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Nimesh A Patel, Parth Shah, Nicolas A Yannuzzi, Zubair Ansari, Jill S Zaveri, Nidhi Relhan, Basil K Williams Jr, Ajay E Kuriyan, Christopher R Henry, Jayanth Sridhar, Luis Haddock, Jorge A Fortun, Thomas A Albini, Janet L Davis, Harry W Flynn JrDepartment of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, FL 33136, USA Purpose: To report the visual outcomes and complications of scleral fixated intraocular lenses (IOLs) using Gore-Tex suture.Methods: The current study is a retrospective noncomparative case series including patients who underwent scleral fixation of IOL (Akreos AO60) using Gore-Tex suture from August 2015 to March 2017 at a university teaching center. Primary outcome measures were visual acuity and complications at last follow-up.Results: The current study included 49 eyes of 48 patients. Mean follow-up duration postsurgery was 6.9 months (range: 0.9–29.4 months). The indications for secondary IOL surgery were dislocated IOL in 16/49 (33%), subluxed IOL in 9/49 (18%), dislocated or subluxed crystalline lens in 9/49 (18%), traumatic cataract in 8/49 (16%), and complicated cataract surgery in 7/49 (14%). Mean best-corrected logMAR visual acuity improved from 1±0.7 (20/200 Snellen equivalent) preoperatively to 0.5±0.5 (20/63 Snellen equivalent) at last follow-up. There were no intraoperative complications noted. Early postoperative complications included significant persistent corneal edema (longer than 1 week) in 4/49 (8.2%), ocular hypertension (intraocular pressure ≥25 mmHg) in 8/49 (16.3%), hypotony (intraocular pressure ≤5 mmHg) in 6/49 (12.2%), cystoid macular edema 3/21 (6.1%), IOL tilt 2/49 (4.1%), hyphema in 2/49 (4.1%), and vitreous hemorrhage in 5/49 (4.8%). There was one case of recurrent retinal detachment. One patient presented with an erosion of the Gore-Tex suture through the conjunctiva resulting in a purulent scleritis 6 months after the initial surgery, and was managed with removal of the IOL, debridement, and cryotherapy. Forty-one of 49 patients completed 3-month follow-up, among which visual acuity improved, deteriorated, or remained same compared to baseline in 27/49 (55.1%), 8/49 (16.3%), and 6/49 (12.2%) eyes, respectively.Conclusion: In the current study, visual acuity outcomes were generally favorable. The complications were largely transient. Significant complications included a suture-related infection, which required removal of the IOL, and a recurrence of a retinal detachment. Keywords: Gore-Tex, secondary intraocular lens, scleral fixation, clinical outcomes

Patel NA, Shah P, Yannuzzi NA, et al. Clin Ophthalmol. 2018;12:2145–2148. The intraocular lens (Akreos AO60) studied in this paper is made of hydrophilic acrylic material instead of hydrophobic acrylic. Therefore, the title of this paper is incorrect and should read “Clinical outcomes of 4-point scleral fixated 1-piece hydrophilic acrylic equiconvex intraocular lens using polytetrafluoroethylene suture”. The authors apologize for this error. Read the original article

