Leishmania spp. possess an additional copy, annotated as the putative S-adenosylmethionine decarboxylase-like proenzyme (ADL). Phylogenetic analysis confirms that ADL is unique to trypanosomatids and has several unique features such as lack of autocatalytic cleavage and a distinct evolutionary lineage, even from trypanosomatid ADs. In Trypanosoma ADL was found to be enzymaticaly dead but plays an essential regulatory role by forming a heterodimer complex with AD. However, no structural or functional information is available about ADL from Leishmania spp. Here, in this study, we report the cloning, expression, purification, structural and functional characterization of Leishmania donovani (L. donovani) ADL using biophysical, biochemical and computational techniques. Biophysical studies show that, L. donovani ADL binds S-adenosylmethionine (SAM) and putrescine which are natural substrates of AD. Computational modeling and docking studies showed that in comparison to the ADs of other organisms including human, residues involved in putrescine binding are partially conserved while the SAM binding residues are significantly different. In silico protein-protein interaction study reveals that L. donovani ADL can interact with AD. These results indicate that L. donovani ADL posses a novel substrate binding property and may play an essential role in polyamine biosynthesis with a different mode of function from known proteins of the S-adenosylmethionine decarboxylase super family.In addition to the S-adenosylmethionine decarboxylase (AD) present in all organisms, trypanosomatids including  Leishmania. It causes an estimated 500,000 new cases of disease and more than 50,000 deaths every year with 90% occurring in Bangladesh, Nepal, India, Sudan, Ethiopia and Brazil Leishmania can be one such pathway Trypanosoma brucei (T. brucei) is eflorinithine, a suicide inhibitor of ornithine decarboxylase (ODC), an enzyme in the polyamine biosynthesis pathway Leishmania, putrescine is synthesized from L-ornithine through a decarboxylation reaction catalyzed by ODC. Subsequently, spermidine is synthesized by the incorporation of an aminopropyl group in a reaction catalyzed by the enzyme spermidine synthase. The aminopropyl group is provided by decarboxylated S-adenosylmethionine, which is produced in a reaction catalyzed by S-adenosylmethionine decarboxylase referred here as AD. Spermidine is subsequently conjugated with glutathione to synthesize a unique polyamine in trypanosomatids i.e. trypanothione, which is essential for cellular redox reactions and nucleotide synthesis Visceral Leishmaniasis or kala-azar is a one of the most neglected diseases caused by the parasitic protozoan Trypanosoma and plants T. brucei putrescine is not necessary for the autoprocessing reaction but it stimulates the decarboxylation reaction, although the decarboxylation reaction is not as efficient as found in human and plant AD AD (E.C 4.1.1.50) belongs to a small group of enzymes that depend on a pyruvoyl cofactor for decarboxylation reaction. AD is expressed as an inactive proenzyme that undergoes an autoprocessing reaction in humans, Leishmania possess another AD like protein annotated in databases as a putative S-adenosylmethionine decarboxylase-like proenzyme, here onwards referred as ADL (NCBI gene accession no. CBZ36337.1). The T. brucei ADL was shown to be paralogous to AD, did not undergo autocatalysis and could not retain its native confirmation in the absence of AD. T. brucei ADL was also found to be enzymatically dead, but playing a significant role as a regulatory subunit by forming a high affinity heterodimer with AD, upregulating the enzyme activity by ∼1200 fold T. brucei AD and ADL revealed that depletion of either protein led to a reduction in the level of spermidine and trypanothione ultimately resulting in the death of the parasite. This implies that ADL is an essential protein for the survival of TrypanosomaL. donovani AD and ADL with human AD suggest that the ADL has a significantly lower sequence identity (∼13%) than L. donovani AD (∼25%). Further, L. donovani AD shares