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1.  Dicer expression is essential for adult midbrain dopaminergic neuron maintenance and survival 
The type III RNAse, Dicer, is responsible for the processing of microRNA (miRNA) precursors into functional miRNA molecules, non-coding RNAs that bind to and target messenger RNAs for repression. Dicer expression is essential for mouse midbrain development and dopaminergic (DAergic) neuron maintenance and survival during the early post-natal period. However, the role of Dicer in adult mouse DAergic neuron maintenance and survival is unknown. To bridge this gap in knowledge, we selectively knocked-down Dicer expression in individual DAergic midbrain areas, including the ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) via viral-mediated expression of Cre in adult floxed Dicer knock-in mice (Dicerflox/flox). Bilateral Dicer loss in the VTA resulted in progressive hyperactivity that was significantly reduced by the dopamine agonist, amphetamine. In contrast, decreased Dicer expression in the SNpc did not affect locomotor activity but did induce motor-learning impairment on an accelerating rotarod. Knock-down of Dicer in both midbrain regions of adult Dicerflox/flox mice resulted in preferential, progressive loss of DAergic neurons likely explaining motor behavior phenotypes. In addition, knock-down of Dicer in midbrain areas triggered neuronal death via apoptosis. Together, these data indicate that Dicer expression and, as a consequence, miRNA function, is essential for DAergic neuronal maintenance and survival in adult midbrain DAergic neuron brain areas.
doi:10.1016/j.mcn.2013.10.009
PMCID: PMC3944994  PMID: 24184162
2.  Activation of GABAergic neurons in the interpeduncular nucleus triggers physical nicotine withdrawal symptoms 
Current biology : CB  2013;23(23):10.1016/j.cub.2013.09.041.
Summary
Background
Chronic exposure to nicotine elicits physical dependence in smokers, yet the mechanism and neuroanatomical bases for withdrawal symptoms are unclear. As in humans, rodents undergo physical withdrawal symptoms after cessation from chronic nicotine characterized by increased scratching, head nods, and body shakes.
Results
Here we show that induction of physical nicotine withdrawal symptoms activates GABAergic neurons within the interpeduncular nucleus (IPN). Optical activation of IPN GABAergic neurons via light stimulation of channel rhodopsin elicited physical withdrawal symptoms in both nicotine-naïve and chronic nicotine-exposed mice. Dampening excitability of GABAergic neurons during nicotine withdrawal through IPN-selective infusion of an NMDA receptor antagonist or through blocking IPN neurotransmission from the medial habenula reduced IPN neuronal activation and alleviated withdrawal symptoms. During chronic nicotine exposure, nicotinic acetylcholine receptors containing the β4 subunit were upregulated in somatostatin interneurons clustered in the dorsal region of the IPN. Blockade of these receptors induced withdrawal signs more dramatically in nicotine-dependent compared to nicotine-naïve mice and activated non-somatostatin neurons in the IPN.
Conclusions
Together, our data indicate that therapeutic strategies to reduce IPN GABAergic neuron excitability during nicotine withdrawal, for example, by activating nicotinic receptors on somatostatin interneurons, may be beneficial for alleviating withdrawal symptoms and facilitating smoking cessation.
doi:10.1016/j.cub.2013.09.041
PMCID: PMC3855889  PMID: 24239118
3.  Endoscopic Endonasal Approach to the Infraorbital Nerve with Nasolacrimal Duct Preservation 
Objectives Infraorbital nerve (ION) decompression, excision to remove intrinsic tumors, and resection with oncological margins in malignancies with perineural invasion or dissemination are usually accomplished with an open approach. The objective is to describe the surgical anatomy, technique, and indications of the endonasal endoscopic approach (EEA) to the ION with nasolacrimal duct preservation.
Design Eleven sides of formalin-fixed specimens were dissected. An anterior maxillary antrostomy was performed. The length of the ION prominence within the sinus and anatomic features of the covering bone were studied. A 45-degree endoscope visualized the infraorbital prominence endonasally. An angled dissector and dural blade allowed for dissection and resection of the ION ipsilaterally and contralaterally.
Results The bone features of the ION prominence allowed for ipsilateral dissection in 10 out of 11 sides. In one case with the ION surrounded by thick cortical bone, the dissection could only be started by drilling contralaterally. The 45-degree endoscope visualized 92.2% and 100% of the length of the nerve using the ipsilateral and contralateral nostrils, respectively. Ipsilaterally, 83% of its length was resected, and 96.3% was resected contralaterally.
