A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways.

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.

Chaperone requirements for de novo folding of Saccharomyces cerevisiae septins.

Polymers of septin protein complexes play cytoskeletal roles in eukaryotic cells. The specific subunit composition within complexes controls functions and higher-order structural properties. All septins have globular GTPase domains. The other eukaryotic cytoskeletal NTPases strictly require assistance from molecular chaperones of the cytosol, particularly the cage-like chaperonins, to fold into oligomerization-competent conformations. We previously identified cytosolic chaperones that bind septins and influence the oligomerization ability of septins carrying mutations linked to human disease, but it was unknown to what extent wild-type septins require chaperone assistance for their native folding. Here we use a combination of in vivo and in vitro approaches to demonstrate chaperone requirements for de novo folding and complex assembly by budding yeast septins. Individually purified septins adopted nonnative conformations and formed nonnative homodimers. In chaperonin- or Hsp70-deficient cells, septins folded slower and were unable to assemble posttranslationally into native complexes. One septin, Cdc12, was so dependent on cotranslational chaperonin assistance that translation failed without it. Our findings point to distinct translation elongation rates for different septins as a possible mechanism to direct a stepwise, cotranslational assembly pathway in which general cytosolic chaperones act as key intermediaries.

Investigating the genetic pathways of insomnia in Autism Spectrum Disorder.

BACKGROUND: Sleep problems are common in children with autism spectrum disorder (autism). There is sparse research to date to examine whether insomnia in people with autism is related to autism genetics or insomnia genetics. Moreover, there is a lack of research examining whether circadian-rhythm related genes share potential pathways with autism. AIMS: To address this research gap, we tested whether polygenic scores of insomnia or autism are related to risk of insomnia in people with autism, and whether the circadian genes are associated with insomnia in people with autism. METHODS AND PROCEDURES: We tested these questions using the phenotypically and genotypically rich MSSNG dataset (N = 1049) as well as incorporating in the analyses data from the Vanderbilt University Biobank (BioVU) (N = 349). OUTCOMES AND RESULTS: In our meta-analyzed sample, there was no evidence of associations between the polygenic scores (PGS) for insomnia and a clinical diagnosis of insomnia, or between the PGS of autism and insomnia. We also did not find evidence of a greater burden of rare and disruptive variation in the melatonin and circadian genes in individuals with autism and insomnia compared to individuals with autism without insomnia. CONCLUSIONS AND IMPLICATIONS: Overall, we did not find evidence for strong effects of genetic scores influencing sleep in people with autism, however, we cannot rule out the possibility that smaller genetic effects may play a role in sleep problems. Our study indicated the need for a larger collection of data on sleep problems and sleep quality among people with autism.

Post-Transcriptional Control of Mating-Type Gene Expression during Gametogenesis in Saccharomyces cerevisiae.

Gametogenesis in diploid cells of the budding yeast Saccharomyces cerevisiae produces four haploid meiotic products called spores. Spores are dormant until nutrients trigger germination, when they bud asexually or mate to return to the diploid state. Each sporulating diploid produces a mix of spores of two haploid mating types, a and α. In asexually dividing haploids, the mating types result from distinct, mutually exclusive gene expression programs responsible for production of mating pheromones and the receptors to sense them, all of which are silent in diploids. It was assumed that spores only transcribe haploid- and mating-type-specific genes upon germination. We find that dormant spores of each mating type harbor transcripts representing all these genes, with the exception of Mata1, which we found to be enriched in a spores. Mata1 transcripts, from a rare yeast gene with two introns, were mostly unspliced. If the retained introns reflect tethering to the MATa locus, this could provide a mechanism for biased inheritance. Translation of pheromones and receptors were repressed at least until germination. We find antisense transcripts to many mating genes that may be responsible. These findings add to the growing number of examples of post-transcriptional regulation of gene expression during gametogenesis.

Saccharomyces spores are born prepolarized to outgrow away from spore-spore connections and penetrate the ascus wall.

