Multiplex shRNA screening of germ cell development by in vivo transfection of mouse testis

Spermatozoa are one of the few mammalian cell types that cannot be fully derived in vitro, severely limiting the application of modern genomic techniques to study germ cell biology. The current gold standard approach of characterizing single-gene knockout mice is slow as generation of each mutant line can take 6–9 months. Here, we describe an in vivo approach to rapid functional screening of germline genes based on a new nonsurgical, nonviral in vivo transfection method to deliver nucleic acids into testicular germ cells. By coupling multiplex transfection of short hairpin RNA (shRNA) constructs with pooled amplicon sequencing as a readout, we were able to screen many genes for spermatogenesis function in a quick and inexpensive experiment. We transfected nine mouse testes with a pilot pool of RNA interference (RNAi) against well-characterized genes to show that this system is highly reproducible and accurate. With a false negative rate of 18% and a false positive rate of 12%, this method has similar performance as other RNAi screens in the well-described Drosophila model system. In a separate experiment, we screened 26 uncharacterized genes computationally predicted to be essential for spermatogenesis and found numerous candidates for follow-up studies. Finally, as a control experiment, we performed a long-term selection screen in neuronal N2a cells, sampling shRNA frequencies at five sequential time points. By characterizing the effect of both libraries on N2a cells, we show that our screening results from testis are tissue-specific. Our calculations indicate that the current implementation of this approach could be used to screen thousands of protein-coding genes simultaneously in a single mouse testis. The experimental protocols and analysis scripts provided will enable other groups to use this procedure to study diverse aspects of germ cell biology ranging from epigenetics to cell physiology. This approach also has great promise as an applied tool for validating diagnoses made from medical genome sequencing, or designing synthetic biological sequences that can act as potent and highly specific male contraceptives

Validation of “Care of the Dying Evaluation” in emergency medicine (CODEEM): pilot phase of End-of-life Management Protocol Offered Within Emergency Room (EMPOWER) study

Background: An increasing number of patients who present to emergency departments are at their endof-life phase and have significant palliative care needs such as in symptom control for pain and dyspnoea. Evaluating quality of care provided is imperative, yet there is no suitable tool validated in the emergency and Asian settings. We aim to examine the face and construct validity, and reliability of a newly developed questionnaire, Care of the Dying Evaluation - Emergency Medicine, for measuring the quality of end-of-life care in an Asian emergency context. Methods: A mixed methods pilot study was conducted. Participants composed of the next-of-kin to thirty dying patients who presented to the emergency departments of three public hospitals in Singapore. Qualitative evaluation, using cognitive "think-aloud" interviews, and quantitative analysis were employed. Percentage agreement and κ statistic were measured to evaluate temporal stability of the questionnaire. Cronbach's α and item-total correlations were used to assess internal consistency within the constructs. Confirmatory factor analysis was performed for construct validity. Results: All participants reported clear understanding of the questionnaire with no ambiguity; a minority felt the questions caused emotional distress (7/30, 23.3%). The questions showed moderate to good testretest reliability. Internal consistencies within the constructs were good for "ENVIRONMENT" and "CARE", and moderate for "COMMUNICATION". Factor loadings range from 0.40 to 0.99. Conclusions: The Care of the Dying Evaluation - Emergency Medicine questionnaire may be valid and reliable for use in an Asian emergency setting. Our prospective multicentre study using this evaluation tool may provide more insight on the quality of care rendered to dying patients and identify areas for improvement. Trial registration: ClinicalTrials.gov (NCT03906747)

BESII Detector Simulation

A Monte Carlo program based on Geant3 has been developed for BESII detector simulation. The organization of the program is outlined, and the digitization procedure for simulating the response of various sub-detectors is described. Comparisons with data show that the performance of the program is generally satisfactory.Comment: 17 pages, 14 figures, uses elsart.cls, to be submitted to NIM

Partial wave analysis of J/\psi \to \gamma \phi \phi

Using $5.8 \times 10^7 J/\psi$ events collected in the BESII detector, the radiative decay $J/\psi \to \gamma \phi \phi \to \gamma K^+ K^- K^0_S K^0_L$ is studied. The $\phi\phi$ invariant mass distribution exhibits a near-threshold enhancement that peaks around 2.24 GeV/$c^{2}$. A partial wave analysis shows that the structure is dominated by a $0^{-+}$ state ($\eta(2225)$) with a mass of $2.24^{+0.03}_{-0.02}{}^{+0.03}_{-0.02}$ GeV/$c^{2}$ and a width of $0.19 \pm 0.03^{+0.06}_{-0.04}$ GeV/$c^{2}$. The product branching fraction is: $Br(J/\psi \to \gamma \eta(2225))\cdot Br(\eta(2225)\to \phi\phi) = (4.4 \pm 0.4 \pm 0.8)\times 10^{-4}$.Comment: 11 pages, 4 figures. corrected proof for journa

