Regulatory guidelines and preclinical tools to study the biodistribution of RNA therapeutics.

The success of the messenger RNA-based COVID-19 vaccines of Moderna and Pfizer/BioNTech marks the beginning of a new chapter in modern medicine. However, the rapid rise of mRNA therapeutics has resulted in a regulatory framework that is somewhat lagging. The current guidelines either do not apply, do not mention RNA therapeutics, or do not have widely accepted definitions. This review describes the guidelines for preclinical biodistribution studies of mRNA/siRNA therapeutics and highlights the relevant differences for mRNA vaccines. We also discuss the role of in vivo RNA imaging techniques and other assays to fulfill and/or complement the regulatory requirements. Specifically, quantitative whole-body autoradiography, microautoradiography, mass spectrometry-based assays, hybridization techniques (FISH, bDNA), PCR-based methods, in vivo fluorescence imaging, and in vivo bioluminescence imaging, are discussed. We conclude that this new and rapidly evolving class of medicines demands a multi-layered approach to fully understand its biodistribution and in vivo characteristics.

In vitro exploration of a myeloid-derived suppressor cell line as vehicle for cancer gene therapy.

Recent research indicates that cell-mediated gene therapy can be an interesting method to obtain intratumoral expression of therapeutic proteins. This paper explores the possibility of using transfected myeloid-derived suppressor cells (MDSCs), derived from a murine cell line, as cellular vehicles for transporting plasmid DNA (pDNA) encoding interleukin-12 (IL-12) to tumors. Transfecting these cells via electroporation caused massive cell death. This was not due to electroporation-induced cell damage, but was mainly the result of the intracellular presence of plasmids. In contrast, pDNA transfection using Lipofectamine 2000 (LF2000) did not result in a significant loss of viability. Differences in delivery mechanism may explain the distinctive effects on cell viability. Indeed, electroporation is expected to cause a rapid and massive influx of pDNA resulting in cytosolic pDNA levels that most likely surpass the activation threshold of the intracellular DNA sensors leading to cell death. In contrast, a more sustained intracellular release of the pDNA is expected with LF2000. After lipofection with LF2000, 56% of the MDSCs were transfected and transgene expression lasted for at least 24 h. Moreover, biologically relevant amounts of IL-12 were produced by the MDSCs after lipofection with an IL-12 encoding pDNA. In addition, IL-12 transfection caused a significant upregulation of CD80 and considerably reduced the immunosuppressive capacity of the MDSCs. IL-12-transfected MDSCs were still able to migrate to tumor cells, albeit that lipofection of the MDSCs seemed to slightly decrease their migration capacity.

Competition between Spin Echo and Spin Self-Rephasing in a Trapped Atom Interferometer.

We perform Ramsey interferometry on an ultracold ^{87}Rb ensemble confined in an optical dipole trap. We use a π pulse set at the middle of the interferometer to restore the coherence of the spin ensemble by canceling out phase inhomogeneities and creating a spin echo in the contrast. However, for high atomic densities, we observe the opposite behavior: the π pulse accelerates the dephasing of the spin ensemble leading to a faster contrast decay of the interferometer. We understand this phenomenon as a competition between the spin-echo technique and an exchange-interaction driven spin self-rephasing mechanism based on the identical spin rotation effect. Our experimental data are well reproduced by a numerical model.

Powers of the likelihood ratio test and the correlation test using empirical bayes estimates for various shrinkages in population pharmacokinetics.

We compared the powers of the likelihood ratio test (LRT) and the Pearson correlation test (CT) from empirical Bayes estimates (EBEs) for various designs and shrinkages in the context of nonlinear mixed-effect modeling. Clinical trial simulation was performed with a simple pharmacokinetic model with various weight (WT) effects on volume (V). Data sets were analyzed with NONMEM 7.2 using first-order conditional estimation with interaction and stochastic approximation expectation maximization algorithms. The powers of LRT and CT in detecting the link between individual WT and V or clearance were computed to explore hidden or induced correlations, respectively. Although the different designs and variabilities could be related to the large shrinkage of the EBEs, type 1 errors and powers were similar in LRT and CT in all cases. Power was mostly influenced by covariate effect size and, to a lesser extent, by the informativeness of the design. Further studies with more models are needed.