How nonspore haploid Saccharomyces cells choose sites of budding and polarize towards pheromone signals in order to mate has been a subject of intense study. Unlike nonspore haploids, sibling spores produced via meiosis and sporulation by a diploid cell are physically interconnected and encased in a sac derived from the old cell wall of the diploid, called the ascus. Nonspore haploids bud adjacent to previous sites of budding, relying on stable cortical landmarks laid down during prior divisions, but because spore membranes are made de novo, it was assumed that, as is known for fission yeast, Saccharomyces spores break symmetry and polarize at random locations. Here, we show that this assumption is incorrect: Saccharomyces cerevisiae spores are born prepolarized to outgrow, prior to budding or mating, away from interspore bridges. Consequently, when spores bud within an intact ascus, their buds locally penetrate the ascus wall, and when they mate, the resulting zygotes adopt a unique morphology reflective of repolarization towards pheromone. Long-lived cortical foci containing the septin Cdc10 mark polarity sites, but the canonical bud site selection programme is dispensable for spore polarity, thus the origin and molecular composition of these landmarks remain unknown. These findings demand further investigation of previously overlooked mechanisms of polarity establishment and local cell wall digestion and highlight how a key step in the Saccharomyces life cycle has been historically neglected.

Effect of operative approach on quality of life following anatomic lung cancer resection.

Patient-reported outcomes (PRO) after lung cancer surgery are of increasing interest to patients and clinicians. A variety of studies have investigated the impact of the surgical approach on quality of life (QOL) after surgery for early non-small-cell lung cancer (NSCLC). Our aim is to review the current evidence on how minimally-invasive approaches, including video-assisted thoracoscopic surgery (VATS) and robotic-assisted thoracoscopic surgery (RATS), versus open thoracotomy for lung cancer affect QOL. We conducted a systematic review of the literature of studies comparing QOL after VATS/RATS versus thoracotomy approach using studies published before 2019 on PubMed and Google Scholar. Studies were assessed for differences in QOL by domains. Fifteen studies met our inclusion criteria including 14 observational studies and one randomized trial. Survey instruments and timing of QOL assessments differed between all studies. A thoracoscopic (VATS or RATS) approach was associated with better general health (3/10 studies), physical functioning (9/14 studies), social functioning (1/12 studies), mental health (3/13 studies), emotional role functioning (4/12 studies), physical role functioning (7/12 studies), and bodily pain (7/12 studies) as compared to open surgery. The open thoracotomy approach was associated with better general health and mental health in one study each. Although QOL assessment in current studies is highly variable, the existing evidence suggests that a thoracoscopic approach is associated with improved QOL, particularly in the areas of physical functioning and pain as compared to open lung cancer surgery.

Evaluation of a Lung Nodule in the Setting of Metastatic Gastrointestinal Stromal Tumor: Surgical Considerations in Carney's Triad.

In 1977, Carney et al. first described an association of gastric epithelioid leiomyosarcoma or gastrointestinal stromal tumor (GIST), pulmonary chondroma, and extra-adrenal paraganglioma. This previously unrecognized disorder came to be known as Carney's triad. We describe a case of a 27-year-old female with metastatic GIST, diagnosed with Carney's triad following pulmonary wedge resection, and highlight the surgical implication of this rare disease association.

Patient Satisfaction After Lung Cancer Surgery: Do Clinical Outcomes Affect Hospital Consumer Assessment of Health Care Providers and Systems Scores?

