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Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS). We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184(+)/CD271(-)/CD44(-)/CD24(+) from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184(-)/CD44(-)/CD15(LOW)/CD24(+) and a population of glia that was CD184(+)/CD44(+) were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo. These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.

Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions. HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions. The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types, identification and isolation of stem cell subpopulations, and generation of single cell clones. Finally, these results demonstrate an additional application of ROCK inhibition to hESC research.

Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116). Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

Pluripotent stem cells (PSCs) have been successfully differentiated to many cell types relevant to human neurodegenerative diseases. Moreover, induced pluripotent stem cell technology offers an unprecedented opportunity to study these diseases in a patient-specific manner. One impediment to this research is that neural differentiations from PSCs often yield heterogeneous cell cultures, which can hamper or preclude studies requiring pure cell populations. Researchers have partially overcome this hurdle by identifying cell surface signatures of neural stem cells, neurons, glia, and neural crest cells and utilized flow cytometry to isolate near-pure populations of these cells. These advancements have enabled groundbreaking research models of neurodegenerative diseases and have been instrumental in animal models of spinal cord injury. In addition, flow cytometry has been employed to facilitate quantification of neural differentiation cultures. The identification of novel cell surface signatures has also been instrumental in a more comprehensive characterization of pluripotent stem cell derivatives, as well as adult and cancer stem cells. All together, these studies have demonstrated that multiparameter flow cytometry is an indispensable tool for studying neurogenesis and other cell lineages utilizing human PSCs.

Patients with symptomatic, paroxysmal, untreated atrial fibrillation were randomly assigned to antiarrhythmic drug therapy or cryoablation. At 1 year, there was a significantly lower rate of recurrence of atrial fibrillation with cryoablation than with drug therapy.

Streptococcus pneumoniae (pneumococcus) can cause a range of diseases. Although there is a robust pneumococcal vaccination program that reduces invasive pneumococcal disease by targeting various polysaccharide capsules, there has been an increase in the isolation of nonvaccine serotypes and nonencapsulated S. pneumoniae (NESp) strains. Streptococcus pneumoniae colonizes the human nasopharynx and causes several diseases. Pneumococcal vaccines target the polysaccharide capsule and prevent most serious disease, but there has been an increase in the prevalence of nonencapsulated S. pneumoniae (NESp). Previously, it was thought that a capsule was necessary to cause invasive disease. NESp strains expressing the oligopeptide transporters AliC and AliD have been isolated from patients with invasive disease. The AliC and AliD oligopeptide transporters regulate the expression of several genes, including choline binding protein AC (CbpAC) (a homolog of PspA), which aids in reducing C3b deposition. It is hypothesized that by altering CbpAC expression, AliC and AliD provide protection from classical complement-mediated clearance by reducing C-reactive protein (CRP) binding. Our study demonstrates that AliC and AliD regulate CbpAC expression in NESp and that AliD found in certain serotypes of encapsulated strains regulates PspA expression. C3b deposition was increased in the NESp Δ aliD and encapsulated mutants in comparison to the wild type. NESp strains expressing AliC and AliD have a significant decrease in C1q and CRP deposition in comparison to the Δ aliC Δ aliD mutant. The complement protein C1q is required for NESp clearance in a murine model and increases opsonophagocytosis. By regulating CbpAC expression, NESp inhibits CRP binding to the bacterial surface and blocks classical complement activation, leading to greater systemic survival and virulence. Due to the increase in the prevalence of NESp, it is important to gain a better understanding of NESp virulence mechanisms that aid in establishing disease and persistence within a host by avoiding clearance by the immune system. IMPORTANCE Streptococcus pneumoniae (pneumococcus) can cause a range of diseases. Although there is a robust pneumococcal vaccination program that reduces invasive pneumococcal disease by targeting various polysaccharide capsules, there has been an increase in the isolation of nonvaccine serotypes and nonencapsulated S. pneumoniae (NESp) strains. While most studies of pneumococcal pathogenesis have focused on encapsulated strains, there is little understanding of how NESp causes disease. NESp lacks a protective capsule but contains novel genes, such as aliC and aliD , which have been shown to regulate the expression of numerous genes and to be required for NESp virulence and immune evasion. Furthermore, NESp strains have high transformation efficiencies and harbor resistance to multiple drugs. This could be deleterious to current treatment strategies employed for pneumococcal disease as NESp can be a reservoir of drug resistance genes. Therefore, deciphering how NESp survives within a host and facilitates disease is a necessity that will allow the fabrication of improved, broad-spectrum treatments and preventatives against pneumococcal disease. Our study provides a better understanding of NESp virulence mechanisms during host-pathogen interactions through the examination of genes directly regulated by the NESp proteins AliC and AliD.

Most human protein-coding genes are expressed as multiple isoforms, which greatly expands the functional repertoire of the encoded proteome. While at least one reliable open reading frame (ORF) model has been assigned for every coding gene, the majority of alternative isoforms remains uncharacterized due to (i) vast differences of overall levels between different isoforms expressed from common genes, and (ii) the difficulty of obtaining full-length transcript sequences. Here, we present ORF Capture-Seq (OCS), a flexible method that addresses both challenges for targeted full-length isoform sequencing applications using collections of cloned ORFs as probes. As a proof-of-concept, we show that an OCS pipeline focused on genes coding for transcription factors increases isoform detection by an order of magnitude when compared to unenriched samples. In short, OCS enables rapid discovery of isoforms from custom-selected genes and will accelerate mapping of the human transcriptome. Most human protein-coding genes are expressed as multiple isoforms. Here the authors present ORF Capture-seq that uses cloned ORFs as probes to capture and sequence full length transcript sequences. This enables highly sensitive characterization of eukaryotic transcriptomes.