Unbalanced expression of CK2 kinase subunits is sufficient to drive epithelial-to-mesenchymal transition by Snail1 induction.

Epithelial-to-mesenchymal transition (EMT) is closely linked to conversion of early-stage tumours into invasive malignancies. Many signalling pathways are involved in EMT, but the key regulatory kinases in this important process have not been clearly identified. Protein kinase CK2 is a multi-subunit protein kinase, which, when overexpressed, has been linked to disease progression and poor prognosis in various cancers. Specifically, overexpression of CK2α in human breast cancers is correlated with metastatic risk. In this article, we show that an imbalance of CK2 subunits reflected by a decrease in the CK2ß regulatory subunit in a subset of breast tumour samples is correlated with induction of EMT-related markers. CK2ß-depleted epithelial cells displayed EMT-like morphological changes, enhanced migration, and anchorage-independent growth, all of which require Snail1 induction. In epithelial cells, Snail1 stability is negatively regulated by CK2 and GSK3ß through synergistic hierarchal phosphorylation. This process depends strongly on CK2ß, thus confirming that CK2 functions upstream of Snail1. In primary breast tumours, CK2ß underexpression also correlates strongly with expression of EMT markers, emphasizing the link between asymmetric expression of CK2 subunits and EMT in vivo. Our results therefore highlight the importance of CK2ß in controlling epithelial cell plasticity. They show that CK2 holoenzyme activity is essential to suppress EMT, and that it contributes to maintaining a normal epithelial morphology. This study also suggests that unbalanced expression of CK2 subunits may drive EMT, thereby contributing to tumour progression.

High molecular gas fractions in normal massive star-forming galaxies in the young Universe.

Stars form from cold molecular interstellar gas. As this is relatively rare in the local Universe, galaxies like the Milky Way form only a few new stars per year. Typical massive galaxies in the distant Universe formed stars an order of magnitude more rapidly. Unless star formation was significantly more efficient, this difference suggests that young galaxies were much more molecular-gas rich. Molecular gas observations in the distant Universe have so far largely been restricted to very luminous, rare objects, including mergers and quasars, and accordingly we do not yet have a clear idea about the gas content of more normal (albeit massive) galaxies. Here we report the results of a survey of molecular gas in samples of typical massive-star-forming galaxies at mean redshifts of about 1.2 and 2.3, when the Universe was respectively 40% and 24% of its current age. Our measurements reveal that distant star forming galaxies were indeed gas rich, and that the star formation efficiency is not strongly dependent on cosmic epoch. The average fraction of cold gas relative to total galaxy baryonic mass at z = 2.3 and z = 1.2 is respectively about 44% and 34%, three to ten times higher than in today's massive spiral galaxies. The slow decrease between z approximately 2 and z approximately 1 probably requires a mechanism of semi-continuous replenishment of fresh gas to the young galaxies.

An almost head-on collision as the origin of two off-centre rings in the Andromeda galaxy.

The unusual morphology of the Andromeda galaxy (Messier 31, the closest spiral galaxy to the Milky Way) has long been an enigma. Although regarded for decades as showing little evidence of a violent history, M31 has a well-known outer ring of star formation at a radius of ten kiloparsecs whose centre is offset from the galaxy nucleus. In addition, the outer galaxy disk is warped, as seen at both optical and radio wavelengths. The halo contains numerous loops and ripples. Here we report the presence of a second, inner dust ring with projected dimensions of 1.5 x 1 kiloparsecs and offset by about half a kiloparsec from the centre of the galaxy (based upon an analysis of previously-obtained data). The two rings appear to be density waves propagating in the disk. Numerical simulations indicate that both rings result from a companion galaxy plunging through the centre of the disk of M31. The most likely interloper is M32. Head-on collisions between galaxies are rare, but it appears nonetheless that one took place 210 million years ago in our Local Group of galaxies.