Conclusion The ION can be approached using an ipsilateral EEA with nasolacrimal duct preservation in most cases. The contralateral approach provides a wider angle to access the ION. This technique is primarily indicated in cases where the EEA can be used for tumor resection and oncological margins within the ION.
doi:10.1055/s-0033-1347372
PMCID: PMC3836806  PMID: 24436942
infraorbital nerve; endoscopic endonasal approach; maxillary sinus; infraorbital nerve decompression; Caldwell-Luc
4.  The Extended Nasoseptal Flap for Skull Base Reconstruction of the Clival Region: An Anatomical and Radiological Study 
Objective Reconstruction of large clival defects after an endoscopic endonasal procedure is challenging. The objective is to analyze the morphology, indications, and limitations of the extended nasoseptal flap, which adds the nasal floor and inferior meatus mucosa, compared with the standard nasoseptal flap, for clival reconstruction.
Design Twenty-seven sides of formalin-fixed anatomical specimens and 13 computed tomography (CT) scans were used. Under 0-degree endoscopic visualization, a standard flap on one side and an extended flap on the other side were performed, as well as exposure of the sella, cavernous sinus, and clival dura mater. Coverage of both flaps was assessed, and they were incised and extracted for measurements.
Results The extended flap has two parts: septal and inferior meatal. The extended flaps are 20 mm longer and add 774 mm2 of mucosal area. They cover a clival defect from tuberculum to foramen magnum in 66.6% cases and from below the sella in 91.6%. They cover both parasellar and paraclival segments of the internal carotid arteries. The lateral inferior limits are the medial aspect of the hypoglossal canals and Eustachian tubes. CT scans can predict the need or limitation of an extended nasoseptal flap.
Conclusions The nasal floor and inferior meatus mucosa adds a significant area for reconstruction of the clivus. A defect laterally beyond the hypoglossal canals is not likely covered with this variation of the flap. Preoperative CT scans are useful to guide the reconstruction techniques.
doi:10.1055/s-0033-1347368
PMCID: PMC3836807  PMID: 24436940
cerebrospinal fluid leak; clival reconstruction; endoscopic endonasal; nasoseptal flap; skull base
5.  Robust Identification of Noncoding RNA from Transcriptomes Requires Phylogenetically-Informed Sampling 
PLoS Computational Biology  2014;10(10):e1003907.
Noncoding RNAs are integral to a wide range of biological processes, including translation, gene regulation, host-pathogen interactions and environmental sensing. While genomics is now a mature field, our capacity to identify noncoding RNA elements in bacterial and archaeal genomes is hampered by the difficulty of de novo identification. The emergence of new technologies for characterizing transcriptome outputs, notably RNA-seq, are improving noncoding RNA identification and expression quantification. However, a major challenge is to robustly distinguish functional outputs from transcriptional noise. To establish whether annotation of existing transcriptome data has effectively captured all functional outputs, we analysed over 400 publicly available RNA-seq datasets spanning 37 different Archaea and Bacteria. Using comparative tools, we identify close to a thousand highly-expressed candidate noncoding RNAs. However, our analyses reveal that capacity to identify noncoding RNA outputs is strongly dependent on phylogenetic sampling. Surprisingly, and in stark contrast to protein-coding genes, the phylogenetic window for effective use of comparative methods is perversely narrow: aggregating public datasets only produced one phylogenetic cluster where these tools could be used to robustly separate unannotated noncoding RNAs from a null hypothesis of transcriptional noise. Our results show that for the full potential of transcriptomics data to be realized, a change in experimental design is paramount: effective transcriptomics requires phylogeny-aware sampling.
Author Summary
We have analysed more than 400 public transcriptomes, generated using RNA-seq, from almost 40 strains of Bacteria and Archaea. We discovered that the capacity to identify noncoding RNA outputs from this data is strongly dependent on phylogenetic sampling. Our results show that, for the full potential of transcriptomics data as a discovery tool to be realized, a change in experimental design is critical: effective comparative transcriptomics requires phylogeny-aware sampling. We also examined how comparative transcriptomics experiments can be used to effectively identify RNA elements. We find that, for RNA element discovery, a phylogeny-informed sampling approach is more effective than analyses of individual species. Phylogeny-informed sampling reveals a narrow ‘Goldilocks Zone’ (where species are not too similar and not too divergent) for RNA identification using clusters of related species. In stark contrast to protein-coding genes, not only is the phylogenetic window for the effective use of comparative methods for noncoding RNA identification perversely narrow, but few existing datasets sit within this Goldilocks Zone: by aggregating public datasets, we were only able to create one phylogenetic cluster where comparative tools could be used to confidently separate unannotated noncoding RNAs from transcriptional noise.
doi:10.1371/journal.pcbi.1003907
PMCID: PMC4214555  PMID: 25357249
6.  Transposition of the Pterygopalatine Fossa during Endoscopic Endonasal Transpterygoid Approaches 
Introduction Complete or partial removal of the pterygoid process provides lateral extension of the endonasal corridor necessary to approach the Meckel cave, infrapetrous skull base, and medial infratemporal fossa. This paper provides the anatomical foundations for the endoscopic endonasal transpterygoid approach with preservation of all neurovascular structures inside the pterygopalatine fossa.
