Two-compartment method for determination of the oxygen transfer rate with electrochemical sensors based on sulfite oxidation

The dissolved oxygen concentration is a crucial parameter in aerobic bioprocesses due to the low solubility of oxygen in water. The present study describes a new method for determining the oxygen transfer rate (OTR) in shaken-culture systems based on the sodium sulfite method in combination with an electrochemical oxygen sensor. The method replaces the laborious titration of the remaining sulfite by an on-line detection of the end point of the reaction. This method is a two-step procedure that can be applied in arbitrary flasks that do not allow the insertion of electrodes. The method does not therefore depend on the type of vessel in which the OTR is detected. The concept is demonstrated by determination of the OTR for standard baffled 1-L shake flasks and for opaque Ultra Yield™ flasks. Under typical shaking conditions, k_La values in the standard baffled flasks reached values up to 220 h^-1, whereas the k_La values of the Ultra Yield flasks were significantly higher (up to 422 h^-1).     

In vitro and in vivo activity of ribavirin against Andes virus infection

Pathogenic hantaviruses are a closely related group of rodent-borne viruses which are responsible for two distinct diseases in humans, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS, otherwise known as hantavirus cardiopulmonary syndrome, HCPS). The antiviral effect of ribavirin against Old World hantaviruses, most notably Hantaan virus, is well documented; however, only a few studies have addressed its inhibitory effect on New World hantaviruses. In the present study, we demonstrate that ribavirin is highly active against Andes virus (ANDV), an important etiological agent of HPS, both in vitro and in vivo using a lethal hamster model of HPS. Treatment of ANDV infected Vero E6 cells with ribavirin resulted in dose-dependent reductions in viral RNA and protein as well as virus yields with a half maximal inhibitory concentration between 5 and 12.5 μg ml(-1). In hamsters, treatment with as little as 5 mg kg(-1) day(-1) was 100% effective at preventing lethal HPS disease when therapy was administered by intraperitoneal injection from day 1 through day 10 post-infection. Significant reductions were observed in ANDV RNA and antigen positive cells in lung and liver tissues. Ribavirin remained completely protective when administered by intraperitoneal injections up to three days post-infection. In addition, we show that daily oral ribavirin therapy initiated 1 day post-infection and continuing for ten days is also protective against lethal ANDV disease, even at doses of 5 mg kg(-1) day(-1). Our results suggest ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses and should be considered in scenarios where exposure to the virus is probable. The similarities between the results obtained in this study and those from previous clinical evaluations of ribavirin against HPS, further validate the hamster model of lethal HPS and demonstrate its usefulness in screening antiviral agents against this disease.     

Comparative pathogenesis of three human and zoonotic SARS-CoV strains in cynomolgus macaques

The severe acute respiratory syndrome (SARS) epidemic was characterized by increased pathogenicity in the elderly due to an early exacerbated innate host response. SARS-CoV is a zoonotic pathogen that entered the human population through an intermediate host like the palm civet. To prevent future introductions of zoonotic SARS-CoV strains and subsequent transmission into the human population, heterologous disease models are needed to test the efficacy of vaccines and therapeutics against both late human and zoonotic isolates. Here we show that both human and zoonotic SARS-CoV strains can infect cynomolgus macaques and resulted in radiological as well as histopathological changes similar to those seen in mild human cases. Viral replication was higher in animals infected with a late human phase isolate compared to a zoonotic isolate. While there were significant differences in the number of host genes differentially regulated during the host responses between the three SARS-CoV strains, the top pathways and functions were similar and only apparent early during infection with the majority of genes associated with interferon signaling pathways. This study characterizes critical disease models in the evaluation and licensure of therapeutic strategies against SARS-CoV for human use.     

31P-NMR STUDIES ON INORGANIC POLYPHOSPHATES IN MICROALGAE1

Using “P nuclear magnetic resonance analysis, total inorganic polyphosphate in algae could be quantitatively estimated, For this purpose the algal suspension, which had been kept in cold trichloroacetic acid, was further treated with 6 mM EDTA, or the cells were kept in 2 N KOH containing 100 mM EDTA for 18 h at 37°C. These simple methods avoid hydrolysis of cellular inorganic polyphosphate and, therefore, are useful for the study of phosphorus metabolism in algae. The effects of these treatments on visualization of the signal for inorganic polyphosphate in nuclear magnetic resonance spectra were discussed in comparison with in vivo, ‘P nuclear magnetic resonance spectra of algae.     

