Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants

The in vivo fate of Autographa californica multiple nucleopolyhedrovirus deletion mutants originating from serial passage in cell culture was investigated by passaging a population enriched in these mutants in insect larvae. The infectivity of polyhedra and occlusion-derived virion content per polyhedron were restored within two passages in vivo. The frequency of occurrence of deletion mutants was determined by real-time PCR. The frequency of the non-homologous region origin (non-HR ori) of DNA replication was reduced to wild-type levels within two passages. The frequency of the polyhedrin gene did not increase and remained below wild-type levels. A low m.o.i. during the initial infection in insect larvae, causing strong purifying selection for autonomously replicating viruses, could explain these observations. The same virus population used in vivo was also passaged once at a different m.o.i. in cell culture. A similar effect (i.e. lower non-HR ori frequency) was observed at low m.o.i. only, indicating that m.o.i. was the key selective conditio

Dioxin Toxicity In Vivo Results from an Increase in the Dioxin-Independent Transcriptional Activity of the Aryl Hydrocarbon Receptor

The Aryl hydrocarbon receptor (Ahr) is the nuclear receptor mediating the toxicity of dioxins -widespread and persistent pollutants whose toxic effects include tumor promotion, teratogenesis, wasting syndrome and chloracne. Elimination of Ahr in mice eliminates dioxin toxicity but also produces adverse effects, some seemingly unrelated to dioxin. Thus the relationship between the toxic and dioxin-independent functions of Ahr is not clear, which hampers understanding and treatment of dioxin toxicity. Here we develop a Drosophila model to show that dioxin actually increases the in vivo dioxin-independent activity of Ahr. This hyperactivation resembles the effects caused by an increase in the amount of its dimerisation partner Ahr nuclear translocator (Arnt) and entails an increased transcriptional potency of Ahr, in addition to the previously described effect on nuclear translocation. Thus the two apparently different functions of Ahr, dioxin-mediated and dioxin-independent, are in fact two different levels (hyperactivated and basal, respectively) of a single function

Fast Spectral Velocity Estimation Using Adaptive Techniques: In-Vivo Results

Adaptive spectral estimation techniques are known to provide good spectral resolution and contrast even when the observation window (OW) is very short. In this paper two adaptive techniques are tested and compared to the averaged periodogram (Welch) for blood velocity estimation. The blood power spectral capon (BPC) method is based on a standard minimum variance technique adapted to account for both averaging over slow-time and depth. The blood amplitude and phase estimation technique (BAPES) is based on finding a set of matched filters (one for each velocity component of interest) and filtering the blood process over slow-time and averaging over depth to find the power spectral density estimate. In this paper, the two adaptive methods are explained, and performance is assessed in controlled steady flow experiments and in-vivo measurements. The three methods were tested on a circulating flow rig with a blood mimicking fluid flowing in the tube. The scanning section is submerged in water to allow ultrasound data acquisition. Data was recorded using a BK8804 linear array transducer and the RASMUS ultrasound scanner. The controlled experiments showed that the OW could be significantly reduced when applying the adaptive methods without compromising spectral resolution or contrast. The in-vivo data was acquired using a BK8812 transducer. OWs of 128, 64, 32 and 16 slow- time samples were tested. Spectrograms with duration of 2 seconds were generated. Welch's method required 128 samples to give a reasonable spectrogram, whereas the BPC only required 32 samples before the SNR became a limiting factor. The BAPES managed to display the spectrogram with sufficient quality at 16 slow-time samples

Disruption of pre-mRNA splicing in vivo results in reorganization of splicing factors

We have examined the functional significance of the organization of pre-mRNA splicing factors in a speckled distribution in the mammalian cell nucleus. Upon microinjection into living cells of oligonucleotides or antibodies that inhibit pre-mRNA splicing in vitro, we observed major changes in the organization of splicing factors in vivo. Interchromatin granule clusters became uniform in shape, decreased in number, and increased in both size and content of splicing factors, as measured by immunofluorescence. These changes were transient and the organization of splicing factors returned to their normal distribution by 24 h following microinjection. Microinjection of these oligonucleotides or antibodies also resulted in a reduction of transcription in vivo, but the oligonucleotides did not inhibit transcription in vitro. Control oligonucleotides did not disrupt splicing or transcription in vivo. We propose that the reorganization of splicing factors we observed is the result of the inhibition of splicing in vivo

A Thiazole Orange Derivative Targeting the Bacterial Protein FtsZ Shows Potent Antibacterial Activity.

The prevalence of multidrug resistance among clinically significant bacteria calls for the urgent development of new antibiotics with novel mechanisms of action. In this study, a new small molecule exhibiting excellent inhibition of bacterial cell division with potent antibacterial activity was discovered through cell-based screening. The compound exhibits a broad spectrum of bactericidal activity, including the methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and NDM-1 Escherichia coli. The in vitro and in vivo results suggested that this compound disrupts the dynamic assembly of FtsZ protein and Z-ring formation through stimulating FtsZ polymerization. Moreover, this compound exhibits no activity on mammalian tubulin polymerization and shows low cytotoxicity on mammalian cells. Taken together, these findings could provide a new chemotype for development of antibacterials with FtsZ as the target

Optical imaging of Tc-99m-based tracers: in vitro and in vivo results.