Methods Eight endoscopic transpterygoid approaches were performed in fresh cadaveric specimens. In all dissections the vidian nerve and the periosteal sac enclosing the pterygopalatine fossa were preserved.
Results We reliably transposed the pterygopalatine fossa to approach the Meckel cave, infrapetrous skull base, and medial infratemporal region, preserving the neurovascular structures inside the pterygopalatine fossa in all specimens.
Conclusions The transposition of the pterygopalatine fossa neurovascular structures for endoscopic endonasal approaches to the skull base is an alternative technique that is both feasible and desirable. The transposition requires no additional technical skills but requires comprehensive knowledge of its anatomy. The anatomical preservation of the neurovascular structures is potentially beneficial to the quality of life of patients. Clinical studies are necessary to prove the real benefits of this technique.
doi:10.1055/s-0033-1347367
PMCID: PMC3774828  PMID: 24436922
endoscopy; endonasal; cranial base; pterygopalatine fossa; transpterygoid approaches
7.  Chicken Wing Training Model for Endoscopic Microsurgery 
Objectives To present and validate a chicken wing model for endoscopic endonasal microsurgical skill development.
Setting A surgical environment was constructed using a Styrofoam box and measurements from radiological studies. Endoscopic visualization and instrumentation were utilized in a manner to mimic operative setting.
Design Five participants were instructed to complete four sequential tasks: (1) opening the skin, (2) exposing the main artery in its neurovascular sheath, (3) opening the neurovascular sheath, and (4) separating the nerve from the artery. Time to completion of each task was recorded.
Participants Three junior attendings, one senior resident, and one medical student were recruited internally.
Main Outcome Measures Time to perform the surgical tasks measured in seconds.
Results The average time of the first training session was 48.8 minutes; by the 10th training session, the average time was 22.4 minutes. The range of improvement was 25.7 minutes to 72.4 minutes. All five participants exhibited statistically significant decrease in time after 10 trials. Kaplan-Meier analysis revealed that an improvement of 50% was achieved by an average of five attempts at the 95% confidence interval.
Conclusions The ex vivo chicken wing model is an inexpensive and relatively realistic model to train endoscopic dissection using microsurgical techniques.
doi:10.1055/s-0033-1348026
PMCID: PMC3774829  PMID: 24436926
chicken wing dissection; endoscopic surgical skills; training model
8.  Molecular characterization of a new member of the lariat capping twin-ribozyme introns 
Mobile DNA  2014;5:25.
Background
Twin-ribozyme introns represent a complex class of mobile group I introns that harbour a lariat capping (LC) ribozyme and a homing endonuclease gene embedded in a conventional self-splicing group I ribozyme (GIR2). Twin-ribozyme introns have so far been confined to nucleolar DNA in Naegleria amoeboflagellates and the myxomycete Didymium iridis.
Results
We characterize structural organization, catalytic properties and molecular evolution of a new twin-ribozyme intron in Allovahlkampfia (Heterolobosea). The intron contains two ribozyme domains with different functions in ribosomal RNA splicing and homing endonuclease mRNA maturation. We found Allovahlkampfia GIR2 to be a typical group IC1 splicing ribozyme responsible for addition of the exogenous guanosine cofactor (exoG), exon ligation and circularization of intron RNA. The Allovahlkampfia LC ribozyme, by contrast, represents an efficient self-cleaving ribozyme that generates a small 2′,5′ lariat cap at the 5′ end of the homing endonuclease mRNA, and thus contributes to intron mobility.
Conclusions
The discovery of a twin-ribozyme intron in a member of Heterolobosea expands the distribution pattern of LC ribozymes. We identify a putative regulatory RNA element (AP2.1) in the Allovahlkampfia LC ribozyme that involves homing endonuclease mRNA coding sequences as an important structural component.
doi:10.1186/1759-8753-5-25
PMCID: PMC4167309  PMID: 25342998
Allovahlkampfia; circular RNA; Heterolobosea; intron splicing; LC ribozyme; Naegleria; RNA structure
9.  A 23-Year-Old Female with a Mixed Germ Cell Tumor of the Pituitary Infundibulum: The Challenge of Differentiating Neoplasm from Lymphocytic Infundibuloneurohypophysitis—A Case Report and Literature Review 
Case Reports in Endocrinology  2014;2014:129471.
