONS: Our study has shown that p53 status is important for CD::UPRT/5-FC treatment. Moreover, this study demonstrated that the three-dimensional spheroid model is a more stringent in vitro model for suicide gene therapy evaluation than are monolayer cultures.     

Temporal shifts of fungal communities in the rhizosphere and on tubers in potato fields

Soil fungal communities perform important ecological roles determining, at least in part, agricultural productivity. This study aimed at examining the fungal community dynamics in the potato rhizosphere across different development stages in two consecutive growing seasons (winter and summer). Microbial fingerprinting of rhizosphere soil samples collected at pre-planting, tuber initiation, flowering and at senescence was performed using ARISA in conjunction with Next Generation Sequencing (Illumina MiSeq). The epiphytic fungal communities on tubers at harvest were also investigated. Alpha-diversity was stable over time within and across the two seasons. In contrast, rhizospheric fungal community structure and composition were different between the two seasons and in the different plant growth stages within a given season, indicating the significance of the rhizosphere in shaping microbial communities. The phylum Ascomycota was dominant in the potato fungal rhizosphere, with Operational Taxonomic Units (OTUs) belonging to the genus Peyronellaea being the most abundant in all samples. Important fungal pathogens of potato, together with potential biological control agents and saprophytic species, were identified as indicator OTUs at different plant growth stages. These findings indicate that potato rhizosphere fungal communities are functionally diverse, which may contribute to soil health.     

Chemotherapeutic Treatments Increase PD-L1 Expression in Esophageal Squamous Cell Carcinoma through EGFR/ERK Activation

The current study reveals the clinicopathological association of PD-L1 in Hong Kong esophageal squamous cell carcinoma (ESCC) patients and the differential regulation of PD-L1 by standard first-line chemotherapy in ESCC. Immunohistochemical analysis of tissue microarray data from 84 Hong Kong ESCC patients shows that PD-L1 was expressed in 21% of the tumors. Positive PD-L1 staining was significantly associated with later disease stage (stages III and IV) (P value = .0379) and lymph node metastasis (P value = .0466) in the Hong Kong cohort. Furthermore, PD-L1 expression was significantly induced in ESCC cell lines after standard chemotherapy treatments, along with EGFR and ERK activation in both in vitro studies and the in vivo esophageal orthotopic model. The endogenous expression of PD-L1 was reduced by treatment with an EGFR inhibitor (erlotinib) or by the knockdown of EGFR. Moreover, the upregulation of PD-L1 by chemotherapy was also attenuated by the treatment with erlotinib and a MAPK/MEK inhibitor (AZD6244), suggesting that PD-L1 is regulated by the EGFR/ERK pathway in ESCC. The regulation of PD-L1 by the EGFR pathway was further supported by the correlation of PD-L1 and EGFR expression observed in the commercially available tissue microarray set (P value = .028). Taken together, the current study was the first to demonstrate the upregulation of PD-L1 by chemotherapy in ESCC and its regulation through the EGFR/ERK pathway. The results suggest the potential usefulness of combined conventional chemotherapy together with anti–PD-L1 immunotherapy to achieve better treatment outcome.     

First report of the fall armyworm,Spodoptera frugiperda (Lepidoptera: Noctuidae), natural enemies from Africa

The fall armyworm (FAW), Spodoptera frugiperda, is a major pest of maize in North and South America. It was first reported from Africa in 2016 and currently established as a major invasive pest of maize. A survey was conducted to explore for natural enemies of the fall armyworm in Ethiopia, Kenya and Tanzania in 2017. Smallholder maize farms were randomly selected and surveyed in the three countries. Five different species of parasitoids were recovered from fall armyworm eggs and larvae, including four within the Hymenoptera and one Dipteran. These species are new associations with FAW and were never reported before from Africa, North and South America. In Ethiopia, Cotesia icipe was the dominant larval parasitoid with parasitism ranging from 33.8% to 45.3%, while in Kenya, the tachinid fly, Palexorista zonata, was the primary parasitoid with 12.5% parasitism. Charops ater and Coccygidium luteum were the most common parasitoids in Kenya and Tanzania with parasitism ranging from 6 to 12%, and 4 to 8.3%, respectively. Although fall armyworm has rapidly spread throughout these three countries, we were encouraged to see a reasonable level of biological control in place. This study is of paramount importance in designing a biological control program for fall armyworm, either through conservation of native natural enemies or augmentative release.     

A Cell-Based Model of Extracellular-Matrix-Guided Endothelial Cell Migration During Angiogenesis

Angiogenesis, the formation of new blood vessels sprouting from existing ones, occurs in several situations like wound healing, tissue remodeling, and near growing tumors. Under hypoxic conditions, tumor cells secrete growth factors, including VEGF. VEGF activates endothelial cells (ECs) in nearby vessels, leading to the migration of ECs out of the vessel and the formation of growing sprouts. A key process in angiogenesis is cellular self-organization, and previous modeling studies have identified mechanisms for producing networks and sprouts. Most theoretical studies of cellular self-organization during angiogenesis have ignored the interactions of ECs with the extra-cellular matrix (ECM), the jelly or hard materials that cells live in. Apart from providing structural support to cells, the ECM may play a key role in the coordination of cellular motility during angiogenesis. For example, by modifying the ECM, ECs can affect the motility of other ECs, long after they have left. Here, we present an explorative study of the cellular self-organization resulting from such ECM-coordinated cell migration. We show that a set of biologically-motivated, cell behavioral rules, including chemotaxis, haptotaxis, haptokinesis, and ECM-guided proliferation suffice for forming sprouts and branching vascular trees.     

