Cell-cell metabolite exchange creates a pro-survival metabolic environment that extends lifespan.

Metabolism is deeply intertwined with aging. Effects of metabolic interventions on aging have been explained with intracellular metabolism, growth control, and signaling. Studying chronological aging in yeast, we reveal a so far overlooked metabolic property that influences aging via the exchange of metabolites. We observed that metabolites exported by young cells are re-imported by chronologically aging cells, resulting in cross-generational metabolic interactions. Then, we used self-establishing metabolically cooperating communities (SeMeCo) as a tool to increase metabolite exchange and observed significant lifespan extensions. The longevity of the SeMeCo was attributable to metabolic reconfigurations in methionine consumer cells. These obtained a more glycolytic metabolism and increased the export of protective metabolites that in turn extended the lifespan of cells that supplied them with methionine. Our results establish metabolite exchange interactions as a determinant of cellular aging and show that metabolically cooperating cells can shape the metabolic environment to extend their lifespan.

Lentiviral Gene Therapy for Artemis-Deficient SCID.

BACKGROUND: The DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C, which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation. METHODS: We carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C, in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months. RESULTS: Marrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor ß chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report. CONCLUSIONS: Infusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.).

Inorganic sulfur fixation via a new homocysteine synthase allows yeast cells to cooperatively compensate for methionine auxotrophy.

The assimilation, incorporation, and metabolism of sulfur is a fundamental process across all domains of life, yet how cells deal with varying sulfur availability is not well understood. We studied an unresolved conundrum of sulfur fixation in yeast, in which organosulfur auxotrophy caused by deletion of the homocysteine synthase Met17p is overcome when cells are inoculated at high cell density. In combining the use of self-establishing metabolically cooperating (SeMeCo) communities with proteomic, genetic, and biochemical approaches, we discovered an uncharacterized gene product YLL058Wp, herein named Hydrogen Sulfide Utilizing-1 (HSU1). Hsu1p acts as a homocysteine synthase and allows the cells to substitute for Met17p by reassimilating hydrosulfide ions leaked from met17Δ cells into O-acetyl-homoserine and forming homocysteine. Our results show that cells can cooperate to achieve sulfur fixation, indicating that the collective properties of microbial communities facilitate their basic metabolic capacity to overcome sulfur limitation.

Lysine harvesting is an antioxidant strategy and triggers underground polyamine metabolism.

Both single and multicellular organisms depend on anti-stress mechanisms that enable them to deal with sudden changes in the environment, including exposure to heat and oxidants. Central to the stress response are dynamic changes in metabolism, such as the transition from the glycolysis to the pentose phosphate pathway-a conserved first-line response to oxidative insults1,2. Here we report a second metabolic adaptation that protects microbial cells in stress situations. The role of the yeast polyamine transporter Tpo1p3-5 in maintaining oxidant resistance is unknown6. However, a proteomic time-course experiment suggests a link to lysine metabolism. We reveal a connection between polyamine and lysine metabolism during stress situations, in the form of a promiscuous enzymatic reaction in which the first enzyme of the polyamine pathway, Spe1p, decarboxylates lysine and forms an alternative polyamine, cadaverine. The reaction proceeds in the presence of extracellular lysine, which is taken up by cells to reach concentrations up to one hundred times higher than those required for growth. Such extensive harvest is not observed for the other amino acids, is dependent on the polyamine pathway and triggers a reprogramming of redox metabolism. As a result, NADPH-which would otherwise be required for lysine biosynthesis-is channelled into glutathione metabolism, leading to a large increase in glutathione concentrations, lower levels of reactive oxygen species and increased oxidant tolerance. Our results show that nutrient uptake occurs not only to enable cell growth, but when the nutrient availability is favourable it also enables cells to reconfigure their metabolism to preventatively mount stress protection.

Coordination between aminoacylation and editing to protect against proteotoxicity.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that ligate amino acids to tRNAs, and often require editing to ensure accurate protein synthesis. Recessive mutations in aaRSs cause various neurological disorders in humans, yet the underlying mechanism remains poorly understood. Pathogenic aaRS mutations frequently cause protein destabilization and aminoacylation deficiency. In this study, we report that combined aminoacylation and editing defects cause severe proteotoxicity. We show that the ths1-C268A mutation in yeast threonyl-tRNA synthetase (ThrRS) abolishes editing and causes heat sensitivity. Surprisingly, experimental evolution of the mutant results in intragenic mutations that restore heat resistance but not editing. ths1-C268A destabilizes ThrRS and decreases overall Thr-tRNAThr synthesis, while the suppressor mutations in the evolved strains improve aminoacylation. We further show that deficiency in either ThrRS aminoacylation or editing is insufficient to cause heat sensitivity, and that ths1-C268A impairs ribosome-associated quality control. Our results suggest that aminoacylation deficiency predisposes cells to proteotoxic stress.

