Tackling feline infectious peritonitis via reverse genetics

Feline infectious peritonitis (FIP) is caused by feline coronaviruses (FCoVs) and represents one of the most important lethal infectious diseases of cats. To date, there is no efficacious prevention and treatment, and our limited knowledge on FIP pathogenesis is mainly based on analysis of experiments with field isolates. In a recent study, we reported a promising approach to study FIP pathogenesis using reverse genetics. We generated a set of recombinant FCoVs and investigated their pathogenicity in vivo. The set included the type I FCoV strain Black, a type I FCoV strain Black with restored accessory gene 7b, two chimeric type I/type II FCoVs and the highly pathogenic type II FCoV strain 79-1146. All recombinant FCoVs and the reference strain isolates were found to establish productive infections in cats. While none of the type I FCoVs and chimeric FCoVs induced FIP, the recombinant type II FCoV strain 79-1146 was as pathogenic as the parental isolate. Interestingly, an intact ORF 3c was confirmed to be restored in all viruses (re)isolated from FIP-diseased animals.

Surgical Technique: Reverse Periacetabular Osteotomy

Acetabular retroversion is the result of an externally rotated hemipelvis rather than a focal overgrowth of the anterior wall and/or hypoplasia of the posterior wall. Acetabular retroversion is a cause of pincer impingement which, if left untreated, can lead to hip pain and osteoarthritis. The causal surgical treatment in hips with acetabular retroversion is acetabular reorientation with a reverse periacetabular osteotomy (PAO). Indication is based on a positive correlation among symptoms (typically groin pain), physical findings on examination (positive anterior impingement test and decreased flexion and internal rotation), and radiographic signs for acetabular retroversion. These include a positive crossover, posterior wall, and ischial spine sign. A reverse PAO is performed with four osteotomies and a controlled fracture. Unlike reorientation of the acetabular fragment in dysplastic hips, correction for acetabular retroversion is achieved by a combined extension and internal rotation of the acetabular fragment. Typically, a small supra-acetabular wedge resection is required to allow sufficient extension of the fragment. The quality of acetabular reorientation is evaluated by intraoperative AP pelvic radiographs. In addition, intraoperative testing of range of motion following acetabular reorientation is mandatory. An arthrotomy and offset correction of the femoral head-neck area is indicated in hips with decreased internal rotation following acetabular reorientation. In a 10-year follow-up study of reverse PAO, a favorable outcome with preservation of all native joints was found. Correct acetabular orientation and, if necessary, a concomitant offset correction were the keys of successful outcome.

Relevant drug to drug interactions between itraconazole and non-nucleoside reverse transcriptase inhibitors in HIV-infected patients on maintenance therapy for disseminated histoplasmosis

Objective. Itraconazole is recommended life-long for preventing relapse of disseminated histoplasmosis in HIV-infected patients. I sought to determine if serum itraconazole levels are affected by the type of Highly Active Anti-Retroviral Therapy (NNRTI or PI) being taken concomitantly to treat HIV. ^ Design. Retrospective cohort. ^ Methods. De-identified data were used from an IRB-approved parent study which identified patients on HAART and maintenance itraconazole for confirmed disseminated histoplasmosis between January 2003 and December 2006. Available itraconazole blood levels were abstracted as well as medications taken by each patient at the time of the blood tests. Mean itraconazole levels were compared using the student's t-test. ^ Results. 11 patients met study criteria. Patient characteristics were: median age 36, 91% men, 18% white, 18% black, 55% Hispanic and 9% Asians, median CD4 cell count 120 cells/mm3. 14 blood levels were available for analysis—8 on PI, 4 on NNRTI and 2 on both. 8/8 itraconazole levels obtained while taking concomitant PI were therapeutic (>0.4 μg/mL) in contrast to 0/4 obtained while taking NNRTI. Two patients switched from NNRTI to PI and reached therapeutic levels. Mean levels on NNRTI (0.05 μg/mL, s.d. 0.0) and on PI (2.45 μg/mL, s.d. 0.21) for these two patients were compared via a paired t-test (t = 16.00, d.f. = 1, P = 0.04). Remaining patient levels were compared using an unpaired t-test. Mean itraconazole on concomitant PI (n = 6) was 1.37 μg/mL (s.d. 0.74), while the mean on concomitant NNRTI was 0.05 μg/mL (s.d. 0.0), t = 2.39, d.f. = 6, P = 0.05. ^ Conclusions. Co-administration of NNRTI and itraconazole results in significant decreases in itraconazole blood levels, likely by inducing the CYP3A4 enzyme system. Itraconazole drug levels should be monitored in patients on concomitant NNRTI. PI-based HAART may be preferred over NNRTI-based HAART when using itraconazole to treat HIV-infected patients with disseminated histoplasmosis. ^