Simian immunodeficiency virus infection in free-ranging sooty mangabeys (Cercocebus atys atys) from the Taï Forest, Côte d'Ivoire: implications for the origin of epidemic human immunodeficiency virus type 2

Simian immunodeficiency virus of sooty mangabeys (SIVsmm) is recognized as the progenitor of human immunodeficiency virus type 2 (HIV-2) and has been transmitted to humans on multiple occasions, yet the epidemiology and genetic diversity of SIVsmm infection in wild-living populations remain largely unknown. Here, we report the first molecular epidemiological survey of SIVsmm in a community of approximately 120 free-ranging sooty mangabeys in the Ta? Forest, C?te d'Ivoire. Fecal samples (n = 39) were collected from 35 habituated animals (27 females and 8 males) and tested for SIVsmm virion RNA (vRNA). Viral gag (800 bp) and/or env (490 bp) sequences were amplified from 11 different individuals (eight females and three males). Based on the sensitivity of fecal vRNA detection and the numbers of samples analyzed, the prevalence of SIVsmm infection was estimated to be 59% (95% confidence interval, 0.35 to 0.88). Behavioral data collected from this community indicated that SIVsmm infection occurred preferentially in high-ranking females. Phylogenetic analysis of gag and env sequences revealed an extraordinary degree of genetic diversity, including evidence for frequent recombination events in both the recent and distant past. Some sooty mangabeys harbored near-identical viruses (<2% interstrain distance), indicating epidemiologically linked infections. These transmissions were identified by microsatellite analyses to involve both related (mother/daughter) and unrelated individuals, thus providing evidence for vertical and horizontal transmission in the wild. Finally, evolutionary tree analyses revealed significant clustering of the Ta? SIVsmm strains with five of the eight recognized groups of HIV-2, including the epidemic groups A and B, thus pointing to a likely geographic origin of these human infections in the eastern part of the sooty mangabey range.     

Modulation of pharmacokinetics of radioiodinated sugar-conjugated somatostatin analogues by variation of peptide net charge and carbohydration chemistry

Sugar conjugation of biooactive peptides has been shown to be a powerful tool to modulate peptide pharmacokinetics. In the case of radiolabeled somatostatin analogues developed for in vivo scintigraphy of somatostatin receptor (sst) expressing tumors, it generally led to tracers with predominant renal excretion and low uptake in nontarget organs, and in some cases also with enhanced tumor accumulation. Especially with respect to endoradiotherapeutic applicability of these tracers, however, understanding the structural requirements for minimal kidney accumulation and maximal tumor uptake is important. The aim of this study was therefore the evaluation of the potential of specific glycoside structures in combination with reduced peptide net charge to reduce kidney accumulation without affecting tumor accumulation. Three glyco analogues of radioiodinated Tyr(3)-octreotate (TOCA) with z = 0 were evaluated in a comparative study using [(125)I]Mtr-TOCA (z = +1), the maltotriose-Amadori analogue of [(125)I]TOCA, as a reference, [(125)I]Glucuron-TOCA, the Amadori conjugate with glucuronic acid, and [(125)I]Gluc-S- and [(125)I]Gal-S-TOCA, the coupling products with glucosyl- and mannosyl-mercaptopropionate. In cells transfected with sst(1)-sst(5), all three new analogues show sst-subtype binding profiles similar to I-Mtr-TOCA with high, but somewhat reduced, affinity for sst(2). In contrast, internalization into sst(2)-expressing cells (in % of [(125)I]Tyr(3)-octreotide ([(125)I]TOC)) as well as the EC(50,R) of unlabeled TOC for internalization determined in dual-tracer experiments are substantially enhanced for [(123)I]Gal-S-TOCA and [(123)I]Gluc-S-TOCA (internalization, 190% +/- 12% and 265% +/- 20%, respectively, vs 168% +/- 6% of [(125)I]TOC for [(123)I]Mtr-TOCA; EC(50,R), 2.62 +/- 0.07 and 2.96 +/- 0.14, respectively, vs 1.81 +/- 0.07 for [(123)I]Mtr-TOCA). The tumor accumulation of [(125)I]Gal-S-TOCA and [(125)I]Gluc-S-TOCA in AR42J tumor-bearing nude mice 1 h p.i. is consequently very high (22.6 +/- 2.2 and 26.2 +/- 5.6%ID/g) and comparable to that of [(125)I]Mtr-TOCA (25.1 +/- 4.4%ID/g). [(125)I]Glucuron-TOCA showed lower uptake in sst-expressing tissues than did [(125)I]Mtr-TOCA, but considerably enhanced accumulation in nontarget organs such as liver, intestine, and kidney. Due to increased lipophilicity, hepatic and intestinal uptake 1 and 4 h p.i. of [(125)I]Gal-S-TOCA and [(125)I]Gluc-S-TOCA was also slightly higher than that of [(125)I]Mtr-TOCA. Kidney accumulation, however, was reduced by approximately 50% for both compounds (2.6 +/- 0.3 and 2.2 +/- 0.4, respectively, vs 4.0 +/- 0.7%ID/g at 1 h p.i.). Because no sugar-specific effect was detected in the latter case, it is concluded that general ligand pharmacokinetics and especially kidney accumulation of the tracers investigated are mainly determined by physicochemical characteristics such as lipophilicity, net charge, and also charge distribution ([(125)I]Glucuron-TOCA vs [(125)I]Gal-S- and [(125)I]Gluc-S-TOCA). With respect to receptor targeting, however, the structure of the carbohydrate moiety plays an important role, leading to dramatically enhanced ligand internalization, especially in the case of [(123)I]Gluc-S-TOCA. Taking into account the combined effects of the Gluc-S-moiety both on kidney and on tumor accumulation, this group seems to be a promising synthon for the synthesis of other radiolabeled peptide analogues with improved pharmacokinetics.     