The pathologic spectrum of diseases that infiltrate the pituitary infundibulum includes a broad variety of clinical entities. There are significant differences in the prevalence of these etiologies depending on the age of presentation. Lymphocytic infundibuloneurohypophysitis (LINH) predominates over other causes of infundibular disease in adults over age 21. Differentiating LINH from other causes of infundibular disease can be difficult because the various etiologies often have similar clinical presentations and radiologic imaging characteristics. We report the first case in an adult of a mixed germ cell tumor comprised of germinoma and embryonal cell carcinoma infiltrating the pituitary infundibulum. In our case, a 23-year-old female was initially misdiagnosed as having LINH. She presented with panhypopituitarism and diabetes insipidus, which is the most common initial presentation in both entities. The two diagnoses are difficult to distinguish based on MRI imaging, CSF findings, and histopathological examination. Our case demonstrates the need for close follow-up of patients with isolated lesions of the pituitary infundibulum and reinforces the need for biopsy of an infundibular lesion when progression of disease is demonstrated. In our case, biopsy with comprehensive immunohistochemical staining was the sole means of making a definitive diagnosis.
doi:10.1155/2014/129471
PMCID: PMC4087301  PMID: 25045548
10.  From Smoking to Lung Cancer: The CHRNA5/A3/B4 Connection 
Oncogene  2010;29(35):4874-4884.
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that modulate key physiological processes ranging from neurotransmission to cancer signaling. These receptors are activated by the neurotransmitter, acetylcholine, and the tobacco alkaloid, nicotine. Recently, the gene cluster encoding the α3, α5 and β4 nAChR subunits received heightened interest after a succession of linkage analyses and association studies identified multiple single nucleotide polymorphisms (SNPs) in these genes that are associated with an increased risk for nicotine dependence and lung cancer. It is not clear whether the risk for lung cancer is direct or an effect of nicotine dependence, as evidence for both scenarios exist. Here, we summarize the body of work implicating nAChRs in the pathogenesis of lung cancer, with special focus on the clustered nAChR subunits and their emerging role in this disease state.
doi:10.1038/onc.2010.256
PMCID: PMC3934347  PMID: 20581870
Nicotine Addiction; Lung Cancer; Nicotinic Acetylcholine Receptors; CHRNA5/A3/B4 gene cluster
11.  Rare Infundibular Tumors: Clinical Presentation, Imaging Findings, and the Role of Endoscopic Endonasal Surgery in Their Management 
Background The spectrum of infundibular lesions is broad and distinct from sellar pathologies. In many cases, histology is needed to establish the correct diagnosis and determine the treatment approach.
Methods Medical files of eight patients with distinct infundibular tumors were reviewed. Histopathologically confirmed diagnosis included three pituicytomas, three granular cell tumors, and two pilocytic astrocytomas.
Results Patients shared similar imaging findings and clinical symptoms, including visual impairment (n = 5), hypopituitarism (n = 4), and headache (n = 4); one patient presented with disseminated disease and symptoms from spinal metastases. All the pituicytomas, two granular cell tumors, and one infundibular pilocytic astrocytoma case underwent endoscopic endonasal surgery; gross total resection was achieved in five patients, three developed postoperative diabetes insipidus, and two developed hypopituitarism. No recurrences were observed. One granular cell tumor patient was treated with gamma-knife radiosurgery after stereotactic biopsy; the tumor remained stable in size for over 9 years. The infundibular pilocytic astrocytoma patient who presented with spinal metastases received radiotherapy and systemic chemotherapy. The overall mean follow-up period was 25.1 months.
Conclusion Infundibular tumors are rare entities that represent a diagnostic challenge. Histopathological examination is essential for definitive diagnosis. Surgery, radiation therapy, and chemotherapy all have a role in the management of these tumors.
doi:10.1055/s-0032-1329619
PMCID: PMC3699169  PMID: 24436883
endoscopic endonasal approach; granular cell tumor; pilocytic astrocytoma; pituicytoma; radiation therapy
12.  Intraoperative Computed Tomography Guidance to Confirm Decompression Following Endoscopic Endonasal Approach for Cervicomedullary Compression 
Introduction Cervicomedullary compression often requires an anterior approach to address the compressive vector. In certain cases an endoscopic endonasal approach (EEA) is ideal for decompression. It is essential that an adequate decompression be achieved and verified before the patient leaves the operating room. The purpose of this study was to evaluate the use intraoperative computed tomography (IO-CT) in assessing the adequacy of decompression.
Methods A retrospective chart review revealed 11 cases of EEA odontoid resection IO-CT verification of decompression. Operative reports and review of imaging was used to determine if further decompression was performed following the intraoperative scan.
Results Out of 11 EEA cases, 4 (36%) patients showed evidence of residual compression following an initial IO-CT. Further operative decompression was undertaken following the first scan in all cases. A second intraoperative scan was then used to confirm complete decompression. No patient left the operating room with residual compression.