It has been recently shown that optical imaging (OI) methods can be used to image the in vivo biodistribution of several radiopharmaceuticals labeled with beta or alpha emitters. In this work particular attention has been focused on investigating the weaker optical signal induced by an almost pure gamma emitter like Tc-99m. Visible light emission measurements of a water solution containing Tc-99m were performed using a small animal OI system. A sequence of images was acquired for 24 h in order to study the decay of the luminescence signal. The difference between the luminescence decay half life and well-known Tc-99m half life was equal to 1%. in vivo imaging was performed by injecting one control nude mice with Tc-99m-MDP. Optical images obtained with equipment designed for bioluminescence imaging showed that a visible light emission was distinguishable and correctly localized in the bladder region where a higher concentration of Tc-99m-MDP was expected. The bladder to background ratio was always greater than 1. We conclude that the experimental data presented in this paper show that it is possible to detect in vivo luminescence optical photons induced by Tc-99m. This is important especially considering the large number of Tc-99m-based radiopharmaceutical currently available

Inhibition of Sonic hedgehog signaling in vivo results in craniofacial neural crest cell death

Background: Sonic hedgehog (Shh) is well known for its role in patterning tissues, including structures of the head. Haploinsufficiency for SHH in humans results in holoprosencephaly, a syndrome characterized by facial and forebrain abnormalities. Shh null mice have cyclopia and loss of branchial arch structures. It is unclear, however, whether these phenotypes arise solely from the early function of Shh in patterning midline structures, or whether Shh plays other roles in head development. Results: To address the role of Shh after floorplate induction, we inhibited Shh signaling by injecting hybridoma cells that secrete a function-blocking anti-Shh antibody into the chick cranial mesenchyme. The antibody subsequently bound to Shh in the floorplate, notochord, and the pharyngeal endoderm. Perturbation of Shh signaling at this stage resulted in a significant reduction in head size after 1 day, loss of branchial arch structures after 2 days, and embryos with smaller heads after 7 days. Cell death was significantly increased in the neural tube and neural crest after 1 day, and neural crest cell death was not secondary to the loss of neural tube cells. Conclusions: Reduction of Shh signaling after neural tube closure resulted in a transient decrease in neural tube cell proliferation and an extensive increase in cell death in the neural tube and neural crest, which in turn resulted in decreased head size. The phenotypes observed after reduction of Shh are similar to those observed after cranial neural crest ablation. Thus, our results demonstrate a role for Shh in coordinating the proliferation and survival of cells of the neural tube and cranial neural crest

Real-Time Optical Coherence Tomography Controlled Microsecond Laser Retinal Microsurgery: First In-vivo Results

Reliable mild photocoagulation and selective retina therapy (SRT) selectively damaging the retinal pigment epithelium (RPE) while sparing the neuroretina, the photoreceptors as well as the choroid are highly demanded. However, due to the inter- and intraindividual variability of RPE and choroidal absorption, optical microsurgery requires reliable real-time laser dosing to prevent unwanted overexposure and extended damage of the neuroretina. In this experiment optical coherence tomography (OCT) was implemented to detect minimal damage, and a laser feedback control algorithm was used for real-time dosing. For the first time in-vivo experiments on rabbits were performed with microsecond laser pulses of varying duration. Pigment rabbit eyes (n=6) were exposed to laser pulses of 4, 8, 12, and 20 μs in duration (wavelength, 532 nm; ramp-mode, maximum 15 pulses; repetition rate, 100 Hz). Therefore, a system with a scanning laser ophthalmoscope and spectral-domain OCT (Heidelberg Engineering) extended with a prototype laser (Meridian Medical) was used. For each laser lesion, the increasing ramp’s end energy was individually controlled in real-time using OCT dosimetry (central wavelength, 870 nm; scan rate, 80 kHz). Within 1 hour after irradiation, retinal changes were assessed with fluorescein angiography (FA), indocyanine green angiography (ICGA), color fundus photography (CFP) and OCT. OCT dosimetry utilizing the control algorithm can interrupt the ramp-mode in real-time for each lesion individually. The preconditioned algorithm enabled treatment with a clearly visible breakdown of the blood-retinal barrier (BRB) according to FA and ICGA imaging and barely visible treatment lesions according to CFP. OCT B-scans through the treated areas provided a first indication of the morphological tissue impact. Preliminary evaluation shows that the algorithm stopped the laser at 4 μs at a ramp end energy of 53 μJ (corresponds to 13/15 pulses), at 8 μs at 68 μJ (5/15 pulses), at 12 μs at 74 μJ (7/15 pulses), and at 20 μs at 100 μJ (1/15 pulses). The novel system with OCT based laser dosing proved to induce minimal visible damage and BRB breakdown in a wide range of pulse durations. The new irradiation scheme and algorithm are being optimized and tested in multiple subjects to further limit unwanted damage and enable pure RPE selective laser microsurgery in real-time. This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually

Efficacy of chloroquine, sulphadoxine-pyrimethamine and amodiaquine for treatment of uncomplicated Plasmodium falciparum malaria in Kajo Keji county, Sudan.

To provide advice on the rational use of antimalarial drugs, Médecins Sans Frontières conducted a randomized, an open label efficacy study in Kajo Keji, an area of high transmission of malaria in southern Sudan. The efficacy of chloroquine (CQ), sulphadoxine-pyrimethamine (SP) and amodiaquine (AQ) were measured in a 28-day in vivo study, with results corrected by PCR genotyping. Of 2010 children screened, 115 children aged 6-59 months with uncomplicated Plasmodium falciparum malaria were randomized into each group to receive a supervised course of treatment. Of these, 114, 103 and 111 were analysed in the CQ, SP and AQ groups, respectively. The overall parasitological failure rates at day 28 were 93.9% [95% confidence interval (CI) 87.3-97.3] for CQ, 69.9% (95% CI 60.0-78.3) for SP, and 25.2% (95% CI 17.7-34.5) for AQ. These results provide important missing data on antimalarial drug efficacy in southern Sudan. They indicate that none of the drugs could be used in monotherapy and suggest that even in combination with artemisinin, cure rates might not be efficacious enough. We recommend a combination of artemether and lumefantrine as first-line treatment for uncomplicated P. falciparum malaria cases in Kajo Keji county