Changes associated with tyrosine phosphorylation during short-term hypoxia in retinal microvascular endothelial cells in vitro

The occlusion of capillary vessels results in low oxygen tension in adjacent tissues which triggers a signaling cascade that culminates in neovascularization. Using bovine retinal capillary endothelial cells (BRCEC), we investigated the effects of short-term hypoxia on DNA synthesis, phosphotyrosine induction, changes in the expression of basic fibroblast growth factor receptor (bFGFR), protein kinase C (PKCα), heat shock protein 70 (HSP70), and SH2-containing protein (SHC). The effect of protein tyrosine kinase (PTK) and phosphatase inhibitors on hypoxia-induced phosphotyrosine was also studied. Capillary endothelial cells cultured in standard normoxic (pO₂ = 20%) conditions were quiesced in low serum containing medium and then exposed to low oxygen tension or hypoxia (pO₂ = 3%) in humidified, 5% CO₂, 37°C, tissue culture chambers, on a time-course of up to 24 h. DNA synthesis was potentiated by hypoxia in a time-dependent manner. This response positively correlated with the cumulative induction of phosphotyrosine and the downregulation of bFGFR (M_r ～ 85 kDa). Protein tyrosine kinase inhibitors, herbimycin-A, and methyl 2,5-dihydroxycinnamate, unlike genistein, markedly blocked hypoxia-induced phosphotyrosine. Prolonged exposure of cells to phosphatase inhibitor, sodium orthovanadate, also blocked hypoxia-induced phosphotyrosine. The expression of HSP70, PKCα, and SHC were not markedly altered by hypoxia. Taken together, these data suggest that short-term hypoxia activates endothelial cell proliferation in part via tyrosine phosphorylation of cellular proteins and changes in the expression of the FGF receptor. Thus, endothelial cell mitogenesis and neovascularization associated with low oxygen tension may be controlled by abrogating signaling pathways mediated by protein tyrosine kinase and phosphatases. © 1995 Wiley-Liss, Inc.     

Achievements and challenges of lymphatic filariasis elimination in Sierra Leone

BackgroundLymphatic filariasis (LF) is targeted for elimination in Sierra Leone. Epidemiological coverage of mass drug administration (MDA) with ivermectin and albendazole had been reported >65% in all 12 districts annually. Eight districts qualified to implement transmission assessment survey (TAS) in 2013 but were deferred until 2017 due to the Ebola outbreak (2014–2016). In 2017, four districts qualified for conducting a repeat pre-TAS after completing three more rounds of MDA and the final two districts were also eligible to implement a pre-TAS.Methodology/Principal findingsFor TAS, eight districts were surveyed as four evaluation units (EU). A school-based survey was conducted in children aged 6–7 years from 30 clusters per EU. For pre-TAS, one sentinel and one spot check site per district (with 2 spot check sites in Bombali) were selected and 300–350 persons aged 5 years and above were selected. For both surveys, finger prick blood samples were tested using the Filariasis Test Strips (FTS).For TAS, 7,143 children aged 6–7 years were surveyed across four EUs, and positives were found in three EUs, all below the critical cut-off value for each EU. For the repeat pre-TAS/pre-TAS, 3,994 persons over five years of age were surveyed. The Western Area Urban had FTS prevalence of 0.7% in two sites and qualified for TAS, while other five districts had sites with antigenemia prevalence >2%: 9.1–25.9% in Bombali, 7.5–19.4% in Koinadugu, 6.1–2.9% in Kailahun, 1.3–2.3% in Kenema and 1.7% - 3.7% in Western Area Rural.Conclusions/SignificanceEight districts in Sierra Leone have successfully passed TAS1 and stopped MDA, with one more district qualified for conducting TAS1, a significant progress towards LF elimination. However, great challenges exist in eliminating LF from the whole country with repeated failure of pre-TAS in border districts. Effort needs to be intensified to achieve LF elimination.     

Impact of Three Rounds of Mass Drug Administration on Lymphatic Filariasis in Areas Previously Treated for Onchocerciasis in Sierra Leone

Background

1974–2005 studies across Sierra Leone showed onchocerciasis endemicity in 12 of 14 health districts (HDs) and baseline studies 2005–2008 showed lymphatic filariasis (LF) endemicity in all 14 HDs. Three integrated annual mass drug administration (MDA) were conducted in the 12 co-endemic districts 2008–2010 with good geographic, programme and drug coverage. Midterm assessment was conducted 2011 to determine impact of these MDAs on LF in these districts.

