Defining synonymous codon compression schemes by genome recoding

Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome

Hip MRI in flexion abduction external rotation for assessment of the ischiofemoral interval in patients with hip pain-a feasibility study.

OBJECTIVES To assess the feasibility of flexion-abduction-external rotation (FABER) magnetic resonance imaging (MRI) of the hip to visualize changes in the ischiofemoral interval and ability to provoke foveal excursion over the acetabular rim. METHODS IRB-approved retrospective single-center study. Patients underwent non-contrast 1.5-T hip MRI in the neutral and FABER position. Two readers measured the ischiofemoral interval at three levels: proximal/distal intertrochanteric distance and ischiofemoral space. Subgroup analysis was performed for hips with/without high femoral torsion, or quadratus femoris muscle edema (QFME), respectively. A receiver operating curve with calculation of the area under the curve (AUC) for the prediction of QFME was calculated. The presence of foveal excursion in both positions was assessed. RESULTS One hundred ten patients (121 hips, mean age 34 ± 11 years, 67 females) were evaluated. FABER-MRI led to narrowing (both p < .001) of the ischiofemoral interval which decreased more at the proximal (mean decrease by 26 ± 7 mm) than at the distal (6 ± 7 mm) intertrochanteric ridge. With high femoral torsion/ QFME, the ischiofemoral interval was significantly narrower at all three measurement locations compared to normal torsion/no QFME (p < .05). Accuracy for predicting QFME was high with an AUC of .89 (95% CI .82-.94) using a threshold of ≤ 7 mm for the proximal intertrochanteric distance. With FABER-MRI foveal excursion was more frequent in hips with QFME (63% vs 25%; p = .021). CONCLUSION Hip MRI in the FABER position is feasible, visualizes narrowing of the ischiofemoral interval, and can provoke foveal excursion. CRITICAL RELEVANCE STATEMENT FABER MRI may be helpful in diagnosing ischiofemoral impingement and detecting concomitant hip instability by overcoming shortcomings of static MR protocols that do not allow visualization of dynamic changes in the ischiofemoral interval and thus may improve surgical decision making. KEY POINTS • FABER MRI enables visualization of narrowing of the ischiofemoral interval proximal to the lesser trochanter. • Proximal intertrochanteric distance of ≤ 7 mm accurately predicts quadratus femoris muscle edema. • Foveal excursion was more frequent in hips with quadratus femoris muscle edema

Defining synonymous codon compression schemes by genome recoding

Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome

Are degenerative findings detected on traction MR arthrography of the hip associated with failure of arthroscopic femoroacetabular impingement surgery?

OBJECTIVES To identify preoperative degenerative features on traction MR arthrography associated with failure after arthroscopic femoroacetabular impingement (FAI) surgery. METHODS Retrospective study including 102 patients (107 hips) undergoing traction magnetic resonance arthrography (MRA) of the hip at 1.5 T and subsequent hip arthroscopic FAI surgery performed (01/2016 to 02/2020) with complete follow-up. Clinical outcomes were assessed using the International Hip Outcome Tool (iHOT-12) score. Clinical endpoint for failure was defined as an iHOT-12 of < 60 points or conversion to total hip arthroplasty. MR images were assessed by two radiologists for presence of 9 degenerative lesions including osseous, chondrolabral/ligamentum teres lesions. Uni- and multivariate Cox regression analysis was performed to assess the association between MRI findings and failure of FAI surgery. RESULTS Of the 107 hips, 27 hips (25%) met at least one endpoint at a mean 3.7 ± 0.9 years follow-up. Osteophytic changes of femur or acetabulum (hazard ratio [HR] 2.5-5.0), acetabular cysts (HR 3.4) and extensive cartilage (HR 5.1) and labral damage (HR 5.5) > 2 h on the clockface were univariate risk factors (all p  2 h on the clockface (HR 3.2, p = 0.01), central femoral osteophyte (HR 3.1, p = 0.02), and femoral cartilage damage with ligamentum teres damage (HR 3.0, p = 0.04). CONCLUSION Joint damage detected by preoperative traction MRA is associated with failure 4 years following arthroscopic FAI surgery and yields promise in preoperative risk stratification. CLINICAL RELEVANCE STATEMENT Evaluation of negative predictors on preoperative traction MR arthrography holds the potential to improve risk stratification based on the already present joint degeneration ahead of FAI surgery. KEY POINTS • Osteophytes, acetabular cysts, and extensive chondrolabral damage are risk factors for failure of FAI surgery. • Extensive acetabular cartilage damage, central femoral osteophytes, and combined femoral cartilage and ligamentum teres damage represent independent negative predictors. • Survival rates following hip arthroscopy progressively decrease with increasing prevalence of these three degenerative findings

Penning-trap mass spectrometry of highly charged, neutron-rich Rb and Sr isotopes in the vicinity of A≈100A\approx100

The neutron-rich mass region around $A\approx100$ presents challenges for modeling the astrophysical $r$-process because of rapid shape transitions. We report on mass measurements using the TITAN Penning trap at TRIUMF-ISAC to attain more reliable theoretical predictions of $r$-process nucleosynthesis paths in this region. A new approach using highly charged ($q=15+$) ions has been applied which considerably saves measurement time and preserves accuracy. New mass measurements of neutron-rich $^{94,97,98}$Rb and $^{94,97-99}$Sr have uncertainties of less than 4 keV and show deviations of up to 11$\sigma$ to previous measurements. An analysis using a parameterized $r$-process model is performed and shows that mass uncertainties for the A=90 abundance region are eliminated

Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.