Natural determinant reference functional theory.

The natural determinant reference (NDR) or principal natural determinant is the Slater determinant comprised of the N most strongly occupied natural orbitals of an N-electron state of interest. Unlike the Kohn-Sham (KS) determinant, which yields the exact ground-state density, the NDR only yields the best idempotent approximation to the interacting one-particle reduced density matrix, but it is well-defined in common atom-centered basis sets and is representation-invariant. We show that the under-determination problem of prior attempts to define a ground-state energy functional of the NDR is overcome in a grand-canonical ensemble framework at the zero-temperature limit. The resulting grand potential functional of the NDR ensemble affords the variational determination of the ground state energy, its NDR (ensemble), and select ionization potentials and electron affinities. The NDR functional theory can be viewed as an "exactification" of orbital optimization and empirical generalized KS methods. NDR functionals depending on the noninteracting Hamiltonian do not require troublesome KS-inversion or optimized effective potentials.

Room-Temperature Stable Ln(II) Complexes Supported by 2,6-Diadamantyl Aryloxide Ligands.

The sterically bulky aryloxide ligand OAr* (OAr* = -OC6H2-Ad2-2,6tBu-4; Ad = 1-adamantyl) has been used to generate Ln(II) complexes across the lanthanide series that are more thermally stable than complexes of any other ligand system reported to date for 4fnd1 Ln(II) ions. The Ln(III) precursors Ln(OAr*)3 (1-Ln) were synthesized by reacting 1.2 equiv of Ln(NR2)3 (R = SiMe3) with 3 equiv of HOAr* for Ln = La, Ce, Nd, Gd, Dy, Yb, and Lu. 1-Ce, 1-Nd, 1-Gd, 1-Dy, and 1-Lu were identified by single-crystal X-ray diffraction studies. Reductions of 1-Ln with potassium graphite (KC8) in tetrahydrofuran in the presence of 2.2.2-cryptand (crypt) yielded the Ln(II) complexes [K(crypt)][Ln(OAr*)3] (2-Ln). The 2-Ln complexes for Ln = Nd, Gd, Dy, and Lu were characterized by X-ray crystallography and found to have Ln-O bond distances 0.038-0.087 Å longer than those of their 1-Ln analogues; this is consistent with 4fn5d1 electron configurations. The structure of 2-Yb has Yb-O distances 0.167 Å longer than those predicted for 1-Yb, which is consistent with a 4f14 electron configuration. Although 2-La and 2-Ce proved to be challenging to isolate, with 18-crown-6 (18-c-6) as the potassium chelator, La(II) and Ce(II) complexes with OAr* could be isolated and crystallographically characterized: [K(18-c-6)][Ln(OAr*)3] (3-Ln). The Ln(II) complexes decompose at room temperature more slowly than other previously reported 4fn5d1 Ln(II) complexes. For example, only 30% decomposition of 2-Dy was observed after 30 h at room temperature compared to complete decomposition of [Dy(OAr')3]- and [DyCp'3]- under similar conditions (OAr' = OC6H2-2,6-tBu2-4-Me; Cp' = C5H4SiMe3).

Hypoglossal Nerve Stimulator Explantation Technique and Outcomes: A Retrospective Case Series.

INTRODUCTION: Upper airway stimulation via the hypoglossal nerve stimulator (HGNS) implant is a surgical method for treating obstructive sleep apnea. However, patients may need the implant removed for a variety of reasons. The purpose of this case series is to assess surgical experiences with HGNS explantation at our institution. We report on surgical approach, overall operative times, operative and postoperative complications, and discuss relevant patient-specific surgical findings when removing the HGNS. METHODS: We performed a retrospective case series of all patients that underwent HGNS implantation at a single tertiary medical center between January 9, 2021, and January 9, 2022. Subjects included adult patients who presented to the sleep surgery clinic of the senior author for surgical management of previously implanted HGNS. Patient clinical history was reviewed to determine the timing of the patient's implant, reasons for explant, and postoperative recovery course. Operative reports were reviewed to determine overall duration of surgery and any associated difficulties or deviations from the general approach. RESULTS: Between January 9, 2021, and January 9, 2022, 5 patients had an explantation of their HGNS implant. Explantation occurred between 8 and 63 months of their original implant surgery. The average operative time from incisional start time to close was 162 min for all cases with a range of 96-345 min. No significant complications were reported including pneumothorax and nerve palsy. CONCLUSION: This reported case series outlines the general steps for Inspire HGNS explantation as well as details the experiences in a case series of 5 subjects explanted over the year at a single institution. The results from the cases suggest that the explantation of the device can be performed efficiently and safely.