Methodology/Principal Findings

The mf prevalence and intensity in the 12 districts were determined using the thick blood film method and results compared with baseline data from 2007–2008. Overall mf prevalence fell from 2.6% (95% CI: 2.3%–3.0%) to 0.3% (95% CI: 0.19%–0.47%), a decrease of 88.5% (p = 0.000); prevalence was 0.0% (100.0% decrease) in four districts: Bo, Moyamba, Kenema and Kono (p = 0.001, 0.025, 0.085 and 0.000 respectively); and seven districts had reductions in mf prevalence of between 70.0% and 95.0% (p = 0.000, 0.060, 0.001, 0.014, 0.000, 0.000 and 0.002 for Bombali, Bonthe, Kailahun, Kambia, Koinadugu, Port Loko and Tonkolili districts respectively). Pujehun had baseline mf prevalence of 0.0%, which was maintained. Only Bombali still had an mf prevalence ≥1.0% (1.58%, 95% CI: 0.80%–3.09%)), and this is the district that had the highest baseline mf prevalence: 6.9% (95% CI: 5.3%–8.8%). Overall arithmetic mean mf density after three MDAs was 17.59 mf/ml (95% CI: 15.64 mf/ml–19.55 mf/ml) among mf positive individuals (65.4% decrease from baseline of 50.9 mf/ml (95% CI: 40.25 mf/ml–61.62 mf/ml; p = 0.001) and 0.05 mf/ml (95% CI: 0.03 mf/ml–0.08 mf/ml) for the entire population examined (96.2% decrease from baseline of 1.32 mf/ml (95% CI: 1.00 mf/ml–1.65 mf/ml; p = 0.000)).

Conclusions/Significance

The results show that mf prevalence decreased to <1.0% in all but one of the 12 districts after three MDAs. Overall mf density reduced by 65.0% among mf-positive individuals, and 95.8% for the entire population.     

Selection of antimalarial drug resistance after intermittent preventive treatment of infants and children (IPTi/c) in Senegal

Senegal has since 2003 used sulphadoxine-pyrimethamine (SP) for Intermittent Preventive Treatment (IPT) of malaria in risk groups. However, the large-scale IPT strategy may result in increasing drug resistance. Our study investigated the possible impact of SP-IPT given to infants and children on the prevalence of SP-resistant haplotypes in the Plasmodium falciparum genes Pfdhfr and Pfdhps, comparing sites with and without IPTi/c. P. falciparum positives samples (n = 352) were collected from children under 5 years of age during two cross-sectional surveys in 2010 and 2011 in three health districts (two on IPTi/c and one without IPTi/c intervention) located in the southern part of Senegal. The prevalence of SP-resistance-related haplotypes in Pfdhfr and Pfdhps was determined by nested PCR followed by sequence-specific oligonucleotide probe (SSOP)–ELISA. The prevalence of the Pfdhfr double mutant haplotypes (CNRN and CICN) was stable between years at < 10% in the control group (P = 0.69), while it rose significantly in the IPTi/c group from 2% in 2010 to 20% in 2011 (P = 0.008). The prevalence of the Pfdhfr triple mutant haplotype (CIRN) increased in both groups, but only significantly in the IPTi/c group from 41% to 65% in 2011 (P = 0.005). Conversely, the Pfdhps 437G mutation decreased in both groups from 44.6% to 28.6% (P = 0.07) and from 66.7% to 47.5% (P = 0.02) between 2010 and 2011 in the control and the IPTi/c groups, respectively. Combined with Pfdhfr, there was a weak trend for decreasing prevalence of quadruple mutants (triple Pfdhfr + Pfdhps 437G) in both groups (P = 0.15 and P = 0.34). During the two cross-sectional surveys, some significant changes were observed in the SP-resistance-related genes. However, since these changes were observed in the two groups, the IPTi/c strategy does only seem to have limited impact on resistance development and other factors as well. However, continuous monitoring will be needed, due to the up-scaling of the IPTi/c strategy in Senegal according to WHO recommendations.     

Opto-Mechanical Coupling in Interfaces under Static and Propagative Conditions and Its Biological Implications

Fluorescent dyes are vital for studying static and dynamic patterns and pattern formation in cell biology. Emission properties of the dyes incorporated in a biological interface are known to be sensitive to their local environment. We report that the fluorescence intensity of dye molecules embedded in lipid interfaces is indeed a thermodynamic observable of the system. Opto-mechanical coupling of lipid-dye system was measured as a function of the thermodynamic state of the interface. The corresponding state diagrams quantify the thermodynamic coupling between intensity I and lateral pressure π. We further demonstrate that the coupling is conserved upon varying the temperature T. Notably, the observed opto-mechanical coupling is not limited to equilibrium conditions, but also holds for propagating pressure pulses. The non-equilibrium data show, that fluorescence is especially sensitive to dynamic changes in state such as the LE-LC phase transition. We conclude that variations in the thermodynamic state (here π and T, in general pH, membrane potential V, etc also) of lipid membranes are capable of controlling fluorescence intensity. Therefore, interfacial thermodynamic state diagrams of I should be obtained for a proper interpretation of intensity data.     