The frequency and richness of the theories developed, tested, and used by researchers in an academic discipline exemplify several pertinent factors, namely, the growth, the maturity, the independence, the legitimacy, and the influence of the discipline. Although organizations have been working on projects for centuries, Project Management (PM) is a considerably new academic discipline with emerging research themes, models, methodologies, frameworks, and paradigms. These PM concepts are anchored on or reinforced by new or existing theories. This exploratory study aims to add to the existing PM body of knowledge by investigating the prevalence of theory use in PM research. A systematic content analysis of 9200 PM research articles published from 2000 to 2019 (20 years) in the leading PM journals identified 248 unique theories. These results reveal that the PM discipline is increasingly embracing the use of theories with game theory, fuzzy theory, agency theory, contingency theory, and stakeholder theory emerging as the most dominant theories in the reviewed research articles. Also, although PM is developing its theories, the results revealed that PM researchers continue to heavily use theories borrowed from other academic disciplines such as psychology, sociology, mathematics, and economics., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Prior studies have found no differences in procedural chest discomfort for patients undergoing manual syringe aspiration or drainage with gravity after thoracentesis. However, whether gravity drainage could protect against chest pain due to the larger negative-pressure gradient generated by wall suction has not been investigated., Research Question: Does wall suction drainage result in more chest discomfort compared with gravity drainage in patients undergoing large-volume thoracentesis?, Study Design and Methods: In this multicenter, single-blinded, randomized controlled trial, patients with large free-flowing effusions of ≥ 500 mL were assigned at a 1:1 ratio to wall suction or gravity drainage. Wall suction was performed with a suction system attached to the suction tubing and with vacuum pressure adjusted to full vacuum. Gravity drainage was performed with a drainage bag placed 100 cm below the catheter insertion site and connected via straight tubing. Patients rated chest discomfort on a 100-mm visual analog scale before, during, and after drainage. The primary outcome was postprocedural chest discomfort at 5 minutes. Secondary outcomes included measures of postprocedure chest discomfort, breathlessness, procedure time, volume of fluid drained, and complication rates., Results: Of the 228 patients initially randomized, 221 were included in the final analysis. The primary outcome of procedural chest discomfort did not differ significantly between the groups (P = .08), nor did the secondary outcomes of postprocedural discomfort and dyspnea. Similar volumes were drained in both groups, but the procedure duration was longer in the gravity arm by approximately 3 minutes. No differences in rate of pneumothorax or reexpansion pulmonary edema were noted between the two groups., Interpretation: Thoracentesis via wall suction and gravity drainage results in similar levels of procedural discomfort and dyspnea improvement., Clinical Trial Registry: ClinicalTrials.gov; No.: NCT05131945; URL: www., Clinicaltrials: gov., Competing Interests: Financial/Nonfinancial Disclosures The authors have reported to CHEST the following: F. M. reports conflicts of interest with Pleural Dynamics, Medtronic, and Rocket Medical. K. K. reports conflicts of interest with Astra Zeneca. L. D. reports conflicts of interest with Intuitive Surgical and Elucent Medical. None declared (S. S., J. Pannu, L. Y., A. F., C. MacR, R. B., L. D., Z. S. DeP., S. A. F., C. A. J., S. K. A., E. V., A. DeM., L. B., T. F., R. A., G. A. E., D. E. O., S. B., A. K., V. S., A. S., R. F. C., S. E. E., K. P., N. C., D. D. B., V. P., R. L., C. P., L. R., J. M., J. Z., J. L., L. V., E. O., G. Y., J. A., C. B., R.-R. A., M. S., J. Pettee, L. Z. M., M. H. A., H. B. G.)., (Copyright © 2024 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the spike protein. Although these differences in spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome that may also affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through interference with innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other nonspike mutations on SARS-CoV-2 pathogenesis, we synthesized both viruses possessing deletions in the accessory genes as well as viruses where the WA-1 spike is replaced by each variant spike gene in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to both WA-1 and the full variant viruses. Our work has revealed that the accessory proteins contribute to SARS-CoV-2 pathogenesis and the nonspike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host. This work suggests that while spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may alter clinical disease presentation.