The Implant-borne Articulation Splint in Fibula Free Flap Mandibular Reconstruction: A Technical Note.

Computer-aided design and computer-aided manufacturing and digitally simulated surgeries have revolutionized maxillomandibular reconstruction. In particular, this technology has increased the accuracy and facilitated the process of dental implantation in fibula free flaps. Despite the efficacy of virtual planning, there is a minor degree of translational difference between digital and intraoperative measurements, which may affect the precision of implant and fibula orientations. This is especially concerning during the last stage of fibula insetting, where the graft segments have the potential to roll, yaw, or pitch. The objective of this study is to describe an advanced prosthodontic technique that ensures the fibula grafts and implants remain in a restorable position during final insetting. We describe the technique and workflow of the implant-borne articulation splint through a case presentation and demonstrate results at 4 months postoperative and postradiotherapy. Given the degree of investment placed in virtual planning, free flap reconstruction, and endosteal implants, a technique that ensures optimal restorability of each implant is pivotal. Larger studies are still required to fully elucidate the cost-effectiveness and long-term results of the implant-borne articulation splint.

Poor T-cell receptor ß repertoire diversity early posttransplant for severe combined immunodeficiency predicts failure of immune reconstitution.

BACKGROUND: Development of a diverse T-cell receptor ß (TRB) repertoire is associated with immune recovery following hematopoietic cell transplantation (HCT) for severe combined immunodeficiency (SCID). High-throughput sequencing of the TRB repertoire allows evaluation of clonotype dynamics during immune reconstitution. OBJECTIVES: We investigated whether longitudinal analysis of the TRB repertoire would accurately describe T-cell receptor diversity and illustrate the quality of T-cell reconstitution following HCT or gene therapy for SCID. METHODS: We used high-throughput sequencing to study composition and diversity of the TRB repertoire in 27 infants with SCID at 3, 6, and 12 months and yearly posttreatment(s). Total RNA from peripheral blood was used as template to amplify TRB rearrangements. RESULTS: TRB sequence analysis showed poor diversity at 3 months, followed by significant improvement by 6 months after cellular therapies. Kinetics of development of TRB diversity were similar in patients with a range of underlying gene defects. However, in patients with RAG and DCLRE1C defects, HCT with no conditioning or immune suppression only resulted in lower diversity than did HCT with conditioning. HCT from a matched donor correlated with higher diversity than did HCT from a mismatched donor. Naive CD4+ T-cell count at 6 months post-HCT correlated with higher TRB diversity. A Shannon index of diversity of 5.2 or lower 3 months after HCT predicted a need for a second intervention. CONCLUSIONS: TRB repertoire after hematopoietic cell therapies for SCID provides a quantitative and qualitative measure of diversity of T-cell reconstitution and permits early identification of patients who may require a second intervention.

Outcomes and Perioperative Risk Factors after Oncologic Free-Flap Scalp Reconstruction.

BACKGROUND: Little is known about the risk factors associated with complications after free flap scalp reconstruction. The purpose of this study was to identify patient, scalp defect, and flap characteristics associated with increased risk of surgical complications. METHODS: A retrospective study was performed of free-flap scalp reconstruction in oncologic patients at Memorial Sloan Kettering Cancer Center from 2002 to 2017. Data collection included patient, defect, flap, and complication characteristics. Complications were classified into major, defined as complications requiring surgical intervention, and minor, defined as complications requiring conservative treatment. Risk factors and outcome variables were compared using chi-square with Fisher's exact test. RESULTS: A total of 63 free flaps to the scalp in 58 patients were performed; average follow-up was 3.5 years. Most flaps were muscle-only or musculocutaneous. One-third of patients with free flaps experienced complications (n = 21, 15 major and 6 minor). Examining risk factors for complications, patients with cardiovascular disease were nearly three times more likely to have suffered a major complication than patients without cardiovascular disease (36.7 vs. 12.1%, p = 0.04). This was the only significant risk factor noted. Perioperative radiotherapy, prior scalp surgery, flap type, and recipient vessel selection were found to be nonsignificant risk factors. CONCLUSION: Cardiovascular disease may be a significant marker of risk for major complications in patients undergoing free-flap reconstruction of the scalp. This information should be used to help guide perioperative counseling and decision making in this challenging patient population.