The Furlow Z-plasty in two-staged palatal repair modifications and complications.

OBJECTIVES: To report the modifications and complications of the Furlow palatoplasty for two-stage closure of the palate. PATIENTS AND METHODS: Prospective study of a consecutive series of 45 primary closures of the soft palate portion of clefts extending into the hard palate; mean (S.D.) age at repair 12 (2) months; median follow-up 4 years 4 months (range 2 months to 9 years). The hard palatal part of the cleft was closed in 18 patients at the mean age of 3 years 11 months. RESULTS: The main modifications that we made were the use of quilting sutures, lateral V-Y closures, and fibrin glue application, and the omission of lateral releasing incisions. Patients stayed in hospital for a median of 4 days (range 3-8 days). Two patients had postoperative partial obstruction of the airway and were given steroids. In six patients, a smaller portion of the oral layer of the wound broke down; it healed by secondary intention in five, but resulted in partial dehiscence in one. There were no oronasal fistulas in the 18 patients who had delayed closure of the hard palate part of the cleft. Secondary pharyngoplasty was not necessary in any patient. CONCLUSION: Furlow's technique has been modified for use in the two-stage closure of complete cleft palates (with or without cleft lip or alveolus) with an acceptable rate of complications.

Constraints on changes in fundamental constants from a cosmologically distant OH absorber or emitter.

We have detected the four 18 cm OH lines from the z approximaetely 0.765 gravitational lens toward PMN J0134-0931. The 1612 and 1720 MHz lines are in conjugate absorption and emission, providing a laboratory to test the evolution of fundamental constants over a large lookback time. We compare the HI and OH main line absorption redshifts of the different components in the z approximately 0.765 absorber and the z approximately 0.685 lens toward B0218 + 357 to place stringent constraints on changes in F triple-bond g(p)[alpha(2)/mu](1.57). We obtain [DeltaF/F] = (0.44 +/- 0.36(stat) +/- 1.0(sys)t) x 10(-5), consistent with no evolution over the redshift range 0 < z < or = 0.7. The measurements have a 2sigma sensitivity of [Deltaalpha/alpha] < 6.7 x 10(-6) or [Deltamu/mu] < 1.4 x 10(-5) to fractional changes in alpha and mu over a period of approximately 6.5 G yr, half the age of the Universe. These are among the most sensitive constraints on changes in mu.

A search for interstellar H3O+.

The P (2,1) line of H3O+, the hydroxonium ion, a key species in ion-molecule chemistry, has been sought in the interstellar medium and in Halley's Comet. In OMC1 and SgrB2, a line was detected which may possibly be attributed to H3O+. Verification of this identification must be accomplished through observation of the P(3,2) line at 364 GHz, or detection of isotopic variants. If we were to assume that the detected line arises from H3O+, we can deduce a fractional abundance X(H3O+) in OMC1 and SgrB2 of approximately 10(-9) and a production rate in Comet Halley of Q(H3O+) 10(28)s-1. These results would place H3O+ among the more abundant molecular ions in the interstellar gas in agreement with theoretical predictions.

[Amputation stump pain: a therapeutic study (author's transl)]. / Essai clinique sur les algies des amputés.

A study has been done in a Thermal Army Hospital, concerning the suffering of amputed persons and their treatment by "tiapride". For 20 observations, it has been noted 11 excellent and 6 good results, by a mean treatment of 300 mg. Interesting results are particularly observed at the first appearance of the soreness, a better integration of this one has been proved, it has also been noticed that amputed patients tolerate much better their prosthesis.