Purpose: Head and neck cancers radiotherapy (RT) is associated with inevitable injury to parotid glands and subsequent xerostomia. We investigated the utility of SUV derived from 18 FDG-PET to develop metabolic imaging biomarkers (MIBs) of RT-related parotid injury., Methods: Data for oropharyngeal cancer (OPC) patients treated with RT at our institution between 2005 and 2015 with available planning computed tomography (CT), dose grid, pre- & first post-RT 18 FDG-PET-CT scans, and physician-reported xerostomia assessment at 3-6 months post-RT (Xero 3-6 ms) per CTCAE, was retrieved, following an IRB approval. A CT-CT deformable image co-registration followed by voxel-by-voxel resampling of pre & post-RT 18 FDG activity and dose grid were performed. Ipsilateral (Ipsi) and contralateral (contra) parotid glands were sub-segmented based on the received dose in 5 Gy increments, i.e. 0-5 Gy, 5-10 Gy sub-volumes, etc. Median and dose-weighted SUV were extracted from whole parotid volumes and sub-volumes on pre- & post-RT PET scans, using in-house code that runs on MATLAB. Wilcoxon signed-rank and Kruskal-Wallis tests were used to test differences pre- and post-RT., Results: 432 parotid glands, belonging to 108 OPC patients treated with RT, were sub-segmented & analyzed. Xero 3-6 ms was reported as: non-severe (78.7%) and severe (21.3%). SUV- median values were significantly reduced post-RT, irrespective of laterality (p = 0.02). A similar pattern was observed in parotid sub-volumes, especially ipsi parotid gland sub-volumes receiving doses 10-50 Gy (p < 0.05). Kruskal-Wallis test showed a significantly higher mean RT dose in the contra parotid in the patients with more severe Xero 3-6mo (p = 0.03). Multiple logistic regression showed a combined clinical-dosimetric-metabolic imaging model could predict the severity of Xero 3-6mo; AUC = 0.78 (95%CI: 0.66-0.85; p < 0.0001)., Conclusion: We sought to quantify pre- and post-RT 18 FDG-PET metrics of parotid glands in patients with OPC. Temporal dynamics of PET-derived metrics can potentially serve as MIBs of RT-related xerostomia in concert with clinical and dosimetric variables., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Objective: The purpose of this study was to estimate prevalence/severity of self-reported trismus, determine association with quality of life (QOL), and examine clinical risk factors in a large population of patients treated for oropharyngeal cancer., Materials and Methods: A cross-sectional survivorship survey was conducted among patients who completed definitive treatment for oropharyngeal carcinoma, disease-free ≥ 1-year post-treatment (median survival, 7 years among 892 survivors). Associations between trismus and QOL were also analyzed using MDASI-HN, EQ-5D, and MDADI. Dietary and feeding tube status were also correlated to trismus status., Results: Trismus was self-reported in 31%. Severity of trismus positively correlated (r = 0.29) with higher mean interference scores reflecting a moderate association with quality of life (p < 0.0001). There was a negative correlation for MDADI composite scores (r = - 0.33) indicating increased perceived dysphagia related to trismus severity (p < 0.0001). EQ-5D VAS scores were also negatively correlated with trismus severity (r = - 0.26, p < 0.0001). Larger T-stage (p ≤ 0.001), larger nodal stage (p = 0.03), tumor sub-site (p = 0.05), and concurrent chemoradiation (p = 0.01) associated with increased prevalence of trismus. Diet negatively correlated (r = - 0.27) with trismus severity (p = < 0.0001), and survivors with severe trismus were also more likely to be feeding tube-dependent., Conclusion: Severity of trismus appears to negatively impact quality of life and associate with various adverse functional outcomes in long-term oropharyngeal cancer survivorship. Trismus remains associated with advanced disease stages, tumor sub-site (tonsil), and addition of chemotherapy. Further investigation is merited for the dose-effect relationship to the muscles of mastication.

Objectives: Evaluate the use of induction chemotherapy (IC) in oropharyngeal cancer (OPC) and its impact on subjective functional outcomes using a validated MD Anderson Symptom Inventory-Head and Neck (MDASI-HN) survey tool., Methods: A single institution retrospective review of OPC patients who received IC, including reasons given for using IC, regimens employed, responses, and patient-reported outcomes (PRO). The latter included pain, distress, dysphagia, xerostomia, and feeding tube placement and dependency. PRO's were assessed using the validated MD Anderson Symptom Inventory-Head and Neck (MDASI-HN) conducted at baseline, during treatment, and at six-month follow up., Results: One hundred and twenty-five patients were evaluable. They were more likely to have large primary and/or bulky or low neck nodal disease as a reason for IC. A taxane-containing regimen was most common. Primary tumor response was seen in 83.2% and the nodal response in 81.6%. Pain and xerostomia improved with IC, dysphagia was not adversely affected with IC. These symptoms all increased with consolidation chemoradiotherapy (CRT) but returned to baseline by 6 months post treatment. Feeding tube placement did not increase with IC but did with CRT, most patients were no longer feeding tube dependent at 6 months., Conclusion: This retrospective review of subjective functional outcomes, especially swallowing and feeding tube dependency, using the MDASI survey tool in 125 oropharyngeal cancer patients with large primary tumors and/or bulky adenopathy treated predominantly with platinum-taxane based induction chemotherapy showed that such outcomes were not adversely impacted. While not standard, such approach may be beneficial in such patients., Level of Evidence: 2., Competing Interests: There are no financial disclosures or conflicts of interest., (© 2020 The Authors. Laryngoscope Investigative Otolaryngology published by Wiley Periodicals LLC on behalf of The Triological Society.)