Outcomes in Pediatric Maxillofacial Reconstruction With Vascularized Fibular Flaps: A Systematic Review.

BACKGROUND: The purpose of this study was to examine the complications and outcomes after maxillofacial reconstruction using the free fibular flap in the pediatric population. METHODS: A systematic review and descriptive analysis were conducted using data variables, including study characteristics; patient characteristics; postoperative complications (major and minor); surgical revision; and dental rehabilitation. RESULTS: The systematic review resulted in 1622 articles, 55 of which met inclusion criteria for this study. The 55 articles consisted of 17 case series and 38 case reports with level III/IV and level V of evidence, respectively. Of the 155 identified pediatric patients, the rate of major complications was 13.5% and minor complications was 24.5%. The most common complication was mild growth distortion (n = 7) at the recipient site. Complications at the donor site were less common. During follow-up, 29 patients (18.7%) underwent or awaited surgical revision, and 43 patients (27.7%) underwent or awaited dental rehabilitation. CONCLUSIONS: Our study suggests that the free fibular flap for pediatric maxillofacial reconstruction is safe and reliable. Additionally, surgical revision to correct the functional impairments resulting from primary reconstruction using the free fibular flap is relatively common.

Clinical Practice Trends and Postoperative Outcomes in Primary Cleft Rhinoplasty.

PURPOSE: Optimal correction of the cleft nasal deformity remains challenging. The purpose of this study was to examine the practice patterns and postoperative course of patients undergoing cleft lip repair with rhinoplasty compared to those who have primary lip repair without rhinoplasty. METHODS AND MATERIALS: A retrospective cohort study was conducted based on the Kids' Inpatient Database. Data were collected from January 2000 to December 2011 and included infants aged 12 months and younger who underwent cleft lip repair. The predictor variable was the addition of rhinoplasty at primary cleft lip repair. Primary outcome variables included hospital setting, year, and admission cost, while secondary outcome variables included length of stay and postoperative complication rate. Independent t-tests and chi-squared tests were performed. Continuous variables were analyzed by multiple linear regression models. RESULTS: The study sample included 4559 infants with 1422 (31.2%) who underwent primary cleft rhinoplasty. Over time, there was a significant increase in the proportion of cleft lip repairs accompanied by a rhinoplasty (p < .01). A greater proportion of patients with unilateral cleft lips received simultaneous rhinoplasty with their lip repairs (33.8 vs 26.0%, p < .01). This cohort had a significantly shorter length of stay (1.6 vs 2.8 days, p < .01) when compared to children that underwent cleft lip repair alone. CONCLUSIONS: Performing primary cleft rhinoplasty is becoming more common among cleft surgeons. Considering comparable costs and complication rates, a rhinoplasty should be considered during the surgical treatment planning of patients with cleft nasal deformities.

Selfconsistent random phase approximation methods.

This Perspective reviews recent efforts toward selfconsistent calculations of ground-state energies within the random phase approximation (RPA) in the (generalized) Kohn-Sham (KS) density functional theory context. Since the RPA correlation energy explicitly depends on the non-interacting KS potential, an additional condition to determine the energy as a functional of the density is necessary. This observation leads to the concept of functional selfconsistency (FSC), which requires that the KS density equals the interacting density defined as the functional derivative of the ground-state energy with respect to the external potential. While all existing selfconsistent RPA schemes violate FSC, the recent generalized KS semicanonical projected RPA (GKS-spRPA) method takes a step toward satisfying it. This leads to systematic improvements in densities, binding energy curves, reference state stability, and molecular properties compared to non-selfconsistent RPA as well as optimized effective potential RPA. GKS-spRPA orbital energies accurately approximate valence and core ionization potentials, and even electron affinities of non-valence bound anions. The computational cost and performance of GKS-spRPA are compared to those of related selfconsistent schemes, including GW and orbital optimization methods, and limitations are discussed. Large differences between KS and interacting densities observed in the absence of FSC and the well-rounded performance of GKS-spRPA suggest that the KS potential as a density functional should be defined via the FSC condition for explicitly potential-dependent density functionals.

Hepatic Artery Microvascular Anastomosis in Liver Transplantation: A Systematic Review of the Literature.