BACKGROUND: Little is known about patients' hospital experience and satisfaction after lung cancer surgery. We sought to determine how length of hospital stay (LOS) and postoperative complications affect hospital consumer assessment of health care providers and systems (HCAHPS) scores. METHODS: Patients undergoing lung resection for cancer at a single academic cancer center between years 2014 and 2018 were analyzed. Clinical data were derived from The Society of Thoracic Surgeons institutional database and supplemented with HCAHPS survey data. Endpoints were "top-box" satisfaction scores and domain-specific scores for physicians and nurses communication. RESULTS: In total, 181 of 478 patients (38%) who underwent pulmonary resection for lung cancer completed HCAHPS surveys. Median age was 65 years, and most patients underwent lobectomy (94%). The top-box rating for the overall hospital experience, physician communication, and nurse communication were 92%, 84%, and 69%, respectively. Overall and major complication rates were 43% and 3%, and were not associated with top-box HCAHPS scores. Increasing length of stay was associated with worse satisfaction with provider communication. Adjusted for patient factors, increasing length of stay was associated with worse patient satisfaction in the domains of communication with physicians and nurses. Patients with length of stay more than 6 days were less likely to endorse that doctors gave understandable explanations (odds ratio 0.15, 95% confidence interval, 0.04 to 0.56) and that nurses listened carefully (odds ratio 0.11, 95% confidence interval, 0.06 to 0.69). CONCLUSIONS: Overall HCAHPS satisfaction scores after lung resection for cancer were high and were negatively associated with increasing length of stay. Patient satisfaction may be affected more by the perception of effective communication during prolonged hospitalizations than by complications.

Understanding the financial cost of robotic lobectomy: calculating the value of innovation?

BACKGROUND: The advantages of a minimally-invasive surgical approach compared to conventional open thoracotomy for lung resection have been previously described. While robot-assisted thoracoscopic surgery (RATS) has shown comparable clinical outcomes for lobectomy as compared with video-assisted thoracic surgery (VATS), the cost and inherent value associated with the robotic technology remains a main concern. METHODS: We conducted a systematic review of the literature on the cost of RATS lobectomy using studies published prior to December 2017 on MEDLINE and EMBASE. RESULTS: Six observational studies met our inclusion criteria. Median cost of RATS lobectomy ranged from $15,440 to $22,582. Operating room (OR) cost was a major contributing factor to overall cost. The lowest per-procedure cost was reported by the highest volume center. Cost definitions were highly variable among studies. The total cost of RATS was similar or lower to open lobectomy, and higher than that of VATS, with cost difference ranging from $2,901 to $4,708. CONCLUSIONS: Assessment of cost for RATS lobectomy varies significantly. High OR costs may be offset by improved outcomes as compared with open lobectomy, but currently the costs exceed that of VATS lobectomy. Further work is needed to define the cost and actual value parameters for RATS lobectomy.

Hospital cost and clinical effectiveness of robotic-assisted versus video-assisted thoracoscopic and open lobectomy: A propensity score-weighted comparison.

OBJECTIVE: To compare cost and perioperative outcomes of robotic, video-assisted thoracoscopic surgery (VATS), and open surgical approaches to pulmonary lobectomy. METHODS: Patients who underwent pulmonary lobectomy between 2012 and 2017 at a single tertiary referral center were reviewed. Propensity score adjustment by inverse probability of treatment weighting (IPTW) was used to balance baseline patient characteristics. The primary outcomes of the study were direct hospital cost and perioperative outcomes, including operative time, complications rates, and length of stay. Indirect cost and charges were secondary financial outcomes. RESULTS: A total of 697 patients underwent pulmonary lobectomy by robotic (n = 296), VATS (n = 161), and open thoracotomy (n = 240). In the IPTW-adjusted analysis, open thoracotomy had the shortest mean operating room time (robotic 278 minutes vs VATS 298 minutes vs open 265 minutes, P = .05), and lowest operating room costs (robotic $9,912 vs VATS $9491 vs open $8698, P = .001). Length of stay was significantly shorter after robotic and VATS lobectomy (robotic 3.8 days vs VATS 3.8 days vs open 5.4 days, P < .001), with significantly fewer events of atelectasis and pneumonia as compared with the open group. In sum, no significant differences were seen in IPTW-adjusted direct cost (robotic $17,223 vs VATS $17,260 vs open $18,075, P = .48), indirect cost, or charges for the total hospital stay. CONCLUSIONS: Robotic and VATS lobectomy were associated with similar cost and improved clinical effectiveness as compared with the open thoracotomy approach. Increased procedural cost of minimally invasive lobectomy can be recovered by postoperative costs reductions, associated with improved postoperative outcomes and shorter hospital stay.

TIGIT signaling restores suppressor function of Th1 Tregs.