Importance: A major goal of primary transoral robotic surgery (TORS) for oropharyngeal cancer is to optimize swallowing outcomes by personalized treatment based on pathologic staging. However, swallowing outcomes after TORS are uncertain, as are the outcomes compared with nonsurgical options., Objectives: To estimate rates of acute dysphagia and recovery after TORS and to compare swallowing outcomes by primary treatment modality (TORS or radiotherapy)., Design, Setting, and Participants: This case series study was a secondary analysis of prospective registry data from 257 patients enrolled from March 1, 2015, to February 28, 2018, at a single academic institution who, according to the AJCC Staging Manual, 7th edition TNM classification, had low- to intermediate-risk human papillomavirus-related oropharyngeal squamous cell carcinoma possibly resectable by TORS., Exposure: Patients were stratified by primary treatment (75 underwent TORS and 182 received radiotherapy)., Main Outcomes and Measures: Modified barium swallow (MBS) studies graded per Dynamic Imaging Grade of Swallowing Toxicity (DIGEST) and the MD Anderson Symptom Inventory-Head and Neck Module (MDASI-HN) questionnaires were administered at standard intervals. Prevalence and severity of dysphagia were estimated per DIGEST before and after TORS and 3 to 6 months after treatment. Moderate-severe dysphagia (DIGEST grade ≥2) was assessed using logistic regression and compared by primary treatment group. The MDASI swallowing symptom severity item scores during and after radiotherapy were compared using generalized estimating equations by treatment status at the start of radiotherapy, after induction, and after TORS., Results: A total of 257 patients (mean [SD] age, 59.54 [9.07] years; 222 [86.4%] male) were included in the study. Dysphagia severity (per DIGEST) was significantly worse after TORS (r = -0.63; 95% CI, -0.78 to -0.44): 17 patients (22.7%; 95% CI, 13.8%-33.8%) had moderate-severe (DIGEST grade ≥2) acute post-TORS dysphagia significantly associated with primary tumor volume (odds ratio, 1.43; 95% CI, 1.11-1.84). DIGEST improved by 3 to 6 months but remained worse than that at baseline; at 3 to 6 months, the number of patients with DIGEST grade 2 or higher dysphagia was 5 (6.7%; 95% CI, 2.2%-14.9%) after primary TORS and 29 (15.9%; 95% CI, 10.9%-22.1%) after radiotherapy. At the start of radiotherapy, MDASI swallowing symptom severity item scores were significantly worse in the post-TORS group compared with postinduction (mean [SD] change, 2.6 [1.1]) and treatment-naive (mean [SD] change, 1.7 [0.3]) patients. This result inverted at radiotherapy end, and all groups converged at 3 to 6 months., Conclusions and Relevance: Subacute swallowing outcomes were similar regardless of primary treatment modality among patients with low- to intermediate-risk oropharyngeal squamous cell carcinoma.

Background: Oropharyngeal cancer survivors experience difficulty returning to work after treatment. To better understand specific barriers to returning to work, we investigated factors associated with discontinuing employment among older and working-age survivors., Methods: The sample included 675 oropharyngeal cancer survivors (median: 6 years posttreatment) diagnosed from 2000 to 2013 and employed at diagnosis. Relative risk models were constructed to examine the independent associations of demographic and health factors, and symptom experiences per the MD Anderson Symptom Inventory - Head and Neck Module (MDASI-HN) with posttreatment employment, overall and by age (<60 years vs ≥60 years at survey)., Results: Symptom interference was not statistically significantly associated with posttreatment employment status among respondents ≥60 years. Among working-age respondents <60 years, symptom interference was strongly associated with posttreatment employment., Conclusions: Efforts to assess and lessen symptom burden in working-age survivors should be evaluated as approaches to support regaining core functions needed for continued employment., (© 2019 Wiley Periodicals, Inc.)