Background: Although the posterior tibial slope (PTS) has been identified as a key factor influencing anterior cruciate ligament (ACL) graft biomechanics and clinical outcomes, little is known regarding which plateau has the greater effect on mechanics of the ACL-reconstructed knee. There are differences in slopes of the medial and lateral tibial plateaus, but this difference has yet to be biomechanically investigated in an ACL-reconstructed setting. Purpose: To investigate the individual effects of medial and lateral PTS differential modifications on ACL graft force, anterior tibial translation (ATT), and internal tibial rotation in a cadaveric model. Study Design: Controlled laboratory study. Methods: Twelve fresh-frozen human cadaveric knees (mean age, 61.6 years) were tested. Native PTS was measured using computed tomography. ACL reconstruction was performed, followed by individual anterior closing-wedge osteotomies of the medial and lateral compartments to achieve a 0° slope. Each compartment was then adjusted in 5° increments with 3-dimensional printed wedges and secured with an external fixator. Biomechanical testing consisted of a 500-N axial load and a 1-N⋅m axial torque across 8 medial-lateral PTS states: native/native, 5°/10°, 5°/5°, 10°/5°, 10°/10°, 10°/15°, 15°/15°, and 15°/10°. ACL graft force, ATT, and internal tibial rotation at full extension were recorded. Linear mixed modeling was used to evaluate the effect of medial and lateral slopes on the outcome variables. Results: The lateral tibial slope contribution had a greater effect on ACL graft force and ATT than the medial tibial slope contribution. Each 1° increase in lateral slope was associated with a 4.9-N (11.1%) increase in graft force and a 0.63-mm (25.2%) increase in ATT (P < .001). Each 1° increase in medial slope was associated with a 1.9-N (4.3%) increase in graft force and a 0.48-mm (19.2%) increase in ATT (P < .001). Internal tibial rotation was not significantly affected by either lateral or medial PTS. Conclusion: Although both slopes significantly affected ACL graft force and ATT, lateral PTS had a greater effect on ACL graft force and ATT compared with the medial PTS and may warrant primary consideration for a slope-correcting osteotomy. Clinical Relevance: While correction of both medial and lateral slopes is important, the lateral PTS contributes more significantly to ACL graft forces and ATT.

Objectives: It is well established that the posterior tibial slope has been identified as a risk factor for ACL ruptures. Slope correcting osteotomies have become an important aspect of the orthopedic surgeon’s armamentarium when faced with revision ACL reconstruction (ACLR) in the setting of increased tibial slope. However, these current procedures produce the same amount of slope correction for the medial and lateral tibial slope and do not account for difference in tibial slope between the compartments. These differences have been shown to affect both risk factors for ACL tears with concomitant meniscal pathology, poorer clinical outcomes of ACLRs with medial or lateral meniscal repairs, and risk of the pivot shift phenomena (anterior tibial translation and internal knee rotation) after ACLR. It is unclear, however, how changes to the tibial slope and the asymmetry between medial and lateral tibial slopes affects ACL graft force, anterior tibial translation, or knee rotation. The aim of this study is to determine how the difference in medial and lateral tibial slope affect anterior tibial translation, knee rotation, and ACL graft force in the setting of an ACL reconstruction. We hypothesize that greater asymmetry between the medial and lateral slopes will result in greater tibial translation, knee rotation, and ACL graft forces. Methods: Twelve fresh frozen human cadaveric knees were acquired, dissected, and imaged via a computed tomography (CT) scanning. Native medial and lateral tibial slopes were measured from the CT scans by an orthopaedic surgeon. Anterior-wedge, slope-reducing osteotomies were performed on the medial and lateral compartments independently utilizing cuts similar to those used for unicompartmental knee arthroplasty such that the slope could be reduced to zero then increased incrementally back to the native slope. A custom external fixture was installed to stabilize the osteotomy, and a soft-tissue ACLR was performed with the proximal end of the graft secured via an interference screw. Custom 3D printed wedges were created to stabilize the osteotomies such that six testing states could be tested (Table 1). Four specimens underwent biomechanical testing at time of submission. Each specimen was first tested with both the medial and lateral compartments in their native slopes, then each testing state was tested in random order (Table 1). Each specimen was mounted in full extension to an axial-torsion testing machine via a custom jig that permitted anterior-posterior tibial translation, and the distal end of the ACLR graft was secured to a force transducer. A 500 N compressive load and 0-N torque was applied to the specimen. Tibial translation was measured using a linear displacement sensor and knee rotation was measured with the testing machine for all testing states. ACL graft forces were measured via the force transducer clamped to the graft. Differences in these outcomes between the native state and the testing states were calculated. Results: Graphical displays of the means and standard deviations of the outcomes for the four specimens can be found in Figure 1. Testing states where the medial compartment was greater than the lateral compartment (Testing states 1, 5, and 6) experienced greater differences in knee rotation, anterior tibial translation, and ACL graft force compared to the native state. These effects appear to be exacerbated when the medial-lateral asymmetry is greatest (Testing States 1 and 6). Testing states where the lateral compartment was greater than the medial compartment (Testing states 2, 3, and 4) experienced lesser differences in knee rotation, anterior tibial translation, and ACL graft force compared to the native state. Conclusions: This study revealed that a greater tibial slope of the medial compartment compared to the lateral compartment may affect knee rotation, anterior tibial translation, and ACL graft force, and that these effects may be exacerbated as the medial-lateral tibial slope asymmetry increases. This biomechanical evidence supports clinical outcomes which report that increased medial-lateral tibial slope asymmetry increases the likelihood of ACL graft failure. This study supports the need to develop a new technique that would allow for independent medial and lateral slope reducing osteotomies. These results indicate that independently reducing the tibial slope of the medial compartment may reduce the likelihood of ACL graft force failure.