Transport costs, distance, and time : evidence from the Japanese Census of Logistics

Geographic distance is a standard proxy for transport costs under the simple assumption that freight fees increase monotonically over space. Using the Japanese Census of Logistics, this paper examines the extent to which transport distance and time affect freight costs across shipping modes, commodity groups, and prefecture pairs. The results show substantial heterogeneity in transport costs and time across shipping modes. Consistent with an iceberg formulation of transport costs, distance has a significantly positive effect on freight costs by air transportation. However, I find the puzzling results that business enterprises are likely to pay more for short-distance shipments by truck, ship, and railroad transportation. As a plausible explanation, I discuss aggregation bias arising from freight-specific premiums for timely, frequent, and small-batch shipments.

Geographical simulation analysis for logistics enhancement in Asia

This paper presents a simulation of the reduction of several components in trade cost for Asia and examines its impact on the economy. Our simulation model based on the new economic geography embraces seven sectors, including manufacturing and non-manufacturing sectors, and 1,715 regions in 18 countries/economies in Asia, in addition to the two economies of the US and the European Union. The geographical course of transactions among regions is modeled as determined based on firms’ modal choice. The model also includes estimates of some border cost measures such as tariff rates, non-tariff barriers, other border clearance costs, transshipment costs and so on. Our simulation analysis for Asia includes several scenarios involving the improvement/development of routes and the reduction of the above-mentioned border cost. We have shown that the contribution of physical and non-physical infrastructure improvements conducted together is larger than the sum of the contribution by each when conducted independently.

Robustness and Recoverability in Transport Logistics

Abstract Transport is the foundation of any economy: it boosts economic growth, creates wealth, enhances trade, geographical accessibility and the mobility of people. Transport is also a key ingredient for a high quality of life, making places accessible and bringing people together. The future prosperity of our world will depend on the ability of all of its regions to remain fully and competitively integrated in the world economy. Efficient transport is vital in making this happen. Operations research can help in efficiently planning the design and operating transport systems. Planning and operational processes are fields that are rich in combinatorial optimization problems. These problems can be analyzed and solved through the application of mathematical models and optimization techniques, which may lead to an improvement in the performance of the transport system, as well as to a reduction in the time required for solving these problems. The latter aspect is important, because it increases the flexibility of the system: the system can adapt in a faster way to changes in the environment (i.e.: weather conditions, crew illness, failures, etc.). These disturbing changes (called disruptions) often enforce the schedule to be adapted. The direct consequences are delays and cancellations, implying many schedule adjustments and huge costs. Consequently, robust schedules and recovery plans must be developed in order to fight against disruptions. This dissertation makes contributions to two different fields: rail and air applications. Robust planning and recovery methods are presented. In the field of railway transport we develop several mathematical models which answer to RENFE’s (the major railway operator in Spain) needs: 1. We study the rolling stock assignment problem: here, we introduce some robust aspects in order to ameliorate some operations which are likely to fail. Once the rolling stock assignment is known, we propose a robust routing model which aims at identifying the train units’ sequences while minimizing the expected delays and human resources needed to perform the sequences. 2. It is widely accepted that the sequential solving approach produces solutions that are not global optima. Therefore, we develop an integrated and robust model to determine the train schedule and rolling stock assignment. We also propose an integrated model to study the rolling stock circulations. Circulations are determined by the rolling stock assignment and routing of the train units. 3. Although our aim is to develop robust plans, disruptions will be likely to occur and recovery methods will be needed. Therefore, we propose a recovery method which aims to recover the train schedule and rolling stock assignment in an integrated fashion all while considering the passenger demand. In the field of air transport we develop several mathematical models which answer to IBERIA’s (the major airline in Spain) needs: 1. We look at the airline-scheduling problem and develop an integrated approach that optimizes schedule design, fleet assignment and passenger use so as to reduce costs and create fewer incompatibilities between decisions. Robust itineraries are created to ameliorate misconnected passengers. 2. Air transport operators are continuously facing competition from other air operators and different modes of transport (e.g., High Speed Rail). Consequently, airline profitability is critically influenced by the airline’s ability to estimate passenger demands and construct profitable flight schedules. We consider multi-modal competition including airline and rail, and develop a new approach that estimates the demand associated with a given schedule; and generates airline schedules and fleet assignments using an integrated schedule design and fleet assignment optimization model that captures the impacts of schedule decisions on passenger demand.

