An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs), with their dual capacity to self-renew and differentiate, are commonly used to study differentiation, epigenetic regulation, lineage choices, and more. Using non-directed retroviral integration of a YFP/Cherry exon into mouse ESCs, we generated a library of over 200 endogenously tagged fluorescent fusion proteins and present several proof-of-concept applications of this library. We show the utility of this library to track proteins in living cells; screen for pluripotency-related factors; identify heterogeneously expressing proteins; measure the dynamics of endogenously labeled proteins; track proteins recruited to sites of DNA damage; pull down tagged fluorescent fusion proteins using anti-Cherry antibodies; and test for interaction partners. Thus, this library can be used in a variety of different directions, either exploiting the fluorescent tag for imaging-based techniques or utilizing the fluorescent fusion protein for biochemical pull-down assays, including immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, and more. Keywords: embryonic stem cells; imaging; live imaging; fluorescence; differentiation; pluripotency; GFP; microscopy; DNA damage; protein dynamicsNational Institutes of Health (U.S.) (Grant HD045022)National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant R01NS088538-01

A Radical Innovative Change in the Practice of Hand Surgery Using Wide Awake Local Anesthesia

Wide-awake local anesthesia no tourniquet (WALANT) technique is characterized by using local anesthetic plus adrenaline administered into the opera­tive field, enabling us to perform longer and complex hand procedures without the need of an anesthetist. We assessed the impact of integrating WALANT on the practice of hand surgery in our center. We retrospectively reviewed charts of all hand surgeries performed in the years 2011 and 2016. The number of trauma cases performed in the ambulatory OR increased from 56 cases in 2011 to 131 in 2016, and elective complex cases increased from 9 to 65. Number of elective procedures conducted in the main OR increased from 67 to 105, and trauma cases performed “off hours” decreased from 53 to 21. We conclude that WALANT enables us to better utilize our OR resources, to treat hand trauma patients on an outpatient basis, and to shorten the wait time for elective hand surgery.

Preliminary Experience with Postmortem Computed Tomography in Military Penetrating Trauma

BACKGROUND:Postmortem examination serves as a tool for confirmation of clinical diagnosis, “quality” assurance, and education. In Israel, mostly because of religious reasons, most families withhold their permission to perform autopsies. To obtain objective information regarding the death of soldiers, the Israel Defense Forces Medical Corps started in September of 1997 to perform postmortem computed tomographic (PMCT) scans. The purpose of our study is to determine what information can be obtained from the PMCT scans. METHODS:In a period of 16 months, 27 soldiers were killed in low-intensity conflicts and PMCT was obtained in 22 cases. Medical data obtained from the field medical care providers were collected and compared with PMCT results. RESULTS:Several examples of patients whose death was determined at the scene either before any medical intervention or after initiation of resuscitative treatment are shown in our study and compared with the clinical impression of the treating physician. Two examples of autopsy results are compared with PMCT results. Gas was detected in various parts of the circulatory system in many cases. The significance of this finding, described in our study for the first time, needs further investigation. CONCLUSION:PMCT scanning has limits in detecting superficial injuries and injuries of the extremities and determining the exact route of fragments through body tissues in penetrating military trauma. It also cannot serve as a tool for examining ammunition or the protection provided by various armors. However, it can provide a substantial amount of evidence that, when reviewed with the clinical information obtained from the physician at the scene, can help in assessing the treatment given at the field and point toward the probable cause of death

Does prehospital fluid administration impact core body temperature and coagulation functions in combat casualties?