The Impact of the West Africa Ebola Outbreak on Obstetric Health Care in Sierra Leone

Background

As Sierra Leone celebrates the end of the Ebola Virus Disease (EVD) outbreak, we can begin to fully grasp its impact on already weak health systems. The EVD outbreak in West Africa forced many hospitals to close down or reduce their activity, either to prevent nosocomial transmission or because of staff shortages. The aim of this study is to assess the potential impact of EVD on nationwide access to obstetric care in Sierra Leone.

Methods and Findings

Community health officers collected weekly data between January 2014—May 2015 on in-hospital deliveries and caesarean sections (C-sections) from all open facilities (public, private for-profit and private non-profit sectors) offering emergency obstetrics in Sierra Leone. This was compared to official data of EVD cases per district. Logistic and Poisson regression analyses were used to compute risk and rate estimates. Nationwide, the number of in-hospital deliveries and C-sections decreased by over 20% during the EVD outbreak. The decline occurred early on in the EVD outbreak and was mainly attributable to the closing of private not-for-profit hospitals rather than government facilities. Due to difficulties in collecting data in the midst of an epidemic, limitations of this study include some missing data points.

Conclusions

Both the number of in-hospital deliveries and C-sections substantially declined shortly after the onset of the EVD outbreak. Since access to emergency obstetric care, like C-sections, is associated with decreased maternal mortality, many women are likely to have died due to the reduced access to appropriate care during childbirth. Future research on indirect health effects of health system breakdown should ideally be nationwide and continue also into the recovery phase. It is also important to understand the mechanisms behind the deterioration so that important health services can be reestablished.     

Neuropilin-1 modulates p53/caspases axis to promote endothelial cell survival

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), one of the crucial pro-angiogenic factors, functions as a potent inhibitor of endothelial cell (EC) apoptosis. Previous progress has been made towards delineating the VPF/VEGF survival signaling downstream of the activation of VEGFR-2. Here, we seek to define the function of NRP-1 in VPF/VEGF-induced survival signaling in EC and to elucidate the concomitant molecular signaling events that are pivotal for our understanding of the signaling of VPF/VEGF. Utilizing two different in vitro cell culture systems and an in vivo zebrafish model, we demonstrate that NRP-1 mediates VPF/VEGF-induced EC survival independent of VEGFR-2. Furthermore, we show here a novel mechanism for NRP-1-specific control of the anti-apoptotic pathway in EC through involvement of the NRP-1-interacting protein (NIP/GIPC) in the activation of PI-3K/Akt and subsequent inactivation of p53 pathways and FoxOs, as well as activation of p21. This study, by elucidating the mechanisms that govern VPF/VEGF-induced EC survival signaling via NRP-1, contributes to a better understanding of molecular mechanisms of cardiovascular development and disease and widens the possibilities for better therapeutic targets.     

Septins at the annulus of mammalian sperm

The annulus is an electron-dense ring structure connecting the midpiece and the principal piece of the mammalian sperm flagellum. Proteins from the septin family have been shown to localize to the annulus. A septin complex is assembled early in spermiogenesis with the cochaperone DNAJB13 and, in mature sperm, associates with Testis Anion Transporter 1; SLC26A8 (Tat1), a transmembrane protein of the SLC26 family. Studies in mice have shown that the annulus acts as a barrier to protein diffusion and controls correct organization of the midpiece. Consistent with these findings, absence of the annulus is associated with flagellum differentiation defects and asthenozoospermia in humans.     

Designing nanoconjugates to effectively target pancreatic cancer cells in vitro and in vivo

Background

Pancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells.Gold nanoparticles (GNPs) are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography). The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells.

Methodology/Principal Findings

The nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent) on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor), all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer.

Conclusion

Our results suggest the specific targeting of tumor cells depends on a number of crucial components 1) targeting agent to nanoparticle ratio 2) availability of reactive surface area on the nanoparticle 3) ability of the nanoconjugate to bind the target and 4) hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle conjugates.