The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.This work was supported by a Wellcome Trust and Cancer Research UK (CRUK) Grand Challenge Award (C98/A24032). P.J.C. is a Wellcome Trust Senior Clinical Fellow (WT088340MA); S.F.B. was supported by the Swiss National Science Foundation (P2SKP3-171753 and P400PB-180790); M.A.S. is supported by a Rubicon fellowship from NWO (019.153LW.038); the Cambridge Human Research Tissue Bank is supported by the NIHR Cambridge Biomedical Research Centre; and M.H. is supported by a CRUK Clinician Scientist Fellowship (C52489/A19924)

Experimental loophole-free violation of a Bell inequality using entangled electron spins separated by 1.3 km

For more than 80 years, the counterintuitive predictions of quantum theory have stimulated debate about the nature of reality. In his seminal work, John Bell proved that no theory of nature that obeys locality and realism can reproduce all the predictions of quantum theory. Bell showed that in any local realist theory the correlations between distant measurements satisfy an inequality and, moreover, that this inequality can be violated according to quantum theory. This provided a recipe for experimental tests of the fundamental principles underlying the laws of nature. In the past decades, numerous ingenious Bell inequality tests have been reported. However, because of experimental limitations, all experiments to date required additional assumptions to obtain a contradiction with local realism, resulting in loopholes. Here we report on a Bell experiment that is free of any such additional assumption and thus directly tests the principles underlying Bell's inequality. We employ an event-ready scheme that enables the generation of high-fidelity entanglement between distant electron spins. Efficient spin readout avoids the fair sampling assumption (detection loophole), while the use of fast random basis selection and readout combined with a spatial separation of 1.3 km ensure the required locality conditions. We perform 245 trials testing the CHSH-Bell inequality $S \leq 2$ and find $S = 2.42 \pm 0.20$. A null hypothesis test yields a probability of $p = 0.039$ that a local-realist model for space-like separated sites produces data with a violation at least as large as observed, even when allowing for memory in the devices. This result rules out large classes of local realist theories, and paves the way for implementing device-independent quantum-secure communication and randomness certification.Comment: Raw data will be made available after publicatio

CD152 (CTLA-4) Determines CD4 T Cell Migration In Vitro and In Vivo

BACKGROUND:Migration of antigen-experienced T cells to secondary lymphoid organs and the site of antigenic-challenge is a mandatory prerequisite for the precise functioning of adaptive immune responses. The surface molecule CD152 (CTLA-4) is mostly considered as a negative regulator of T cell activation during immune responses. It is currently unknown whether CD152 can also influence chemokine-driven T cell migration. METHODOLOGY/PRINCIPAL FINDINGS:We analyzed the consequences of CD152 signaling on Th cell migration using chemotaxis assays in vitro and radioactive cell tracking in vivo. We show here that the genetic and serological inactivation of CD152 in Th1 cells reduced migration towards CCL4, CXCL12 and CCL19, but not CXCL9, in a G-protein dependent manner. In addition, retroviral transduction of CD152 cDNA into CD152 negative cells restored Th1 cell migration. Crosslinking of CD152 together with CD3 and CD28 stimulation on activated Th1 cells increased expression of the chemokine receptors CCR5 and CCR7, which in turn enhanced cell migration. Using sensitive liposome technology, we show that mature dendritic cells but not activated B cells were potent at inducing surface CD152 expression and the CD152-mediated migration-enhancing signals. Importantly, migration of CD152 positive Th1 lymphocytes in in vivo experiments increased more than 200% as compared to CD152 negative counterparts showing that indeed CD152 orchestrates specific migration of selected Th1 cells to sites of inflammation and antigenic challenge in vivo. CONCLUSIONS/SIGNIFICANCE:We show here, that CD152 signaling does not just silence cells, but selects individual ones for migration. This novel activity of CD152 adds to the already significant role of CD152 in controlling peripheral immune responses by allowing T cells to localize correctly during infection. It also suggests that interference with CD152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge

Penning-trap Q-value determination of the Ga-71(v, e(-))Ge-71 reaction using threshold charge breeding of on-line produced isotopes

<p>We present a first direct Q-value measurement of the Ga-71(v, e(-))Ge-71 reaction using the TITAN mass-measurement facility at ISAC/TRIUMF. The measurements were performed in a Penning trap on neon-like Ga-71(21+) and Ge-71(22+) using isobar separation of the on-line produced mother and daughter nuclei through threshold charge breeding in an electron-beam ion trap. In addition, isoionic samples of Ga-71(21+) and Ge-71(21+) were stored concurrently in the Penning trap and provided a separate Q-value measurement. Both independent measurements result in a combined Q-value of 233.5 +/- 1.2 keV, which is in agreement with the previously accepted Q-value for the v cross-section calculations. Together with a recent measurement of the v-response from the excited states in Ge-71, we conclude that there are no further uncertainties in the nuclear structure, which could remove the persistent discrepancy between the SAGE and GALLEX calibration measurements performed with neutrinos from reactor-produced Cr-51 and Ar-37 sources and the theoretical expectation. (c) 2013 Elsevier B.V. All rights reserved.</p>