Background: Two patient-reported outcomes (PROs) of swallowing and their correlation to quality of life (QOL) were compared in long-term survivors of oropharyngeal cancer (OPC)., Methods: Scores on the single dysphagia item from the 28-item, multisymptom MD Anderson Symptom Inventory-Head and Neck (MDASI-HN-S) were compared with scores on the dysphagia-specific composite MD Anderson Dysphagia Inventory (MDADI) and the EuroQol visual analog scale (EQ-VAS) in 714 patients who had received definitive radiotherapy ≥12 months before the survey. An MDASI-HN-S score ≥6 and an MDADI composite score <60 were considered representative of moderate/severe swallowing dysfunction., Results: Moderate/severe dysphagia was reported by 17% and 16% of respondents on the MDASI-HN-S and the composite MDADI, respectively. Both swallow PROs were predictive of QOL, and the MDASI-HN-S model was slightly more parsimonious for the discrimination of EQ-VAS scores compared with MDADI scores (Bayesian information criteria, 6062 vs 6076, respectively). An MDASI-HN-S cutoff score of ≥6 correlated best with a declining EQ-VAS score (P < .0001) and was associated with increased radiotherapy dose to several normal swallowing structures., Conclusions: In this cohort, the single-item MDASI-HN-S performed favorably for the discrimination of QOL compared with the multi-item MDADI. A time-efficient model for PRO measurement of swallowing is proposed in which the MDADI may be reserved for patients who score ≥6 on the MDASI-HN-S., (© 2019 American Cancer Society.)

Purpose: To explore treatment-related changes in symptom burden and quality of life (QOL) in oropharyngeal squamous cell cancer (OPSCC) patients treated surgically and non-surgically., Patients and Methods: Eighty-six patients with human papillomavirus-associated OPSCC treated at the Head and Neck Center at The University of Texas MD Anderson Cancer Center were recruited to a prospective registry study between 2014 and 2016 and completed the core, head and neck-specific, and symptom interference sections of the MD Anderson symptom inventory (MDASI) multi-symptom questionnaire and the EQ-5D health status assessment as a measure of QOL at four time points., Results: Longitudinal improvements from post-treatment nadir were observed across all groups. For patients treated with single modality, symptom interference, but not core and head and neck specific, MDASI scores were significantly better at 6 months in patients treated with surgery than radiation (P = 0.04). For patients treated with multiple modalities, scores for each of the three domains (i.e., core, head and neck -specific, and interference MDASI) were significantly better in the surgical group than the nonsurgical group at treatment completion (P = 0.0003, P = 0.0006 and P = 0.02) and 6 weeks (P = 0.001, P = 0.05 and P = 0.04), but not 6 months (P = 0.11, P = 0.16 and P = 0.040). No significant differences in EQ5D health status were observed between groups at any time point, reflecting similar overall QOL in all groups., Conclusion: Symptom burden and QOL improves after treatment in OPSCC survivors over time regardless of whether primary surgical or nonsurgical treatment is used, although acute symptom profiles may differ., (Copyright © 2019. Published by Elsevier Ltd.)