Measurements of the observed cross sections for e+e−→e^+e^-\to exclusive light hadrons containing π0π0\pi^0\pi^0 at s=3.773\sqrt s= 3.773, 3.650 and 3.6648 GeV

By analyzing the data sets of 17.3, 6.5 and 1.0 pb$^{-1}$ taken, respectively, at $\sqrt s= 3.773$, 3.650 and 3.6648 GeV with the BES-II detector at the BEPC collider, we measure the observed cross sections for $e^+e^-\to \pi^+\pi^-\pi^0\pi^0$, $K^+K^-\pi^0\pi^0$, $2(\pi^+\pi^-\pi^0)$, $K^+K^-\pi^+\pi^-\pi^0\pi^0$ and $3(\pi^+\pi^-)\pi^0\pi^0$ at the three energy points. Based on these cross sections we set the upper limits on the observed cross sections and the branching fractions for $\psi(3770)$ decay into these final states at 90% C.L..Comment: 7 pages, 2 figure

Measurements of the observed cross sections for exclusive light hadron production in e^+e^- annihilation at \sqrt{s}= 3.773 and 3.650 GeV

By analyzing the data sets of 17.3 pb$^{-1}$ taken at $\sqrt{s}=3.773$ GeV and 6.5 pb$^{-1}$ taken at $\sqrt{s}=3.650$ GeV with the BESII detector at the BEPC collider, we have measured the observed cross sections for 12 exclusive light hadron final states produced in $e^+e^-$ annihilation at the two energy points. We have also set the upper limits on the observed cross sections and the branching fractions for $\psi(3770)$ decay to these final states at 90% C.L.Comment: 8 pages, 5 figur

Direct Measurements of Absolute Branching Fractions for D0 and D+ Inclusive Semimuonic Decays

By analyzing about 33 $\rm pb^{-1}$ data sample collected at and around 3.773 GeV with the BES-II detector at the BEPC collider, we directly measure the branching fractions for the neutral and charged $D$ inclusive semimuonic decays to be $BF(D^0 \to \mu^+ X) =(6.8\pm 1.5\pm 0.7)$ and $BF(D^+ \to \mu^+ X) =(17.6 \pm 2.7 \pm 1.8)$, and determine the ratio of the two branching fractions to be $\frac{BF(D^+ \to \mu^+ X)}{BF(D^0 \to \mu^+ X)}=2.59\pm 0.70 \pm 0.25$

Search for the Rare Decays J/Psi --> Ds- e+ nu_e, J/Psi --> D- e+ nu_e, and J/Psi --> D0bar e+ e-

We report on a search for the decays J/Psi --> Ds- e+ nu_e + c.c., J/Psi --> D- e+ nu_e + c.c., and J/Psi --> D0bar e+ e- + c.c. in a sample of 5.8 * 10^7 J/Psi events collected with the BESII detector at the BEPC. No excess of signal above background is observed, and 90% confidence level upper limits on the branching fractions are set: B(J/Psi --> Ds- e+ nu_e + c.c.)<4.8*10^-5, B(J/Psi --> D- e+ nu_e + c.c.) D0bar e+ e- + c.c.)<1.1*10^-5Comment: 10 pages, 4 figure

Measurements of psi(2S) decays to octet baryon-antibaryon pairs

With a sample of 14 million psi(2S) events collected by the BESII detector at the Beijing Electron Positron Collider (BEPC), the decay channels psi(2S)->p p-bar, Lambda Lambda-bar, Sigma0 Sigma0-bar, Xi Xi-bar are measured, and their branching ratios are determined to be (3.36+-0.09+-0.24)*10E-4, (3.39+-0.20+-0.32)*10E-4, (2.35+-0.36+-0.32)*10E-4, (3.03+-0.40+-0.32)*10E-4, respectively. In the decay psi(2S)->p p-bar, the angular distribution parameter alpha is determined to be 0.82+-0.17+-0.04.Comment: 8 pages, 8 figure

A study of charged kappa in J/ψ→K±Ksπ∓π0J/\psi \to K^{\pm} K_s \pi^{\mp} \pi^0

Based on $58 \times 10^6$ $J/\psi$ events collected by BESII, the decay $J/\psi \to K^{\pm} K_s \pi^{\mp} \pi^0$ is studied. In the invariant mass spectrum recoiling against the charged $K^*(892)^{\pm}$, the charged $\kappa$ particle is found as a low mass enhancement. If a Breit-Wigner function of constant width is used to parameterize the kappa, its pole locates at $(849 \pm 77 ^{+18}_{-14}) -i (256 \pm 40 ^{+46}_{-22})$ MeV/$c^2$. Also in this channel, the decay $J/\psi \to K^*(892)^+ K^*(892)^-$ is observed for the first time. Its branching ratio is $(1.00 \pm 0.19 ^{+0.11}_{-0.32}) \times 10^{-3}$.Comment: 14 pages, 4 figure