Th1 Tregs are characterized by the acquisition of proinflammatory cytokine secretion and reduced suppressor activity. Th1 Tregs are found at increased frequency in autoimmune diseases, including type 1 diabetes and multiple sclerosis (MS). We have previously reported that in vitro stimulation with IL-12 recapitulates the functional and molecular features of MS-associated Th1 Tregs, revealing a central role for hyperactivation of the Akt pathway in their induction. TIGIT is a newly identified coinhibitory receptor that marks Tregs that specifically control Th1 and Th17 responses. Here, we report that signaling through TIGIT counteracts the action of IL-12 in inducing the Th1 program. Specifically, TIGIT signaling represses production of IFN-γ and T-bet expression and restores suppressor function in Tregs treated with IL-12. FoxO1 functional inhibition abolishes the protective effect of TIGIT, indicating that TIGIT signaling promotes FoxO1 nuclear localization. Consistent with this observation, signaling through TIGIT leads to a rapid suppression of Akt function and FoxO1 phosphorylation. Finally, TIGIT stimulation reduces the production of IFN-γ and corrects the suppressor defect of Tregs from patients with MS. Our results indicate an important role for TIGIT in controlling the functional stability of Tregs through repression of Akt, suggesting that the TIGIT pathway could be targeted for immunomodulatory therapies in human autoimmune disorders.

Postoperative complications decrease the cost-effectiveness of robotic-assisted lobectomy.

BACKGROUND: Cost of robotic-assisted (RATS) lobectomy remains a major concern. We sought to define variability in cost and factors associated with increased hospital expenses after RATS lobectomy for early stage non-small cell lung cancer. METHODS: We performed a retrospective review of patients who underwent RATS lobectomy for stages I-IIIA non-small cell lung cancer at a single institution between 2012 and 2014. Clinical outcomes were linked to hospital financial data. Linear regression analysis was used to test the impact of patient factors and postoperative outcomes on cost. RESULTS: A total of 137 patients underwent RATS lobectomy, predominantly for stage IA (73%, n = 100). Overall in-hospital morbidity was 29.2% (n = 40), median length of stay was 5 days (range 1-27 days). Postoperative cost accounted for approximately 50% of total cost of hospitalization and varied significantly (mean $9,618.38 ± $10,779.65), resulting in an average total hospital cost of $19,565 (±$11,620.42). Male sex and upper lobe predominant disease were independently associated with increased cost, whereas higher preoperative diffusing capacity of lung for carbon monoxide (DLCO) was cost-protective. Hospital expenses associated with prolonged hospitalization were $2,376.23 per day (95% CI $2,178-2,573.60). The most common complication associated with increased cost was atrial fibrillation ($5,609.13; 95% CI $2,095.42-$9,122.84). Postoperative atelectasis requiring bronchoscopy, respiratory failure, pulmonary embolism, and reoperation were seen less frequently in this cohort of patients but were associated with significant additional cost. CONCLUSION: Hospital cost of RATS lobectomy can vary significantly. In addition to patient risk factors, differences in cost are mainly driven by postoperative events. Initiatives aimed to reduce common yet expensive complications have the potential to improve overall cost-effectiveness of RATS lobectomy.

Surgical Technical Evidence Review of Hip Fracture Surgery Conducted for the AHRQ Safety Program for Improving Surgical Care and Recovery.

BACKGROUND: Enhanced recovery pathways (ERPs) have been shown to improve patient outcomes in a variety of contexts. This review summarizes the evidence and defines a protocol for perioperative care of patients with hip fracture and was conducted for the Agency for Healthcare Research and Quality safety program for improving surgical care and recovery. STUDY DESIGN: Perioperative care was divided into components or "bins." For each bin, a semisystematic review of the literature was conducted using MEDLINE with priority given to systematic reviews, meta-analyses, and randomized controlled trials. Observational studies were included when higher levels of evidence were not available. Existing guidelines for perioperative care were also incorporated. For convenience, the components of care that are under the auspices of anesthesia providers will be reported separately. Recommendations for an evidence-based protocol were synthesized based on review of this evidence. RESULTS: Eleven bins were identified. Preoperative risk factor bins included nutrition, diabetes mellitus, tobacco use, and anemia. Perioperative management bins included thromboprophylaxis, timing of surgery, fluid management, drain placement, early mobilization, early alimentation, and discharge criteria/planning. CONCLUSIONS: This review provides the evidence basis for an ERP for perioperative care of patients with hip fracture.

