The Potential of TaqMan Array Cards for Detection of Multiple Biological Agents by Real-Time PCR

The TaqMan Array Card architecture, normally used for gene expression studies, was evaluated for its potential to detect multiple bacterial agents by real-time PCR. Ten PCR assays targeting five biological agents (Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis) were incorporated onto Array Cards. A comparison of PCR performance of each PCR in Array Card and singleplex format was conducted using DNA extracted from pure bacterial cultures. When 100 fg of agent DNA was added to Array Card channels the following levels of agent detection (where at least one agent PCR replicate returned a positive result) were observed: Y. pestis 100%, B. mallei & F. tularensis 93%; B. anthracis 71%; B. pseudomallei 43%. For B. mallei & pseudomallei detection the BPM2 PCR, which detects both species, outperformed PCR assays specific to each organism indicating identification of the respective species would not be reproducible at the 100 fg level. Near 100% levels of detection were observed when 100 fg of DNA was added to each PCR in singleplex format with singleplex PCRs also returning sporadic positives at the 10 fg per PCR level. Before evaluating the use of Array Cards for the testing of environmental and clinical sample types, with potential levels of background DNA and PCR inhibitors, users would therefore have to accept a 10-fold reduction in sensitivity of PCR assays on the Array Card format, in order to benefit for the capacity to test multiple samples for multiple agents. A two PCR per agent strategy would allow the testing of 7 samples for the presence of 11 biological agents or 3 samples for 23 biological agents per card (with negative control channels)

Multidifferential study of identified charged hadron distributions in ZZ-tagged jets in proton-proton collisions at s=\sqrt{s}=13 TeV

Jet fragmentation functions are measured for the first time in proton-proton collisions for charged pions, kaons, and protons within jets recoiling against a $Z$ boson. The charged-hadron distributions are studied longitudinally and transversely to the jet direction for jets with transverse momentum 20 $< p_{\textrm{T}} < 100$ GeV and in the pseudorapidity range $2.5 < \eta < 4$. The data sample was collected with the LHCb experiment at a center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 1.64 fb$^{-1}$. Triple differential distributions as a function of the hadron longitudinal momentum fraction, hadron transverse momentum, and jet transverse momentum are also measured for the first time. This helps constrain transverse-momentum-dependent fragmentation functions. Differences in the shapes and magnitudes of the measured distributions for the different hadron species provide insights into the hadronization process for jets predominantly initiated by light quarks.Comment: All figures and tables, along with machine-readable versions and any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-013.html (LHCb public pages

Study of the B−→Λc+Λˉc−K−B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-} decay

The decay $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ is studied in proton-proton collisions at a center-of-mass energy of $\sqrt{s}=13$ TeV using data corresponding to an integrated luminosity of 5 $\mathrm{fb}^{-1}$ collected by the LHCb experiment. In the $\Lambda_{c}^+ K^{-}$ system, the $\Xi_{c}(2930)^{0}$ state observed at the BaBar and Belle experiments is resolved into two narrower states, $\Xi_{c}(2923)^{0}$ and $\Xi_{c}(2939)^{0}$, whose masses and widths are measured to be $$m(\Xi_{c}(2923)^{0}) = 2924.5 \pm 0.4 \pm 1.1 \,\mathrm{MeV}, \\ m(\Xi_{c}(2939)^{0}) = 2938.5 \pm 0.9 \pm 2.3 \,\mathrm{MeV}, \\ \Gamma(\Xi_{c}(2923)^{0}) = \phantom{000}4.8 \pm 0.9 \pm 1.5 \,\mathrm{MeV},\\ \Gamma(\Xi_{c}(2939)^{0}) = \phantom{00}11.0 \pm 1.9 \pm 7.5 \,\mathrm{MeV},$$ where the first uncertainties are statistical and the second systematic. The results are consistent with a previous LHCb measurement using a prompt $\Lambda_{c}^{+} K^{-}$ sample. Evidence of a new $\Xi_{c}(2880)^{0}$ state is found with a local significance of $3.8\,\sigma$, whose mass and width are measured to be $2881.8 \pm 3.1 \pm 8.5\,\mathrm{MeV}$ and $12.4 \pm 5.3 \pm 5.8 \,\mathrm{MeV}$, respectively. In addition, evidence of a new decay mode $\Xi_{c}(2790)^{0} \to \Lambda_{c}^{+} K^{-}$ is found with a significance of $3.7\,\sigma$. The relative branching fraction of $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ with respect to the $B^{-} \to D^{+} D^{-} K^{-}$ decay is measured to be $2.36 \pm 0.11 \pm 0.22 \pm 0.25$, where the first uncertainty is statistical, the second systematic and the third originates from the branching fractions of charm hadron decays.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-028.html (LHCb public pages