Purpose: Our primary aim was to prospectively validate retrospective dose-response models of chronic radiation-associated dysphagia (RAD) after intensity modulated radiotherapy (IMRT) for oropharyngeal cancer (OPC). The secondary aim was to validate a grade ≥2 cut-point of the published videofluoroscopic dysphagia severity (Dynamic Imaging Grade for Swallowing Toxicity, DIGEST) as radiation dose-dependent., Material and Methods: Ninety-seven patients enrolled on an IRB-approved prospective registry protocol with stage I-IV OPC underwent pre- and 3-6 month post-RT videofluoroscopy. Dose-volume histograms (DVH) for swallowing regions of interest (ROI) were calculated. Dysphagia severity was graded per DIGEST criteria (dichotomized with grade ≥2 as moderate/severe RAD). Recursive partitioning analysis (RPA) and Bayesian Information Criteria (BIC) were used to identify dose-volume effects associated with moderate/severe RAD., Results: 31% developed moderate/severe RAD (i.e. DIGEST grade ≥2) at 3-6 months after RT. RPA found DVH-derived dosimetric parameters of geniohyoid/mylohyoid (GHM), superior pharyngeal constrictor (SPC), and supraglottic region were associated with DIGEST grade ≥2 RAD. V61 ≥ 18.57% of GHM demonstrated optimal model performance for prediction of DIGEST grade ≥2., Conclusion: The findings from this prospective longitudinal registry validate prior observations that dose to submental musculature predicts for increased burden of dysphagia after oropharyngeal IMRT. Findings also support dichotomization of DIGEST grade ≥2 as a dose-dependent split for use as an endpoint in trials or predictive dose-response analysis of videofluoroscopy results., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Although Langerhans cell histiocytosis (LCH) is a familiar entity to most radiologists and to pediatric radiologists in particular, it is but one of a group of disorders caused by the overproduction of histiocytes, a subtype of white blood cells. Other less familiar diseases in this category are Erdheim-Chester disease (ECD), juvenile xanthogranuloma (JXG), Rosai-Dorfman disease (RDD), and hemophagocytic lymphohistiocytosis (HLH). This review describes the classification system, clinical manifestations, and pathophysiology of each disease, with particular attention to differential radiographic findings, including typical locations of involvement and varying appearances at radiography, computed tomography, magnetic resonance imaging, ultrasonography, and nuclear medicine imaging. Although LCH has a wide variety of manifestations and appearances, classic imaging findings include vertebra plana, skull lesions with a beveled edge, the "floating tooth" sign, bizarre lung cysts, and an absent posterior pituitary bright spot with infundibular thickening. The classic imaging findings of ECD are a perirenal rind of soft tissue and patchy long bone osteosclerosis. RDD has more nonspecific imaging findings, including lymphadenopathy (most commonly cervical) and intracranial lesions. Imaging findings in HLH are broad, with the most common abnormalities being hepatosplenomegaly, cerebral volume loss, and periventricular white matter abnormalities. JXG can manifest at imaging, but radiology does not play a major role in diagnosis. Familiarity with these disorders and their associated imaging findings facilitates correct and timely diagnosis. Imaging also features prominently in the assessment of treatment response., (©RSNA, 2014.)

Toward achieving rapid and large scale genome modification directly in a target organism, we have developed a new genome engineering strategy that uses a combination of bioinformatics aided design, large synthetic DNA and site-specific recombinases. Using Cre recombinase we swapped a target 126-kb segment of the Escherichia coli genome with a 72-kb synthetic DNA cassette, thereby effectively eliminating over 54 kb of genomic DNA from three non-contiguous regions in a single recombination event. We observed complete replacement of the native sequence with the modified synthetic sequence through the action of the Cre recombinase and no competition from homologous recombination. Because of the versatility and high-efficiency of the Cre-lox system, this method can be used in any organism where this system is functional as well as adapted to use with other highly precise genome engineering systems. Compared to present-day iterative approaches in genome engineering, we anticipate this method will greatly speed up the creation of reduced, modularized and optimized genomes through the integration of deletion analyses data, transcriptomics, synthetic biology and site-specific recombination., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Canaliculitis is an uncommon, often misdiagnosed diagnosis because canaliculitis can mimic many other common ocular conditions. Canaliculitis should be appropriately diagnosed and treated to avoid recurrent inflammation and possible obstruction of the upper portion of the lacrimal system. This review will serve as a concise resource to aid in diagnosis and provide updated management options.

During the 2009 H1N1 influenza pandemic, vaccines for the virus became available in large quantities only after human infections peaked. To accelerate vaccine availability for future pandemics, we developed a synthetic approach that very rapidly generated vaccine viruses from sequence data. Beginning with hemagglutinin (HA) and neuraminidase (NA) gene sequences, we combined an enzymatic, cell-free gene assembly technique with enzymatic error correction to allow rapid, accurate gene synthesis. We then used these synthetic HA and NA genes to transfect Madin-Darby canine kidney (MDCK) cells that were qualified for vaccine manufacture with viral RNA expression constructs encoding HA and NA and plasmid DNAs encoding viral backbone genes. Viruses for use in vaccines were rescued from these MDCK cells. We performed this rescue with improved vaccine virus backbones, increasing the yield of the essential vaccine antigen, HA. Generation of synthetic vaccine seeds, together with more efficient vaccine release assays, would accelerate responses to influenza pandemics through a system of instantaneous electronic data exchange followed by real-time, geographically dispersed vaccine production.