Burden of Care of Various Infant Orthopedic Protocols for Improvement of Nasolabial Esthetics in Patients With CUCLP.

OBJECTIVES: 1. To evaluate the orthodontic burden of care of nasoalveolar molding (NAM) and modified McNeil for the treatment of patients with complete unilateral cleft lip and palate (CUCLP). 2. To compare the esthetic outcomes of each with those of centers not utilizing infant orthopedics (IO). DESIGN: Retrospective cohort study. SETTING: Institutional. PARTICIPANTS: Four cohorts with repaired CUCLP (n = 149) from 3 centers. INTERVENTIONS: Two cohorts were treated in the same center and had either traditional infant orthopedics (TIO) or NAM and 2 were treated in centers not employing IO. MAIN OUTCOME MEASURES: Burden of care data for the IO groups were compared using t tests. Frontal and profile photographs at approximately age 5 were collected for ratings of nasolabial esthetics, using a modification of the Asher-McDade method. Intrarater and interrater reliabilities were determined using weighted κ statistics. Median ratings were compared using a Kruskal-Wallis test. RESULTS: The burden of care of NAM was significantly greater than TIO for both the number of visits (9.9 vs 6.6, [ P < .001]); and treatment duration (127 vs 112 days, [ P < .05]). Significant differences in nasolabial esthetic ratings were noted among the 3 centers. No significant differences were observed in the nasolabial esthetic outcomes between the NAM and TIO groups. CONCLUSIONS: 1. NAM required more visits and longer overall duration compared with TIO. 2. The center employing IO showed favorable nasolabial esthetics compared to those not utilizing IO. 3. No significant differences were found in the nasolabial esthetics of patients who have received NAM compared with TIO.

Surgical Technical Evidence Review for Elective Total Joint Replacement Conducted for the AHRQ Safety Program for Improving Surgical Care and Recovery.

BACKGROUND: Use of enhanced recovery pathways (ERPs) can improve patient outcomes, yet national implementation of these pathways remains low. The Agency for Healthcare Research and Quality (AHRQ; funder), the American College of Surgeons, and the Johns Hopkins Medicine Armstrong Institute for Patent Safety and Quality have developed the Safety Program for Improving Surgical Care and Recovery-a national effort to catalyze implementation of practices to improve perioperative care and enhance recovery of surgical patients. This review synthesizes evidence that can be used to develop a protocol for elective total knee arthroplasty (TKA) and total hip arthroplasty (THA). STUDY DESIGN: This review focuses on potential components of the protocol relevant to surgeons; anesthesia components are reported separately. Components were identified through review of existing pathways and from consultation with technical experts. For each, a structured review of MEDLINE identified systematic reviews, randomized trials, and observational studies that reported on these components in patients undergoing elective TKA/THA. This primary evidence review was combined with existing clinical guidelines in a narrative format. RESULTS: Sixteen components were reviewed. Of the 10 preoperative components, most were focused on risk factor assessment including anemia, diabetes mellitus, tobacco use, obesity, nutrition, immune-modulating therapy, and opiates. Preoperative education, venous thromboembolism (VTE) prophylaxis, and bathing/Staphylococcus aureus decolonization were also included. The routine use of drains was the only intraoperative component evaluated. The 5 postoperative components included early mobilization, continuous passive motion, extended duration VTE prophylaxis, early oral alimentation, and discharge planning. CONCLUSION: This review synthesizes the evidence supporting potential surgical components of an ERP for elective TKA/THA. The AHRQ Safety Program for Improving Surgical Care and Recovery aims to guide hospitals and surgeons in identifying the best practices to implement in the surgical care of TKA and THA patients.

Regulatory T Cells: From Discovery to Autoimmunity.

