WiCell

Sickle Cell Disease (SCD) is a blood disorder affecting millions worldwide. Emerging evidence reveals that SCD pathophysiology increases risk of myeloid malignancies and hematopoietic stem cell (HSC) dysfunction, possibly due to pathological stress on bone marrow. To investigate this further, we interrogated mice and individuals with SCD and observed extended cell cycle times, oxidative stress, DNA damage, senescence, and dysregulation of molecular programs associated with these processes in bone marrow hematopoietic stem and progenitor cells (HSPCs). Human SCD HSPCs displayed poor hematopoietic potential ex vivo . SCD mice displayed a dramatic loss of transplantable bone marrow HSPCs, which was reversed upon treatment of SCD mice with the senolytic agent, ABT-263 (navitoclax). Thus, senolytics restore bone marrow function during SCD in mice and represent a novel strategy to improve bone marrow health in individuals with SCD and improve the safety of potentially curative gene therapies that utilize autologous HSPCs from individuals with SCD.

Like all canonical PUF proteins, C. elegans FBF-2 binds to specific RNAs via tripartite recognition motifs (TRMs). Here we report that an FBF-2 mutant protein that cannot bind to RNA, is nonetheless biologically active and maintains stem cells. This unexpected result challenges the conventional wisdom that RBPs must bind to RNAs to achieve biological activity. Also unexpectedly, FBF-2 interactions with partner proteins can compensate for loss of RNA-binding. FBF-2 only loses biological activity when its RNA-binding and partner interactions are both defective. These findings highlight the complementary contributions of RNA-binding and protein partner interactions to activity of an RNA-binding protein.

Culture-acquired variants in human pluripotent stem cells (hPSCs) hinder their applications in research and clinic. However, the mechanisms that underpin selection of variants remain unclear. Here, through analysis of comprehensive karyotyping datasets from over 23,000 hPSC cultures of more than 1,500 lines, we explored how culture conditions shape variant selection. Strikingly, we identified an association of chromosome 1q gains with feeder-free cultures and noted a rise in its prevalence in recent years, coinciding with increased usage of feeder-free regimens. Competition experiments of multiple isogenic lines with and without a chromosome 1q gain confirmed that 1q variants have an advantage in feeder-free (E8/vitronectin), but not feeder-based, culture. Mechanistically, we show that overexpression of MDM4, located on chromosome 1q, drives variants' advantage in E8/vitronectin by alleviating genome damage-induced apoptosis, which is lower in feeder-based conditions. Our study explains condition-dependent patterns of hPSC aberrations and offers insights into the mechanisms of variant selection.

Droplet microcompartments linked by lipid bilayers show great promise in the construction of synthetic minimal tissues. Central to controlling the flow of information in these systems are membrane proteins, which can gate in response to specific stimuli in order to control the molecular flux between membrane separated compartments. This has been demonstrated with droplet interface bilayers (DIBs) using several different membrane proteins combined with electrical, mechanical, and/or chemical activators. Here we report the activation of the bacterial mechanosensitive channel of large conductance (MscL) in a dioleoylphosphatidylcholine:dioleoylphosphatidylglycerol DIB by controlling membrane asymmetry. We show using electrical measurements that the incorporation of lysophosphatidylcholine (LPC) into one of the bilayer leaflets triggers MscL gating in a concentration-dependent manner, with partial and full activation observed at 10 and 15 mol% LPC respectively. Our findings could inspire the design of new minimal tissues where flux pathways are dynamically defined by lipid composition.

USDA, FSIS thermal processing guidelines (e.g. Appendix A for cooked beef, roast beef, and cooked corned beef, and Time-Temperature Tables for Cooking Ready-to-Eat Poultry Products) are widely used as validation support for cooking processes, but these procedures were developed and validated only for Salmonella in a limited number of products. To determine the extent to which Appendix A can safely be applied to other pathogens and products, a study was conducted to compare the thermal-death times of Salmonella, Listeria monocytogenes, and shiga toxin-producing E. coli (STEC) in model products representing roast beef, turkey breast, and boneless ham. Raw batter for each of the 3 products was inoculated with 8 log CFU/g of a multi-strain mixture of L. monocytogenes, Salmonella, or STEC. One-gram portions of inoculated roast beef, turkey breast, or ham batter were flattened into a thin film in moisture-impermeable pouches, vacuum-packaged, and heated at 54.4, 60.0, 65.6, or 71.1°C in a water bath. Triplicate samples were removed at predetermined time points and enumerated for surviving pathogens. The time needed to yield a 6.5-log reduction of Salmonella and STEC at 60.0, 65.6, or 71.1°C for the three product types was comparable to the times prescribed by USDA, FSIS Appendix A for Salmonella inactivation; however, at 54.4°C similar inactivation levels were not observed. L. monocytogenes showed greater thermotolerance than Salmonella and STEC for all 3 product types. These data suggest that current USDA, FSIS thermal processing guidelines are acceptable tools for ensuring the safety of cooking processes at 60.0°C or higher to inactivate Salmonella and STEC in the product types, but longer dwell times may be necessary to yield comparable log reduction of L. monocytogenes.

Clostridium strains from six phylogenetic groups, C. botulinum groups I to IV, C. baratii, and C. butyricum, display the capacity to produce botulinum neurotoxin. Here, we present the genome sequence of a C. butyricum isolate, the neurotoxigenic strain 5521, which encodes the type E botulinum neurotoxin.

Human fibrinogen is a biomaterial used in surgical tissue sealants, scaffolding for tissue engineering, and wound healing. Here we report on the post-translational structure and functionality of recombinant human FI (rFI) made at commodity levels in the milk of transgenic dairy cows. Relative to plasma-derived fibrinogen (pdFI), rFI predominantly contained a simplified, neutral carbohydrate structure and >4-fold higher levels of the γ'-chain transcriptional variant that has been reported to bind thrombin and Factor XIII. In spite of these differences, rFI and pdFI were kinetically similar with respect to the thrombin-catalyzed formation of protofibrils and Factor XIIIa-mediated formation of cross-linked fibrin polymer. However, electron microscopy showed rFI produced fibrin with much thicker fibers with less branching than pdFI. In vivo studies in a swine liver transection model showed that, relative to pdFI, rFI made a denser, more strongly wound-adherent fibrin clot that more rapidly established hemostasis.