BACKGROUND: The operating microscope is used in many centers for microvascular hepatic arterial reconstruction in living as well as deceased donor liver transplantation in adult and pediatric recipients. To date, a systematic review of the literature examining this topic is lacking. METHODS: This systematic review of the literature was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Three different electronic databases (PubMed, Embase OVID, and Cochrane CENTRAL) were queried. RESULTS: A total of 34 studies were included. The rate of hepatic artery thrombosis (HAT) in noncomparative studies (28) ranged from 0% to 10%, with 8 studies reporting patient deaths resulting from HAT. Within comparative studies, the rate of HAT in patients who underwent arterial reconstruction using the operating microscope ranged from 0% to 5.3%, whereas the rate of HAT in patients who underwent arterial reconstruction using loupe magnification ranged from 0% up to 28.6%, and 2 studies reported patient deaths resulting from HAT. Two comparative studies did not find statistically significant differences between the 2 groups. CONCLUSIONS: Our comprehensive systematic review of the literature seems to suggest that overall, rates of HAT may be lower when the operating microscope is used for hepatic arterial reconstruction in liver transplantation. However, matched comparisons are lacking and surgical teams need to be mindful of the learning curve associated with the use of the operating microscope as compared with loupe magnification, as well as the logistical and time constraints associated with setup of the operating microscope.

Comparing outcomes between stacked/conjoined and non-stacked/conjoined abdominal microvascular unilateral breast reconstruction.

BACKGROUND: Stacked and conjoined free flaps are increasingly utilized in autologous breast reconstruction to augment tissue transfer volume. However, there is a paucity of comparative data on abdominally-based stacked/conjoined versus non-stacked/conjoined flaps. The purpose of this study was to compare ability to match native breast size, complications, recovery, and symmetrizing procedures between these two cohorts in unilateral breast reconstruction. METHODS: A retrospective review of all stacked (two separate hemiabdominal)/conjoined (bipedicled full abdominal) flaps and non-stacked/conjoined (unipedicled hemiabdominal) flaps in unilateral abdominally-based autologous breast reconstructions was performed from 2011 to 2018. Variables including demographics, operative characteristics, complications, and revisions were compared in 36 stacked/conjoined patients versus 146 non-stacked/conjoined patients. RESULTS: The stacked/conjoined cohort had more DIEP flaps (91.7 vs. 65.1%) and the non-stacked/conjoined group more MS-TRAMs (34.2 vs. 6.9%, p = .000). Additionally, non-stacked/conjoined flaps had greater utilization of combined medial and lateral row perforators (p = .000). Mean flap weight was significantly higher than mastectomy weight in stacked/conjoined flaps (+110.7 g) when compared to non-stacked/conjoined flaps (-40.2) (p = .023). Average follow-up was 54.7 ± 27.5 and 54.6 ± 29.3 months, respectively. Stacked/conjoined flaps had lower fat necrosis rates (8.3 vs. 25.4%, p = .039) and had a decreased risk of fat necrosis on multivariable regression analysis (OR 0.278, p = 0.045). There were otherwise no differences in flap, breast, or donor-site complications. Stacked/conjoined flaps also had a lower rate of contralateral breast reduction (p = .041). CONCLUSION: Stacked/conjoined flaps were associated with a lower risk of fat necrosis compared with non-stacked/conjoined flaps and had a lower rate of contralateral symmetrizing reductions in patients undergoing unilateral abdominally-based breast reconstruction.

Analysis of 20-Year Trends in Medicare Reimbursement for Reconstructive Microsurgery.

BACKGROUND: Microsurgery is being increasingly utilized across surgical specialties, including plastic surgery. Microsurgical techniques require greater time and financial investment compared with traditional methods. This study aimed to evaluate 20-year trends in Medicare reimbursement and utilization for commonly billed reconstructive microsurgery procedures from 2000 to 2019. METHODS: Microsurgical procedures commonly billed by plastic surgeons were identified. Reimbursement data were extracted from The Physician Fee Schedule Look-Up Tool from the Centers for Medicare and Medicaid Services for each current procedural terminology (CPT) code. All monetary data were adjusted for inflation to 2019 U.S. dollars. The average annual and total percentage changes in reimbursement were calculated based on these adjusted trends. To assess utilization trends, CMS physician/supplier procedure summary files were queried for the number of procedures billed by plastic surgeons from 2010 to 2018. RESULTS: After adjusting for inflation, the average reimbursement for all procedures decreased by 26.92% from 2000 to 2019. The greatest mean decrease was observed in CPT 20969 free osteocutaneous flaps with microvascular anastomosis (-36.93%). The smallest mean decrease was observed in repair of blood vessels with vein graft (-9.28%). None of the included procedures saw an increase in reimbursement rate over the study period. From 2000 to 2019, the adjusted reimbursement rate for all procedures decreased by an average of 1.35% annually. Meanwhile, the number of services billed to Medicare by plastic surgeons across the included CPT codes increased by 42.17% from 2010 to 2018. CONCLUSION: This is the first study evaluating 20-year trends in inflation-adjusted Medicare reimbursement and utilization in reconstructive microsurgery. Reimbursement for all included procedures decreased over 20% during the study period, while number of services increased. Increased consideration of these trends will be important for U.S. policymakers, hospitals, and surgeons to assure continued access and reconstructive options for patients.