Increasing the accuracy of positive displacement meters for the measurement of volume in liquid hydrocarbon logistics

Accuracy in the liquid hydrocarbons custody transfer is mandatory because it has a great economic impact. By far the most accurate meter is the positive displacement (PD) meter. Increasing such an accuracy may adversely affect the cost of the custody transfer, unless simple models are developed in order to lower the cost, which is the purpose of this work. PD meter consists of a fixed volume rotating chamber. For each turn a pulse is counted, hence, the measured volume is the number of pulses times the volume of the chamber. It does not coincide with the real volume, so corrections have to be made. All the corrections are grouped by a meter factor. Among corrections highlights the slippage flow. By solving the Navier-Stokes equations one can find an analytical expression for this flow. It is neither easy nor cheap to apply straightforward the slippage correction; therefore we have made a simple model where slippage is regarded as a single parameter with dimension of time. The model has been tested for several PD meters. In our careful experiments, the meter factor grows with temperature at a constant pace of 8?10?5?ºC?1. Be warned

Both DNA gyrase and reverse gyrase are present in the hyperthermophilic bacterium Thermotoga maritima

Like all hyperthermophiles yet tested, the bacterium Thermotoga maritima contains a reverse gyrase. Here we show that it contains also a DNA gyrase. The genes top2A and top2B encoding the two subunits of a DNA gyrase-like enzyme have been cloned and sequenced. The Top2A (type II DNA topoisomerase A protein) is more similar to GyrA (DNA gyrase A protein) than to ParC [topoisomerase IV (Topo IV) C protein]. The difference is especially striking at the C-terminal domain, which differentiates DNA gyrases from Topo IV. DNA gyrase activity was detected in T. maritima and purified to homogeneity using a novobiocin-Sepharose column. This hyperhermophilic DNA gyrase has an optimal activity around 82–86°C. In contrast to plasmids from hyperthermophilic archaea, which are from relaxed to positively supercoiled, we found that the plasmid pRQ7 from Thermotoga sp. RQ7 is negatively supercoiled. pRQ7 became positively supercoiled after addition of novobiocin to cell cultures, indicating that its negative supercoiling is due to the DNA gyrase of the host strain. The findings concerning DNA gyrase and negative supercoiling in Thermotogales put into question the role of reverse gyrase in hyperthermophiles.

Binding of RNA template to a complex of HIV-1 reverse transcriptase/primer/template

HIV-1 reverse transcriptase (RT) catalyzes the synthesis of DNA from DNA or RNA templates. During this process, it must transfer its primer from one template to another RNA or DNA template. Binary complexes made of RT and a primer/template bind an additional single-stranded RNA molecule of the same nucleotide sequence as that of the DNA or RNA template. The additional RNA strand leads to a 10-fold decrease of the off-rate constant, koff, of RT from a primer/DNA template. In a binary complex of RT and a primer/template, the primer can be cross-linked to both the p66 and p51 subunits. Depending on the location of the photoreactive group in the primer, the distribution of the cross-linked primers between subunits is dependent on the nature of the template and of the additional single-stranded molecule. Greater cross-linking of the primer to p51 occurs with DNA templates, whereas cross-linking to p66 predominates with RNA templates. Excess single-stranded DNA shifts the distribution of cross-linking from p66 to p51 with RNA templates, and excess single-stranded RNA shifts the cross-linking from p51 to p66 with DNA templates. RT thus uses two primer/template binding modes depending on the nature of the template.