Multiple sclerosis (MS) is a genetically mediated autoimmune disease of the central nervous system. Allelic variants lead to lower thresholds of T-cell activation resulting in activation of autoreactive T cells. Environmental factors, including, among others, diet, vitamin D, and smoking, in combination with genetic predispositions, play a substantial role in disease development and activation of autoreactive T cells. FoxP3+ regulatory T cells (Tregs) have emerged as central in the control of autoreactive T cells. A consistent finding in patients with MS is defects in Treg cell function with reduced suppression of effector T cells and production of proinflammatory cytokines. Emerging data suggests that functional Tregs become effector-like T cells with loss of function associated with T-bet expression and interferon γ (IFN-γ) secretion.

The Americleft Project: A Modification of Asher-McDade Method for Rating Nasolabial Esthetics in Patients With Unilateral Cleft Lip and Palate Using Q-sort.

The purpose of this study was to investigate ways to improve rater reliability and satisfaction in nasolabial esthetic evaluations of patients with complete unilateral cleft lip and palate (UCLP), by modifying the Asher-McDade method with use of Q-sort methodology. Blinded ratings of cropped photographs of one hundred forty-nine 5- to 7-year-old consecutively treated patients with complete UCLP from 4 different centers were used in a rating of frontal and profile nasolabial esthetic outcomes by 6 judges involved in the Americleft Project's intercenter outcome comparisons. Four judges rated in previous studies using the original Asher-McDade approach. For the Q-sort modification, rather than projection of images, each judge had cards with frontal and profile photographs of each patient and rated them on a scale of 1 to 5 for vermillion border, nasolabial frontal, and profile, using the Q-sort method with placement of cards into categories 1 to 5. Inter- and intrarater reliabilities were calculated using the Weighted Kappa (95% confidence interval). For 4 raters, the reliabilities were compared with those in previous studies. There was no significant improvement in inter-rater reliabilities using the new method. Intrarater reliability consistently improved. All raters preferred the Q-sort method with rating cards rather than a PowerPoint of photos, which improved internal consistency in rating compared to previous studies using the original Asher-McDade method. All raters preferred this method because of the ability to continuously compare photos and adjust relative ratings between patients.

Sustained Growth of a University-Based Endocrine Surgery Program Over 10 Years.

BACKGROUND: Endocrine surgery continues to mature as a subspecialty field. We describe the clinical performance of an academic endocrine surgery program (ESP) over its first 10 years. METHODS: We examined all endocrine procedures performed during the 10-year period (2006-2015) following the inception of the ESP. Institutional and state-level data on case volume, patient geographic origin, and hospital-side costs were obtained. RESULTS: Endocrine case volume increased by approximately ninefold over the study period (from 102 cases in 2006 to 919 cases in 2015). The rate of growth remained approximately linear, and was driven by geographic expansion of referral regions coupled with transitioning low- to moderate-acuity operations to venues outside of the main tertiary care hospital. Market share across the eight-county Southern California region grew by more than twofold over the study period. Increased utilization of outpatient surgery led to cost reductions, averaging 11.1% per case by 2015. CONCLUSIONS: Establishment of an academic ESP can lead to sustained clinical growth and a fundamental shift in regional referral patterns. The nation's continued need for skilled high-volume endocrine surgeons represents opportunities for medical centers to institute their own dedicated endocrine surgery programs.

Imaging the Dynamics of Cell Wall Polymer Deposition in the Unicellular Model Plant, Penium margaritaceum.

The unicellular green alga, Penium margaritaceum, represents a novel and valuable model organism for elucidating cell wall dynamics in plants. This organism's cell wall contains several polymers that are highly similar to those found in the primary cell walls of land plants. Penium is easily grown in laboratory culture and is effectively manipulated in various experimental protocols including microplate assays and correlative microscopy. Most importantly, Penium can be live labeled with cell wall-specific antibodies or other probes and returned to culture where specific cell wall developmental events can be monitored. Additionally, live cells can be rapidly cryo-fixed and cell wall surface microarchitecture can be observed with variable pressure scanning electron microscopy. Here, we describe the methodology for maintaining Penium for experimental cell wall enzyme studies.