CRISPR-Knockout Screen Identifies Dmap1 as a Regulator of Chemically Induced Reprogramming and Differentiation of Cardiac Progenitors.

Direct in vivo reprogramming of cardiac fibroblasts into myocytes is an attractive therapeutic intervention in resolving myogenic deterioration. Current transgene-dependent approaches can restore cardiac function, but dependence on retroviral delivery and persistent retention of transgenic sequences are significant therapeutic hurdles. Chemical reprogramming has been established as a legitimate method to generate functional cell types, including those of the cardiac lineage. Here, we have extended this approach to generate progenitor cells that can differentiate into endothelial cells and cardiomyocytes using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells result in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle. Deployment of a genome-wide knockout screen with clustered regularly interspaced short palindromic repeats-guide RNA library to identify novel mediators that regulate the reprogramming revealed the involvement of DNA methyltransferase 1-associated protein 1 (Dmap1). Loss of Dmap1 reduced promoter methylation, increased the expression of Nkx2-5, and enhanced the retention of self-renewal, although further differentiation is inhibited because of the sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. Stem Cells 2019;37:958-972.

Genome-wide CRISPR-Cas9 screening in mammalian cells.

Forward genetic screens are a powerful and unbiased approach for uncovering the genetic basis behind a specific phenotype. Genome-wide mutagenesis followed by phenotypic screening represents the ultimate manifestation of this method, directly linking biological phenomena to its corresponding genetic cause. Whilst this has been successful in lower organisms, deployment of genome-wide screens in mammalian systems has been hampered by both limitations of scale and inefficient bi-allelic mutagenesis. CRISPR-Cas9 technology has now largely resolved these issues, whereby delivery of genome-scale gRNA libraries in the presence of gRNA-guided Cas9 endonuclease enables the generation of mutant cell libraries; the perfect platform for performing phenotypic screens. Although the tools are now available for virtually any molecular biology laboratory to conduct such screens, many researchers are daunted by the sheer complexity and scale at which such experiments are performed. This Review will address these concerns, presenting a contextual and practical guide to deploying CRISPR-KO screens in mammalian systems. We will discuss key considerations required in all aspects of screening from initiation to conclusion, which will enable researchers to conduct screens of their own, maximising the potential of this powerful technology.

TURBOMOLE: Modular program suite for ab initio quantum-chemical and condensed-matter simulations.

TURBOMOLE is a collaborative, multi-national software development project aiming to provide highly efficient and stable computational tools for quantum chemical simulations of molecules, clusters, periodic systems, and solutions. The TURBOMOLE software suite is optimized for widely available, inexpensive, and resource-efficient hardware such as multi-core workstations and small computer clusters. TURBOMOLE specializes in electronic structure methods with outstanding accuracy-cost ratio, such as density functional theory including local hybrids and the random phase approximation (RPA), GW-Bethe-Salpeter methods, second-order Møller-Plesset theory, and explicitly correlated coupled-cluster methods. TURBOMOLE is based on Gaussian basis sets and has been pivotal for the development of many fast and low-scaling algorithms in the past three decades, such as integral-direct methods, fast multipole methods, the resolution-of-the-identity approximation, imaginary frequency integration, Laplace transform, and pair natural orbital methods. This review focuses on recent additions to TURBOMOLE's functionality, including excited-state methods, RPA and Green's function methods, relativistic approaches, high-order molecular properties, solvation effects, and periodic systems. A variety of illustrative applications along with accuracy and timing data are discussed. Moreover, available interfaces to users as well as other software are summarized. TURBOMOLE's current licensing, distribution, and support model are discussed, and an overview of TURBOMOLE's development workflow is provided. Challenges such as communication and outreach, software infrastructure, and funding are highlighted.