Determination of telithromycin in human plasma and microdialysates by high-performance liquid chromatography

A high-performance liquid chromatography method for the quantitative determination of telithromycin in biological fluids is described. The method is suitable for plasma and microdialysates from the interstitial space fluid of skeletal muscle and subcutaneous adipose tissue. Plasma samples were deproteinised with trichloroacetic acid and neutralised with sodium hydroxide. Microdialysates were analysed without further preparation step. Telithromycin was separated isocratically on a reverse-phase column using acetonitrile-0.03 M ammonium acetate, pH 5.2 (43:57, v/v) at a flow rate of 0.8 mlmin(-1), and fluorescence detection (excitation 263 nm, emission 460 nm). The calibration curve was linear from 0.01 to 5 microgml(-1). Within- and between-day imprecision and inaccuracy was < or =10%. The limits of quantification were 0.02 and 0.015 microgml(-1) for plasma and microdialysates, respectively. Since telithromycin is decomposed in aqueous solution at ambient temperature, it is strongly recommended to store samples frozen at -80 degrees C, to maintain the temperature at 4 degrees C during all preparation steps, and to analyse samples within 120 min after thawing.     

A Novel Model of Lethal Hendra Virus Infection in African Green Monkeys and the Effectiveness of Ribavirin Treatment