Discussion IO-CT provided valuable utility in 36% of the cases after the initial resection was incomplete. The standard fluoroscopic guidance may not provide adequate resolution and enhanced utility like IO-CT.
doi:10.1055/s-0032-1329627
PMCID: PMC3699170  PMID: 24436887
endoscopy; endonasal; odontoid; rheumatoid disease
13.  Conserved Chromosome 2q31 Conformations Are Associated with Transcriptional Regulation of GAD1 GABA Synthesis Enzyme and Altered in Prefrontal Cortex of Subjects with Schizophrenia 
The Journal of Neuroscience  2013;33(29):11839-11851.
Little is known about chromosomal loopings involving proximal promoter and distal enhancer elements regulating GABAergic gene expression, including changes in schizophrenia and other psychiatric conditions linked to altered inhibition. Here, we map in human chromosome 2q31 the 3D configuration of 200 kb of linear sequence encompassing the GAD1 GABA synthesis enzyme gene locus, and we describe a loop formation involving the GAD1 transcription start site and intergenic noncoding DNA elements facilitating reporter gene expression. The GAD1-TSS-50kbLoop was enriched with nucleosomes epigenetically decorated with the transcriptional mark, histone H3 trimethylated at lysine 4, and was weak or absent in skin fibroblasts and pluripotent stem cells compared with neuronal cultures differentiated from them. In the prefrontal cortex of subjects with schizophrenia, GAD1-TSS-50kbLoop was decreased compared with controls, in conjunction with downregulated GAD1 expression. We generated transgenic mice expressing Gad2 promoter-driven green fluorescent protein-conjugated histone H2B and confirmed that Gad1-TSS-55kbLoop, the murine homolog to GAD1-TSS-50kbLoop, is a chromosomal conformation specific for GABAergic neurons. In primary neuronal culture, Gad1-TSS-55kbLoop and Gad1 expression became upregulated when neuronal activity was increased. We conclude that 3D genome architectures, including chromosomal loopings for promoter-enhancer interactions involved in the regulation of GABAergic gene expression, are conserved between the rodent and primate brain, and subject to developmental and activity-dependent regulation, and disordered in some cases with schizophrenia. More broadly, the findings presented here draw a connection between noncoding DNA, spatial genome architecture, and neuronal plasticity in development and disease.
doi:10.1523/JNEUROSCI.1252-13.2013
PMCID: PMC3713726  PMID: 23864674
14.  Endoscopic Endonasal Pituitary Surgery: Impact of Surgical Education on Operation Length and Patient Morbidity 
Objectives To determine the difference in operative times and associated complications for cases performed solely by attending-level surgeons versus cases assisted by surgeons-in-training for endoscopic endonasal pituitary surgeries.
Design Retrospective chart review.
Setting Tertiary-care academic medical center.
Participants A total of 228 patients having undergone endoscopic endonasal pituitary surgery from 2005 to 2011.
Main Outcome Measure Duration of surgery comparing attending only (AO) and trainee-assisted (TA) surgeries.
Results Thirty-seven (19%) of 198 cases were identified as AO surgeries, the remaining 161 (81%) were TA. Operative times (minutes) for the AO group were significantly shorter than the TA group (149.1 ± 54.8 vs 219.5 ± 83.7, p < 0.001). The AO group had fewer intraoperative cerebrospinal fluid leaks (30% vs 39%, p = 0.318), decreased estimated blood loss (408 mL vs 523 mL, p = 0.176), fewer postoperative complications (27% vs 37%, p = 0.268), and shorter length of stay (3.5 vs 4.3 days, p = 0.294).
Conclusions This is the first study in otolaryngology or neurosurgery to compare operative times and outcomes for AO versus TA cases at a single academic institution. Operative times were significantly decreased and a trend toward a decrease in patient morbidity was noted for cases performed solely by attendings. The valuation of teaching activities in the operating room is a necessary first step toward optimizing the allocation of resources and funding of surgical education.
doi:10.1055/s-0032-1329620
PMCID: PMC3578591  PMID: 24294558
skull base surgery; pituitary surgery; residency training; surgical education; endoscopic endonasal approach
15.  Nicotinic acetylcholine receptors mediate lung cancer growth 
Ion channels modulate ion flux across cell membranes, activate signal transduction pathways, and influence cellular transport—vital biological functions that are inexorably linked to cellular processes that go awry during carcinogenesis. Indeed, deregulation of ion channel function has been implicated in cancer-related phenomena such as unrestrained cell proliferation and apoptotic evasion. As the prototype for ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs) have been extensively studied in the context of neuronal cells but accumulating evidence also indicate a role for nAChRs in carcinogenesis. Recently, variants in the nAChR genes CHRNA3, CHRNA5, and CHRNB4 have been implicated in nicotine dependence and lung cancer susceptibility. Here, we silenced the expression of these three genes to investigate their function in lung cancer. We show that these genes are necessary for the viability of small cell lung carcinomas (SCLC), the most aggressive type of lung cancer. Furthermore, we show that nicotine promotes SCLC cell viability whereas an α3β4-selective antagonist, α-conotoxin AuIB, inhibits it. Our findings posit a mechanism whereby signaling via α3/α5/β4-containing nAChRs promotes lung carcinogenesis.