Administration of large amounts of fluids to trauma patients, in the absence of surgical control, may increase bleeding, cause hypothermia and coagulopathy which may worsen the bleeding and increase morbidity and mortality. The purpose of our study is to examine the impact of prehospital fluid administration to military combat casualties on core body temperature and coagulation functions. Prospective data were collected on all cases of moderately (9 or = 16) injured victims wounded in South Lebanon, treated by Israeli military physicians and evacuated to hospitals in Israel, over a two-year period. Data regarding prehospital phase of injury (timetables, amount of fluids) and upon hospital arrival (initial core body temperature, prothrombin time [PT], partial thromboplastin time [PTT]) were examined for monotonic relation using Spearman's non-parametric test. Fifty-three moderately injured and 31 severely injured patients were included in the study. The average evacuation time for the moderately injured group was 109.3 +/- 44.8 min, and for the severely injured 100.3 +/- 38.4 min (P value=NS). The mean volume of fluids administered was 2.39 +/- 1.52 and 2.49 +/- 1.47 l, respectively (P=NS). No statistical correlation was found between core body temperature, PT or PTT, measured upon hospital arrival, and prehospital fluid treatment. In addition, no correlation was found between core body temperature on hospital arrival and prehospital time, or between prehospital fluid volumes and prehospital time. The mean core body temperature of the moderately injured patients was 36.8 degrees C, and that of severely injured was 35.8 degrees C (P=0.026). With proper control of blood loss and avoidance of excessive fluid administration, moderately and severely injured combat casualties in 'low intensity conflict' in South Lebanon can be resuscitated with fluid volumes that do not result in a coagulation deficit or hypothermia. The core body temperature on arrival at the hospital is related to the severity of the injury

Dynamic Proteomics of Human Protein Level and Localization across the Cell Cycle

<div><p>Regulation of proteins across the cell cycle is a basic process in cell biology. It has been difficult to study this globally in human cells due to lack of methods to accurately follow protein levels and localizations over time. Estimates based on global mRNA measurements suggest that only a few percent of human genes have cell-cycle dependent mRNA levels. Here, we used dynamic proteomics to study the cell-cycle dependence of proteins. We used 495 clones of a human cell line, each with a different protein tagged fluorescently at its endogenous locus. Protein level and localization was quantified in individual cells over 24h of growth using time-lapse microscopy. Instead of standard chemical or mechanical methods for cell synchronization, we employed in-silico synchronization to place protein levels and localization on a time axis between two cell divisions. This non-perturbative synchronization approach, together with the high accuracy of the measurements, allowed a sensitive assay of cell-cycle dependence. We further developed a computational approach that uses texture features to evaluate changes in protein localizations. We find that 40% of the proteins showed cell cycle dependence, of which 11% showed changes in protein level and 35% in localization. This suggests that a broader range of cell-cycle dependent proteins exists in human cells than was previously appreciated. Most of the cell-cycle dependent proteins exhibit changes in cellular localization. Such changes can be a useful tool in the regulation of the cell-cycle being fast and efficient.</p> </div

Schematic overview of dynamic proteomics for exploring cell cycle dependent changes in level and localization.

<p>(A) CD tagging was used to insert YFP as an exon into the introns of genes on the chromosome of a human cell line clone (H1299), resulting in a full length protein fused to YFP expressed from its endogenous locus. (B) A panel of 6 representative clones with different tagged proteins from the LARC library (C) Time-lapse microscopy and automated image analysis allow capturing proteins levels and localizations in individual cells over time. Yellow arrow indicates a cell in mitosis, green arrow indicates cells post mitosis. (D) Fluorescence traces of individual cells over a 40 hours movie (tagged protein is DDX5). Sharp decreases are at division events (E) In silico synchronization is done by plotting cell dynamics on a time axis which indicates time from previous or next division. Time is divided by mean cell cycle duration, to provide fraction of cell cycle elapsed. G, S and G2 phases are estimated from Sigal <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048722#pone.0048722-Sigal2" target="_blank">[15]</a>. Grey lines- cells with two mitosis events in the movie, blue, red lines: cells with one mitotic event. The fluorescence level is normalized to the maximal level before cell division. (F) In silico synchronized dynamics are used to examine cell-cycle dependence on levels and localizations. On the top panel, Protein profile (blue) that is significantly different from the average profile (black) is considered cell cycle dependent. On the bottom panel : nuclear protein shows a nuclear ratio (nuc/total) profile close to 1 most of the cell cycle, while cytoplasmic protein, shows a nuclear ratio close to 0 most of the cell cycle (besides during the mitosis, where the nucleus and cytoplasm are hard to segment apart). Protein that change its localization from the cytoplasm to the nucleus in a cell cycle dependent manner, present a nuclear ratio that is variable across the cell cycle.</p