Concomitant combination therapy for HIV infection preferable over sequential therapy with 3TC and non-nucleoside reverse transcriptase inhibitors

Exposure to 3TC of HIV-1 mutant strains containing non-nucleoside reverse transcriptase inhibitor (NNRTI)-specific mutations in their reverse transcriptase (RT) easily selected for double-mutant viruses that had acquired the characteristic 184-Ile mutation in their RT in addition to the NNRTI-specific mutations. Conversely, exposure of 3TC-resistant 184-Val mutant HIV-1 strains to nine different NNRTIs resulted in the rapid emergence of NNRTI-resistant virus strains at a time that was not more delayed than when wild-type HIV-1(IIIB) was exposed to the same compounds. The RTs of these resistant virus strains had acquired the NNRTI-characteristic mutations in addition to the preexisting 184-Val mutation. Surprisingly, when the 184-Ile mutant HIV-1 was exposed to a variety of NNRTIs, the 188-His mutation invariably occurred concomitantly with the 184-Ile mutation in the HIV-1 RT. Breakthrough of this double-mutant virus was markedly accelerated as compared with the mutant virus selected from the wild-type or 184-Val mutant HIV-1 strain. The double (184-Ile + 188-His) mutant virus showed a much more profound resistance profile against the NNRTIs than the 188-His HIV-1 mutant. In contrast with the sequential chemotherapy, concomitant combination treatment of HIV-1-infected cells with 3TC and a variety of NNRTIs resulted in a dramatic delay of virus breakthrough and resistance development.

Transgenic cattle produced by reverse-transcribed gene transfer in oocytes

A critical requirement for integration of retroviruses, other than HIV and possibly related lentiviruses, is the breakdown of the nuclear envelope during mitosis. Nuclear envelope breakdown occurs during mitotic M-phase, the envelope reforming immediately after cell division, thereby permitting the translocation of the retroviral preintegration complex into the nucleus and enabling integration to proceed. In the oocyte, during metaphase II (MII) of the second meiosis, the nuclear envelope is also absent and the oocyte remains in MII arrest for a much longer period of time compared with M-phase in a somatic cell. Pseudotyped replication-defective retroviral vector was injected into the perivitelline space of bovine oocytes during MII. We show that reverse-transcribed gene transfer can take place in an oocyte in MII arrest of meiosis, leading to production of offspring, the majority of which are transgenic. We discuss the implications of this mechanism both as a means of production of transgenic livestock and as a model for naturally occurring recursive transgenesis.

Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism

HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3′ end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3′-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri- or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template (Ki ≈ 5 μM), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3′ nucleotide from the primer to the pyrophosphate moiety in the nucleoside di- or triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the in vivo activity of many nucleoside antiretroviral drugs.

Hybrid Ty1/HIV-1 elements used to detect inhibitors and monitor the activity of HIV-1 reverse transcriptase

We previously demonstrated that hybrid retrotransposons composed of the yeast Ty1 element and the reverse transcriptase (RT) of HIV-1 are active in the yeast Saccharomyces cerevisiae. The RT activity of these hybrid Ty1/HIV-1 (his3AI/AIDS RT; HART) elements can be monitored by using a simple genetic assay. HART element reverse transcription depends on both the polymerase and RNase H domains of HIV-1 RT. Here we demonstrate that the HART assay is sensitive to inhibitors of HIV-1 RT. (−)-(S)-8-Chloro-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione monohydrochloride (8 Cl-TIBO), a well characterized non-nucleoside RT inhibitor (NNRTI) of HIV-1 RT, blocks propagation of HART elements. HART elements that express NNRTI-resistant RT variants of HIV-1 are insensitive to 8 Cl-TIBO, demonstrating the specificity of inhibition in this assay. HART elements carrying NNRTI-resistant variants of HIV-1 RT can be used to identify compounds that are active against drug-resistant viruses.