We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.

We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.

LMX1B is a LIM-homeodomain transcription factor required for the normal development of dorsal limb structures, the glomerular basement membrane, the anterior segment of the eye, and dopaminergic and serotonergic neurons. Heterozygous loss-of-function mutations in LMX1B cause nail patella syndrome (NPS). To further understand LMX1B gene regulation and to identify pathogenic mutations within the coding region, a detailed analysis of LMX1B gene structure was undertaken. 5' -RACE and primer extension identified a long 5' -untranslated region of 1.3 kb that contains two upstream open-reading frames (uORFs). Transient transfection assays showed that sequences required for basal promoter activity extend no further than 112 bp upstream. An additional 47 mutations have been identified in the coding region, as well as nine deletions of large portions of the gene, but not in the promoter or highly conserved intronic sequences. The range of mutations and the identification of uORFs suggest further complexity in the regulation of LMX1B expression.

The high degree of similarity between the mouse and human genomes is demonstrated through analysis of the sequence of mouse chromosome 16 (Mmu 16), which was obtained as part of a whole-genome shotgun assembly of the mouse genome. The mouse genome is about 10% smaller than the human genome, owing to a lower repetitive DNA content. Comparison of the structure and protein-coding potential of Mmu 16 with that of the homologous segments of the human genome identifies regions of conserved synteny with human chromosomes (Hsa) 3, 8, 12, 16, 21, and 22. Gene content and order are highly conserved between Mmu 16 and the syntenic blocks of the human genome. Of the 731 predicted genes on Mmu 16, 509 align with orthologs on the corresponding portions of the human genome, 44 are likely paralogous to these genes, and 164 genes have homologs elsewhere in the human genome; there are 14 genes for which we could find no human counterpart.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

Immunoreactive follicle-stimulating hormone (IR-FSH) is detected in sections of formalin-fixed and paraffin-embedded gastric mucosal tissue of normal men, using the immunoperoxidase staining technique and specific antisera to hFSH (NIDDK, NIH). Positive staining for IR-FSH was detected in the parietal cells lining the gastric glands of the intermediate zone. The staining was intracytoplasmic and distributed throughout the cytoplasm. IR-FSH was also found to be present in the basal part of the foveolar epithelium. Stromal tissue and nuclei were devoid of the stain. The zymogen cells in the deeper region of the mucosa did not show any detectable staining for IR-FSH. The presence of IR-FSH in gastric mucosa was also detected by radioimmunoassay. Gel chromatography of the gastric tissue extract showed a single peak of FSH immunoreactivity that coeluted with the 125I-labeled highly purified FSH preparation (NIDDK, NIH). Furthermore, the FSH in the pituitary tissue extract had a chromatographic profile similar to that of IR-FSH from gastric tissue, and 125I-FSH labeled highly purified FSH, indicating a close resemblance in their molecular sizes. These results demonstrate that IR-FSH is present in the normal human gastric mucosa. The role of this regulatory petpide in gastric tissue, if any, needs to be investigated.

Using indirect immuno-peroxidase staining technique, localization of immunoreactive follicle-stimulating hormone (IR-FSH) is demonstrated in the cytoplasm of the epithelial cells of normal human stomach. In view of their triangular shape and central nucleus and their predominance in the intermediate glands of the gastric mucosa, these cells are identified as parietal cells. The stromal tissue is devoid of staining reaction.

Inhibin biosynthesis by human prostatic tissue was investigated in vitro. Serum levels of inhibin as well as tissue concentrations in different cells and zones of the normal and benign hyperplastic prostates were determined. Immunocytochemical localization of inhibin identified the involvement of epithelial but not stromal cells in the synthesis and release of prostatic inhibin into the circulation. The endocrine and paracrine functions of prostatic inhibin remain to be elucidated.