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are emerging zoonotic paramyxoviruses that can cause severe and often lethal neurologic and/or respiratory disease in a wide variety of mammalian hosts, including humans. There are presently no licensed vaccines or treatment options approved for human or veterinarian use. Guinea pigs, hamsters, cats, and ferrets, have been evaluated as animal models of human HeV infection, but studies in nonhuman primates (NHP) have not been reported, and the development and approval of any vaccine or antiviral for human use will likely require efficacy studies in an NHP model. Here, we examined the pathogenesis of HeV in the African green monkey (AGM) following intratracheal inoculation. Exposure of AGMs to HeV produced a uniformly lethal infection, and the observed clinical signs and pathology were highly consistent with HeV-mediated disease seen in humans. Ribavirin has been used to treat patients infected with either HeV or NiV; however, its utility in improving outcome remains, at best, uncertain. We examined the antiviral effect of ribavirin in a cohort of nine AGMs before or after exposure to HeV. Ribavirin treatment delayed disease onset by 1 to 2 days, with no significant benefit for disease progression and outcome. Together our findings introduce a new disease model of acute HeV infection suitable for testing antiviral strategies and also demonstrate that, while ribavirin may have some antiviral activity against the henipaviruses, its use as an effective standalone therapy for HeV infection is questionable.Hendra virus (HeV) and Nipah virus (NiV) are members of the genus Henipavirus (family Paramyxoviridae) that can cause severe respiratory illness and/or encephalitis in a wide variety of mammals, including horses, pigs, and humans (7, 23). HeV was identified as the causative agent of an acute respiratory disease in horses in 1994 in Queensland, Australia (23), and to date there have been 14 outbreaks in Australia since, with at least one occurrence per year since 2006, most recently in May 2010 (ProMed-mail no. 20100522.1699 [International Society for Infectious Diseases, http://www.promedmail.org]). Every outbreak of HeV has involved horses as the initial infected host, and there have been a total of seven human cases arising from exposure to infected horses. Four human fatalities have occurred (22), with the most recent occurring in August of 2009 (ProMed-mail no. 20090826.2998 and 20090903.3098). All patients initially presented with influenza-like illnesses (ILIs) after an incubation period of 7 to 16 days. While two individuals recovered from ILI, one patient developed pneumonitis and died from multiorgan failure. Three of the lethal cases developed encephalitic manifestations (mild confusion and ataxia), with two patients experiencing seizures (22, 23, 27).Data on the histopathology of fatal human HeV cases are limited, but the pathology includes small necrotic plaques in the cerebrum and cerebellum, in addition to mild parenchymal inflammation (21, 27). Severe parenchymal inflammation and necrosis were observed in the lungs. More extensive histopathologic data are available from 32 autopsies of fatal human NiV cases (28). Similarly to the HeV cases, pathology was characterized by systemic vasculitis and parenchymal necrosis in the central nervous system (CNS), while in the lung, pathological findings mainly included vasculitis, fibrinoid necrosis, alveolar hemorrhage, pulmonary edema, and aspiration pneumonia. Other organs that were affected included heart, kidney, and spleen and showed generally mild or focal inflammation. The development of syncytial multinucleated endothelial cells is characteristic of both HeV and NiV (27, 28). At present, the details of the pathogenesis and histopathological changes mediated by either HeV or NiV infection in humans are naturally derived from only the late phases of the disease course, and therefore a relevant animal model is needed that mimics the disease progression seen in humans.Pteropid fruit bats, commonly known as flying foxes in the family Pteropodidae, are the principle natural reservoirs for both NiV and HeV (reviewed in reference 3). However, these henipaviruses display a broad species tropism, and in addition to bats, horses and humans, natural and/or experimental infection of HeV has been demonstrated in guinea pigs, hamsters, pigs, cats, and ferrets (25). Experimental infections of Syrian hamsters with HeV is lethal, and animals show disease similar to that of human cases, including respiratory and neurological symptoms, depending on the dose (11; unpublished data). In this model, viral RNA can be detected in various organs of infected hamsters, including brain, lung, kidney, heart, liver, and spleen. The main histopathological findings included parenchymal infection in various organs, including the brain, with vasculitis and syncytial multinucleated endothelial cells in many blood vessels (11). While this model is useful in studying pathogenesis, it is limited in the availability of reagents to do so.There are currently no vaccines or treatments licensed for human use. Several in vitro studies have shown that ribavirin is effective against both HeV and NiV infection (1, 2, 29). An open-label ribavirin treatment trial was run during an outbreak of NiV in Malaysia in 1998 and reported to reduce mortality by 36% (6). Of the seven recorded human HeV cases, three patients were treated with ribavirin, one of whom survived (22). In the most recent outbreak of HeV in Australia, three additional people received ribavirin treatment in combination with chloroquine after suspected exposure to HeV-contaminated secretions from infected horses. While all three individuals survived, infection was not confirmed, and therefore it remains unknown whether the treatment had any beneficiary effect (ProMed-mail no. 20090826.2998). In addition, two animal studies in hamsters showed that ribavirin treatment delays but does not prevent death from NiV or HeV infection (8, 10). Therefore, an animal model with greater relevance to humans and that recapitulates the disease processes seen in human cases of HeV is needed to get a better answer to whether ribavirin might be effective against henipavirus infections. In addition, the U.S. FDA implemented the “Animal Efficacy Rule,” which specifically applies to the development of therapeutic products when human efficacy studies are not possible or ethical, such as is often the case with highly virulent pathogens like HeV (24). Essentially, this rule allows for the evaluation of vaccines or therapeutics using data derived from studies carried out in at least two animal models. The licensure of any therapeutic modalities for HeV will require a thorough evaluation of HeV pathogenesis in nonhuman primates (NHPs).In the present study, we report the development and characterization of a new nonhuman primate (NHP) model of lethal HeV infection in the African green monkey (AGM). The pathogenesis and disease progression in the AGM upon HeV infection essentially mirrored the lethal disease episodes seen among human cases of HeV. Using this new model, the efficacy of ribavirin treatment against lethal challenge with HeV was examined. Here we have shown that ribavirin treatment can significantly delay but not prevent death of AGMs from lethal HeV infection. In addition to severe respiratory symptoms in all animals, prolonged disease progression in ribavirin-treated animals was also marked by the appearance of neurological symptoms.     

Insights into Peroxisome Function from the Structure of PEX3 in Complex with a Soluble Fragment of PEX19

The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.