doi:10.3389/fphys.2013.00251
PMCID: PMC3774984  PMID: 24062692
nicotinic acetylcholine receptor; ligand-gated ion channel; lung cancer; small cell lung carcinoma; CHRNA5
16.  Genome and Transcriptome Adaptation Accompanying Emergence of the Definitive Type 2 Host-Restricted Salmonella enterica Serovar Typhimurium Pathovar 
mBio  2013;4(5):e00565-13.
ABSTRACT
Salmonella enterica serovar Typhimurium definitive type 2 (DT2) is host restricted to Columba livia (rock or feral pigeon) but is also closely related to S. Typhimurium isolates that circulate in livestock and cause a zoonosis characterized by gastroenteritis in humans. DT2 isolates formed a distinct phylogenetic cluster within S. Typhimurium based on whole-genome-sequence polymorphisms. Comparative genome analysis of DT2 94-213 and S. Typhimurium SL1344, DT104, and D23580 identified few differences in gene content with the exception of variations within prophages. However, DT2 94-213 harbored 22 pseudogenes that were intact in other closely related S. Typhimurium strains. We report a novel in silico approach to identify single amino acid substitutions in proteins that have a high probability of a functional impact. One polymorphism identified using this method, a single-residue deletion in the Tar protein, abrogated chemotaxis to aspartate in vitro. DT2 94-213 also exhibited an altered transcriptional profile in response to culture at 42°C compared to that of SL1344. Such differentially regulated genes included a number involved in flagellum biosynthesis and motility.
IMPORTANCE
Whereas Salmonella enterica serovar Typhimurium can infect a wide range of animal species, some variants within this serovar exhibit a more limited host range and altered disease potential. Phylogenetic analysis based on whole-genome sequences can identify lineages associated with specific virulence traits, including host adaptation. This study represents one of the first to link pathogen-specific genetic signatures, including coding capacity, genome degradation, and transcriptional responses to host adaptation within a Salmonella serovar. We performed comparative genome analysis of reference and pigeon-adapted definitive type 2 (DT2) S. Typhimurium isolates alongside phenotypic and transcriptome analyses, to identify genetic signatures linked to host adaptation within the DT2 lineage.
doi:10.1128/mBio.00565-13
PMCID: PMC3760250  PMID: 23982073
17.  Value of Free-Run Electromyographic Monitoring of Lower Cranial Nerves in Endoscopic Endonasal Approach to Skull Base Surgeries 
Objective The main objective of this study was to evaluate the value of free-run electromyography (f-EMG) monitoring of cranial nerves (CNs) VII, IX, X, XI, and XII in skull base surgeries performed using endoscopic endonasal approach (EEA) to reduce iatrogenic CN deficits.
Design We retrospectively identified 73 patients out of 990 patients who had EEA in our institution who had at least one CN monitored. In each CN group, we classified patients who had significant (SG) f-EMG activity as group I and those who did not as group II.
Results We monitored a total of 342 CNs. A total of 62 nerves had SG f-EMG activity including CN VII = 18, CN IX = 16, CN X = 13, CN XI = 5, and CN XII = 10. No nerve deficit was found in the nerves that had significant activity during procedure. A total of five nerve deficits including (CN IX = 1, CN X = 2, CN XII = 2) were observed in the group that did not display SG f-EMG activity during surgery.
Conclusions f-EMG seems highly sensitive to surgical manipulations and in locating CNs. It seems to have limited value in predicting postoperative neurological deficits. Future studies to evaluate the EMG of lower CNs during EEA procedures need to be done with both f-EMG and triggered EMG.
doi:10.1055/s-0032-1312712
PMCID: PMC3424031  PMID: 23904999
electromyography; lower cranial nerves; endoscopic endonasal approach; skull base surgery
19.  Value of Free-Run Electromyographic Monitoring of Extraocular Cranial Nerves during Expanded Endonasal Surgery (EES) of the Skull Base 
Objective To evaluate the value of free-run electromyography (f-EMG) monitoring of extraocular cranial nerves (EOCN) III, IV, and VI during expanded endonasal surgery (EES) of the skull base in reducing iatrogenic cranial nerve (CN) deficits.