Antibacterial copper–nickel bilayers and multilayer coatings by pulsed laser deposition on titanium

Biofouling, especially microfouling, is a major concern with the use of titanium (Ti) in the marine environment as a condenser material in cooling water systems. Earlier, copper–nickel (Cu/Ni) alloys were extensively used in marine environments due to their high corrosion and biofouling resistance. However, the choice of condenser material for the new fast breeder reactor in Kalpakkam is Ti to avoid steam side corrosion problems, which may pose a threat to steam generator parts having sodium as the secondary coolant. This study evaluates the surface modification of Ti using nano films of copper (Cu) and nickel (Ni) to utilize the antibacterial property of copper ions in reducing microfouling. The surface modification of Ti was carried out by the deposition of a Cu/Ni bilayer and (Cu/Ni)₁₀ multilayer films using a pulsed laser deposition technique. Various surface characterization studies revealed that the deposited Cu/Ni films were thin and nanocrystalline in nature. The antibacterial properties were evaluated using total viable count and epifluorescence microscopic techniques. The results showed an apparent decrease in bacterial attachment on multilayered and bilayered Cu/Ni thin films on Ti surfaces. Comparative studies between the two types of films showed a bigger reduction in numbers of microorganisms on the multilayers.     

Synthesis of acylated glycoconjugates as templates to investigate in vitro biopharmaceutical properties

A series of novel glycopyranosyl azides were synthesised wherein the carbohydrate moiety was peracylated with four acetyl, propionyl, butanoyl, pentanoyl (valeryl) or 3-methylbutanoyl (isovaleryl) ester linked groups. A panel of glycoconjugates was synthesised from these glycopyranosyl azides using copper-catalysed azide–alkyne cycloaddition. The in vitro metabolic stability, plasma stability and plasma protein binding was then measured to establish the impact of the different acyl group when presented on a common scaffold. The acetyl, propionyl and butanoyl esters exhibited metabolism consistent with esterase processing, and various mono-, di- and tri-acylated hydrolysis products as well as the fully hydrolysed compound were detected. In contrast, the pentanoyl and 3-methylbutanoyl esters were stable.     

Prolonged Minocycline Treatment Impairs Motor Neuronal Survival and Glial Function in Organotypic Rat Spinal Cord Cultures

Background

Minocycline, a second-generation tetracycline antibiotic, exhibits anti-inflammatory and neuroprotective effects in various experimental models of neurological diseases, such as stroke, Alzheimer’s disease, amyotrophic lateral sclerosis and spinal cord injury. However, conflicting results have prompted a debate regarding the beneficial effects of minocycline.

Methods

In this study, we analyzed minocycline treatment in organotypic spinal cord cultures of neonatal rats as a model of motor neuron survival and regeneration after injury. Minocycline was administered in 2 different concentrations (10 and 100 µM) at various time points in culture and fixed after 1 week.

Results

Prolonged minocycline administration decreased the survival of motor neurons in the organotypic cultures. This effect was strongly enhanced with higher concentrations of minocycline. High concentrations of minocycline reduced the number of DAPI-positive cell nuclei in organotypic cultures and simultaneously inhibited microglial activation. Astrocytes, which covered the surface of the control organotypic cultures, revealed a peripheral distribution after early minocycline treatment. Thus, we further analyzed the effects of 100 µM minocycline on the viability and migration ability of dispersed primary glial cell cultures. We found that minocycline reduced cell viability, delayed wound closure in a scratch migration assay and increased connexin 43 protein levels in these cultures.

Conclusions

The administration of high doses of minocycline was deleterious for motor neuron survival. In addition, it inhibited microglial activation and impaired glial viability and migration. These data suggest that especially high doses of minocycline might have undesired affects in treatment of spinal cord injury. Further experiments are required to determine the conditions for the safe clinical administration of minocycline in spinal cord injured patients.     

Adherence to Antiretroviral Therapy in Jinja,Uganda: A Six-Year Follow-Up Study

Introduction

We report on the adherence experience of a group of people living with HIV on ART over six years in Uganda.

Methods

Between 2005 and 2009, we followed up 41 participants who were also part of a clinical trial comparing home and facility based delivery of ART in Jinja, eastern Uganda. We conducted qualitative in-depth interviews at enrolment, 3, 6, 18 and 30 months to capture experiences with adherence over time. In 2011 we returned to these participants to find out how they were fairing with long term adherence. We managed to retrace 24 participants and interviewed them about their experience. We thematically analysed the data and compared findings over time.

Results

Initially there were few barriers to adherence and many followed the adherence guidance closely. By year six, relaxation of these rules was noticeable although self-reported adherence continued to be high. Alcohol consumption was more common than before. Some relatives of the participants who had died claimed that some deaths were a result of alcohol. While participants reported that ART had allowed them to reclaim independence and return to work the changes in work and social routines created new challenges for adherence. Side effects like lipodystrophy were not only causing some stigma but for some tested their faith in the drugs. Many participants reported resumption of sexual lives but apart from those who selected same status partners, disclosure to new partners was minimal.

Conclusion

Good adherence practice to ART wanes over the long-term, and people who may have disclosed at initiation find it difficult to do so to new partners once they are healthy. Further adherence interventions and support with disclosure over the course of therapy may need to be considered. (Words: 283)