High Voltage Electron Microscopy and Low Voltage Scanning Electron Microscopy of Human Neoplastic Cell Culture

Improved procedures were developed to correlate cell culture data with the images provided by advanced ultrastructural technologies. These procedures were compatible with the two main types of cellular behavior: adherent, spreading (melanomas, rhabdomyosarcomas) and non-adherent in suspension (leukemias). The ultrastructure and function of spreading neoplastic cells primarily depend on surface properties of the attaching substrates. Therefore, the films used for cultured cell whole-mount ultrastructural analysis must have adherence features identical to those of standard cell culture vessels. Improved procedures were developed to produce the polystyrene films of required qualities. These films allowed processing of cells for electron microscopy including chemical fixation, cryoimmobilization, and immunolabelling. Furthermore, these polystyrene films permitted observations of the same cell in the high voltage electron microscope to reveal the internal organization and in the low voltage scanning electron microscope to reveal the surface topography. Neoplastic cells in suspension may dramatically change their ultrastructure as a result of interactions with substrates or other cells. Therefore, immobilization of cellular processes must occur rapidly while cells remain in suspension. These processes were cryoimmobilized by high pressure freezing through the use of the newly designed specimen carrier. Procedures allowing high yield attachment of cryofixed neoplastic cells to amino-propyl-derived glass carriers enabled observations of cell surface topography. Furthermore, freeze-substitution and drying of freeze-fractured cells revealed their three-dimensional internal organization in the low voltage scanning electron microscope

Preparation of Plasmid DNA in Transfection Complexes for Fluorescence and Electron Spectroscopic Imaging

The aim of this project was to develop procedures necessary to study mechanisms of receptor mediated gene transfer by means of integrated microscopy. Plasmid DNA was incorporated into a transfection complex consisting of poly(L)lysine and transferrin to which the nuclear localization signal was conjugated. This complex was presented to cultured glioma cells. Preparation of the transfected DNA for imaging was pursued by two methods. In the first method tetramethylrhodamine, nanogold, and ferritin were linked through streptavidin to the biotinylated plasmid DNA. Trafficking of the fluorescent derivatives was studied in living cells with fluorescence microscopy. Then, selected cells were rapidly cryo-immobilized. Ultrastructural distribution of the transfected DNA was imaged with energy filtering transmission electron microscopy. In the second method, the unmodified transfected DNA was detected in cryo-immobilized cells by in situ polymerase chain reaction and in situ hybridization. For laser scanning fluorescence microscopy probes were labeled with tetramethylrhodamine. For ultrastructural analysis by electron spectroscopic imaging, probes containing incorporated digoxigenin were labeled with anti-digoxigenin boronated antibodies. Based upon the developed procedures, it has been demonstrated that the presence of the nuclear localization signal in the transfection complex resulted in rapid nuclear import of the transfected DNA

Scanning Electron Microscopy of High-Pressure-Frozen Sea Urchin Embryos

High-pressure-freezing permits direct cryo-fixation of sea urchin embryos having a defined developmental state without the formation of large ice crystals. We have investigated preparation protocols for observing high-pressure-frozen and freeze-fractured samples in the scanning electron microscope. High-pressure-freezing was superior to other freezing protocols, because the whole bulk sample was reasonably well frozen and the overall three-dimensional shape of the embryos was well preserved. The samples were either dehydrated by freeze-substitution and critical-point-drying, or imaged in the partially hydrated state, using a cold stage in the SEM. During freeze-substitution the samples were stabilized by fixatives. The disadvantage of this method was that shrinking and extraction effects, caused by the removal of the water, could not be avoided. These disadvantages were avoided when. the sample was imaged in the frozen-hydrated state using a cold-stage in the SEM. This would be the method of choice for morphometric studies. Frozen-hydrated samples, however, were very beam sensitive and many structures remained covered by the ice and were not visible. Frozen-hydrated samples were partially freeze-dried to make visible additional structures that had been covered by ice. However, this method also caused drying artifacts when too much water was removed

Novel mutation-deletion in the PHOX2B gene of the patient diagnosed with Neuroblastoma, Hirschsprung's Disease, and Congenital Central Hypoventilation Syndrome (NB-HSCR-CCHS) Cluster

INTRODUCTION: Neuroblastoma (NB), Hirschsprung disease (HSCR), Congenital Central Hypoventilation Syndrome (CCHS), clinically referred as the NB-HSCR-CCHS cluster, are genetic disorders linked to mutations in the PHOX2B gene on chromosome 4p12. SPECIFIC AIM: The specific aim of this project is to define the PHOX2B gene mutations as the genomic basis for the clinical manifestations of the NB-HSCR-CCHS cluster. PATIENT: A one day old male patient presented to the Jagiellonian University Medical College (JUMC), American Children Hospital, neonatal Intensive Care Unit (ICU) due to abdominal distention, vomiting, and severe apneic episodes. With the preliminary diagnosis of the NB-HSCR-CCHS, the blood and tissue samples were acquired from the child, as well as from the child’s parents. All procedures were pursued in accordance with the Declaration of Helsinki, with the patient’s Guardian Informed Consent and the approval from the Institutional Review Board. GENETIC/GENOMIC METHODS: Karyotyping was analyzed based upon Giemsa banding. The patient’s genomic DNA was extracted from peripheral blood and amplified by polymerase chain reaction. Direct microfluidic Sanger sequencing was performed on the genomic DNA amplicons. These procedures were pursued in addition to the routine clinical examinations and tests. RESULTS: G-banding showed the normal 46 XY karyotype. However, genomic sequencing revealed a novel, heterozygous deletion (8 nucleotides: c.699–706, del8) in exon 3 of the PHOX2B gene on chromosome 4. This led to the frame-shift mutation and malfunctioning gene expression product. CONCLUSION: Herein, we report a novel PHOX2B gene mutation in the patient diagnosed with the NB-HSCR-CCHS cluster. The resulting gene expression product may be a contributor to the clinical manifestations of these genetic disorders. It adds to the library of the mutations linked to this syndrome. Consequently, we suggest that screening for the PHOX2B mutations becomes an integral part of genetic counseling, genomic sequencing of fetal circulating nucleic acids and / or genomes of circulating fetal cells prenatally, while preparing supportive therapy upon delivery, as well as on neonates' genomes of intubated infants, when breathing difficulties occur upon extubation. Further, we hypothesize that PHOX2B may be considered as a potential target for gene therapy

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

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