Design We retrospectively identified 200 patients out of 990 who had at least one EOCN monitored during EES. We further separated patients into groups according to the specific CN monitored. In each CN group, we classified patients who had significant (SG) f-EMG activity as Group I and those who did not as Group II.
Results A total of 696 EOCNs were monitored. The number of muscles supplied by EOCNs that had SG f-EMG activity was 88, including CN III = 46, CN IV = 21, and CN VI = 21. There were two deficits involving CN VI in patients who had SG f-EMG activity during surgery. There were 14 deficits observed, including CN III = 3, CN IV = 2, and CN VI = 9 in patients who did not have SG f-EMG activity during surgery.
Conclusions f-EMG monitoring of EOCN during EES can be useful in identifying the location of the nerve. It seems to have limited value in predicting postoperative neurological deficits. Future studies to evaluate the EMG of EOCN during EES need to be done with both f-EMG and triggered EMG.
doi:10.1055/s-0033-1346975
PMCID: PMC3713562  PMID: 23943720
electromyography; lower cranial nerves; expanded endonasal surgery
20.  A comparison of dense transposon insertion libraries in the Salmonella serovars Typhi and Typhimurium 
Nucleic Acids Research  2013;41(8):4549-4564.
Salmonella Typhi and Typhimurium diverged only ∼50 000 years ago, yet have very different host ranges and pathogenicity. Despite the availability of multiple whole-genome sequences, the genetic differences that have driven these changes in phenotype are only beginning to be understood. In this study, we use transposon-directed insertion-site sequencing to probe differences in gene requirements for competitive growth in rich media between these two closely related serovars. We identify a conserved core of 281 genes that are required for growth in both serovars, 228 of which are essential in Escherichia coli. We are able to identify active prophage elements through the requirement for their repressors. We also find distinct differences in requirements for genes involved in cell surface structure biogenesis and iron utilization. Finally, we demonstrate that transposon-directed insertion-site sequencing is not only applicable to the protein-coding content of the cell but also has sufficient resolution to generate hypotheses regarding the functions of non-coding RNAs (ncRNAs) as well. We are able to assign probable functions to a number of cis-regulatory ncRNA elements, as well as to infer likely differences in trans-acting ncRNA regulatory networks.
doi:10.1093/nar/gkt148
PMCID: PMC3632133  PMID: 23470992
21.  Letter from the Editor 
RNA Biology  2013;10(1):1.
doi:10.4161/rna.23502
PMCID: PMC3590227  PMID: 23392241
22.  Pseudomeningoceles of the sphenoid sinus masquerading as sinus pathology 
The Laryngoscope  2011;121(12):2507-2513.
Objective
To describe the clinical presentation, pathophysiology and treatment of spontaneous cerebrospinal fluid (CSF) leaks of the sphenoid bone, with an emphasis on a previously undescribed form in this location, in which CSF is trapped under the mucosa of the sinonasal cavity or in the soft tissue of the skull base.
Study design
Case series and literature review. Level of evidence 4.
Methods
Analysis of cases through medical records and literature review.
Results
Four examples of unusual spontaneous CSF leaks of the skull base are presented. In each case, a CSF collection was contained behind the sinonasal mucosa of the sphenoid sinus, resembling a nasal polyp or mucocele on exam or imaging. In one case, the fluid collection was also associated with significant bone resorption and extravasation into the soft tissue of the infratemporal fossa. In each case, small defects of the ventral skull base (sphenoid bone) were the source of the CSF leaks. Successful treatment was achieved after transnasal endoscopic repair of the skull base defects using a combination of free abdominal fat grafts, free fascial grafts and pedicled nasoseptal flaps. Postoperatively, a ventriculoperitoneal shunt was placed if the intracranial pressure was elevated.
Conclusions
Spontaneous CSF leaks arising in the sphenoid sinus may not always present with overt CSF rhinorrhea but with a submucosal fluid collection (pseudomeningocele) that may mimic a mucocoele or nasal polyp. These bona fide pseudomeningoceles of the skull base may be associated with elevated intracranial pressure and can be managed using endoscopic endonasal surgery.
doi:10.1002/lary.21876
PMCID: PMC3223265  PMID: 22052361
CSF leak; mucocele; meningocele; skull base; sinus
23.  Rfam 11.0: 10 years of RNA families 
Nucleic Acids Research  2012;41(Database issue):D226-D232.