Measurement of the ratios of branching fractions R(D∗)\mathcal{R}(D^{*}) and R(D0)\mathcal{R}(D^{0})

The ratios of branching fractions $\mathcal{R}(D^{*})\equiv\mathcal{B}(\bar{B}\to D^{*}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(\bar{B}\to D^{*}\mu^{-}\bar{\nu}_{\mu})$ and $\mathcal{R}(D^{0})\equiv\mathcal{B}(B^{-}\to D^{0}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(B^{-}\to D^{0}\mu^{-}\bar{\nu}_{\mu})$ are measured, assuming isospin symmetry, using a sample of proton-proton collision data corresponding to 3.0 fb${ }^{-1}$ of integrated luminosity recorded by the LHCb experiment during 2011 and 2012. The tau lepton is identified in the decay mode $\tau^{-}\to\mu^{-}\nu_{\tau}\bar{\nu}_{\mu}$. The measured values are $\mathcal{R}(D^{*})=0.281\pm0.018\pm0.024$ and $\mathcal{R}(D^{0})=0.441\pm0.060\pm0.066$, where the first uncertainty is statistical and the second is systematic. The correlation between these measurements is $\rho=-0.43$. Results are consistent with the current average of these quantities and are at a combined 1.9 standard deviations from the predictions based on lepton flavor universality in the Standard Model.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-039.html (LHCb public pages

Effect of sequence on the folding and stability of DNA G-quadruplexes

Oligonucleotide sequences rich in guanines are known to fold into four-stranded structures that are based on stacks of hydrogen-bonded G-quartets. Sequences with the potential to adopt these structures are found at the end of chromosomes in telomeric DNA, as well as in a number of biologically significant genomic locations, including gene promoter regions. There is considerable interest in establishing whether G- quadruplexes have a natural, regulatory role and also whether they could be targets for therapeutic intervention. G-rich sequences can form an extremely diverse range of quadruplex structures, which may vary in terms of the number of strands, the strand polarity and the conformation of the loop regions that join the G-tracts. Despite the frequency with which potential quadruplex-forming sequences occur within the genome, there is presently a limited understanding of the rules that govern the formation of these structures and their stability. This work has focused on investigating the effect of sequence on the formation and stability of DNA G-quadruplexes. Loop length is known to be an important criterion in determining quadruplex stability and topology. This work first examines the properties of a series of model quadruplex- forming sequences that contain short loops, long loops or combinations of the two, investigating the resultant effects on quadruplex folding, stability and kinetics. Utilising a variety of biophysical techniques, the results highlight the importance of single nucleotide loops in determining quadruplex topology. In the sequences studied, the presence of one single-thymidine loop was sufficient to promote the other loops into an identical conformation, resulting in the formation of parallel-stranded structures. This may be significant given the frequency with which single-nucleotide loops are observed amongst genomic quadruplex-forming sequences. Besides loop-length, loop sequence can also moderate quadruplex stability. The sequence effects of single nucleotide loops have been examined in both model and biologically relevant promoter sequences. The results show that quadruplex stability is sensitive to changes in single-nucleotide loop identity, with adenines significantly disfavoured over pyrimidine loops. Finally, the importance of the loop regions on quadruplex folding is well documented, however little is known regarding the length of the G-tract. The properties of intramolecular G-quadruplexes that are formed by sequences with increasing G-tract lengths have been examined. The results reveal that there is no simple relationship between quadruplex stability and the length of the G-tracts, and that sequences containing longer G-tracts are likely to form heterogeneous populations of folded structures. When challenged with their complementary strand, several G-rich sequences preferentially form quadruplex over duplex.</p

Effect of sequence on the folding and stability of DNA G-quadruplexes

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Schematic representation of detection of 100 fg of <i>Y. pestis</i> DNA in seven Array Card channels across one Array Card.

a<p>Numbered PCR represents assays evaluated in an alternate study. Named PCRs this study.</p>b<p>C_T value: PCR cycle number at which fluorescence first detected. # = negative result.</p

Summary of PCR results from Array Card and Singleplex PCR experiments.

a<p><i>B. mallei</i> genome calculation performed using ATCC 23344 genome size. <i>B. pseudomallei</i> calucalation using 1106B genome size. Other genome calculations performed using genome sizes of stated strains.</p>b<p>No. of PCR positives from <i>n</i> replicates. nt - Not tested.</p>c<p>C_T value: PCR cycle number at which fluorescence first detected. Mean of positives only. Variance of mean in paranthesis.</p>d<p>Detection rate defined as at least one agent PCR positive replicate in the Array Card channel.</p

Quantification of B. anthracis (Ames) DNA by pXO2 MGB PCR replicates incorporated within the TaqMan Array Cards.

<p>Trendline and R2 value generated from mean CT values by Microsoft Excel.</p