The Rfam database (available via the website at http://rfam.sanger.ac.uk and through our mirror at http://rfam.janelia.org) is a collection of non-coding RNA families, primarily RNAs with a conserved RNA secondary structure, including both RNA genes and mRNA cis-regulatory elements. Each family is represented by a multiple sequence alignment, predicted secondary structure and covariance model. Here we discuss updates to the database in the latest release, Rfam 11.0, including the introduction of genome-based alignments for large families, the introduction of the Rfam Biomart as well as other user interface improvements. Rfam is available under the Creative Commons Zero license.
doi:10.1093/nar/gks1005
PMCID: PMC3531072  PMID: 23125362
24.  A VapBC Toxin-Antitoxin Module Is a Posttranscriptional Regulator of Metabolic Flux in Mycobacteria 
Journal of Bacteriology  2012;194(9):2189-2204.
The largest family of toxin-antitoxin (TA) modules are encoded by the vapBC operons, but their roles in bacterial physiology remain enigmatic. Microarray analysis in Mycobacterium smegmatis overexpressing VapC/VapBC revealed a high percentage of downregulated genes with annotated roles in carbon transport and metabolism, suggesting that VapC was targeting specific metabolic mRNA transcripts. To validate this hypothesis, purified VapC was used to identify the RNA cleavage site in vitro. VapC had RNase activity that was sequence specific, cleaving single-stranded RNA substrates at AUAU and AUAA in vitro and in vivo (viz., MSMEG_2121 to MSMEG_2124). A bioinformatic analysis of these regions suggested that an RNA hairpin 3′ of the AUA(U/A) motif is also required for efficient cleavage. VapC-mediated regulation in vivo was demonstrated by showing that MSMEG_2124 (dhaF) and MSMEG_2121 (dhaM) were upregulated in a ΔvapBC mutant growing on glycerol. The ΔvapBC mutant had a specific rate of glycerol consumption that was 2.4-fold higher than that of the wild type during exponential growth. This increased rate of glycerol consumption was not used for generating bacterial biomass, suggesting that metabolism by the ΔvapBC mutant was uncoupled from growth. These data suggest a model in which VapC regulates the rate of glycerol utilization to match the anabolic demands of the cell, allowing for fine-tuning of the catabolic rate at a posttranscriptional level.
doi:10.1128/JB.06790-11
PMCID: PMC3347065  PMID: 22366418
25.  Comparative Analysis of RNA Families Reveals Distinct Repertoires for Each Domain of Life 
PLoS Computational Biology  2012;8(11):e1002752.
The RNA world hypothesis, that RNA genomes and catalysts preceded DNA genomes and genetically-encoded protein catalysts, has been central to models for the early evolution of life on Earth. A key part of such models is continuity between the earliest stages in the evolution of life and the RNA repertoires of extant lineages. Some assessments seem consistent with a diverse RNA world, yet direct continuity between modern RNAs and an RNA world has not been demonstrated for the majority of RNA families, and, anecdotally, many RNA functions appear restricted in their distribution. Despite much discussion of the possible antiquity of RNA families, no systematic analyses of RNA family distribution have been performed. To chart the broad evolutionary history of known RNA families, we performed comparative genomic analysis of over 3 million RNA annotations spanning 1446 families from the Rfam 10 database. We report that 99% of known RNA families are restricted to a single domain of life, revealing discrete repertoires for each domain. For the 1% of RNA families/clans present in more than one domain, over half show evidence of horizontal gene transfer (HGT), and the rest show a vertical trace, indicating the presence of a complex protein synthesis machinery in the Last Universal Common Ancestor (LUCA) and consistent with the evolutionary history of the most ancient protein-coding genes. However, with limited interdomain transfer and few RNA families exhibiting demonstrable antiquity as predicted under RNA world continuity, our results indicate that the majority of modern cellular RNA repertoires have primarily evolved in a domain-specific manner.
Author Summary
In cells, DNA carries recipes for making proteins, and proteins perform chemical reactions, including replication of DNA. This interdependency raises questions for early evolution, since one molecule seemingly cannot exist without the other. A resolution to this problem is the RNA world, where RNA is postulated to have been both genetic material and primary catalyst. While artificially selected catalytic RNAs strengthen the chemical plausibility of an RNA world, a biological prediction is that some RNAs should date back to this period. In this study, we ask to what degree RNAs in extant organisms trace back to the common ancestor of cellular life. Using the Rfam RNA families database, we systematically screened genomes spanning the three domains of life (Archaea, Bacteria, Eukarya) for RNA genes, and examined how far back in evolution known RNA families can be traced. We find that 99% of RNA families are restricted to a single domain. Limited conservation within domains implies ongoing emergence of RNA functions during evolution. Of the remaining 1%, half show evidence of horizontal transfer (movement of genes between organisms), and half show an evolutionary history consistent with an RNA world. The oldest RNAs are primarily associated with protein synthesis and export.
doi:10.1371/journal.pcbi.1002752
PMCID: PMC3486863  PMID: 23133357

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