six residues which are involved in SAM positioning and binding and two among the three putrescine binding residues with the human AD. This indicates that in comparison to AD, ADL may be a better target for any rational drug development approach. Here, in this study we report the cloning, expression, purification, structural and functional characterization of L. donovani ADL, to ascertain its viability in drug development.Apart from AD, trypanosomatids including L. major, L. infantum, L. donovani, L. brazilensis T. brucei and T. cruzi were retrieved from the Swiss-Prot gene database and domain architecture was predicted using CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) http://bip.weizmann.ac.il/fldbin/findex) http://npsapbil.ibcp.fr) http://www.modelling.leeds.ac.uk/qsitefinder/) was used to identify conserved functional residues The amino acid sequences of ADs and ADLs of kinetoplastida, such as L. donovani ADL gene was amplified from the genomic DNA of L. donovani using specific sense “5ATTGGATCCATGTCGCTGTGGGGAGGGTTTTCGAACCC3” and anti-sense “5GGGAAGCTTATCTGCACTGCGGGCGAATAGTAGTTGGTG3” primers designed using Oligo software, with sites for BamHI and HindIII restriction enzymes (underlined) respectively. The amplified gene was cloned in T/A vector pTZ57R/T  and then sub-cloned downstream of the T5 promoter expression vector pQE30 (Qiagen) between BamHI and HindIII sites. E. coli TG1 host cell was transformed with the recombinant plasmid pQE30-ADL and used for over-expression.The 894 bp long E. coli containing the pQE30-ADL plasmid and cultured overnight at 37°C. Fresh LB broth containing ampicillin (100 µg/ml) was inoculated with 1∶100 dilution of this seed culture and incubated at 37°C to an OD600 of ∼0.6. Over-expression was then induced by adding 1 mM isopropyl-1-thio-β-galactopyranoside (IPTG) and allowed to grow overnight at the same temperature. The culture was subsequently harvested by centrifugation, resuspended in a buffer containing 50 mM Tris-HCl pH 7.0 and 200 mM NaCl. Cells were lysed by sonication with a 20 second on and 15 second off pulse for 30 minutes. Cell debris was removed by centrifugation at 13,000 rpm for 30 minutes and the supernatant loaded on an IMAC column pre-equilibrated with the same buffer. The column was incubated for an hour and subsequently washed with buffers containing 10 mM and 60 mM imidazole. Protein was then eluted with 300 mM imidazole in the same buffer. The eluted protein was dialyzed overnight into 50 mM Tris-HCl pH 7.0, 50 mM NaCl, 1 mM putrescine and 3 mM β-me, concentrated using 10 kDa cutoff centricon (Amicon) and loaded on size exclusion chromatography for the second step purification and oligomerization analysis.Luria-Bertani (LB) broth containing ampicillin (100 µg/ml) was inoculated with the Size exclusion chromatography was performed on the Superdex™ 75 10/300 prepacked column (manufacturer's exclusion limit 70 kDa for proteins) on an ΔKTA-FPLC . The dialyzed and concentrated ADL protein (1 mg/ml) was loaded onto the column pre-equilibrated with a buffer containing 50 mM Tris-HCl pH 7.0, 50 mM NaCl, and 3 mM β- me. The elution was carried out isocratically at a flow rate of 0.3–0.4 ml/min and monitored using the absorbance at 280 nm. All measurements were made at 25°C. Calibration of the column was performed using the low molecular weight standard kit  containing conalbumin, ovalbumin, carbonic anhydrase, RNAase and aprotinine as reference proteins.The protein concentration was determined by using Bradford method Trypsin digestion was used to analyze the domain architecture of ADL protein. Trypsin was added to ADL in a 200∶1 ratio and reactions were set up for 10 min, 30 min, 1 hour and overnight and, stopped by adding 1 mM PMSF. The effect of substrates such as SAM and putrescine on limited proteolysis was observed in the concentration range 0–80 µM and 0–90 µM respectively in an overnight reaction. All samples were analyzed on a 12% SDS-PAGE.The fluorescence emission spectra using intrinsic (Tryptophan) and extrinsic  fluorophores were recorded on a Perkin Elmer LS50b luminescence spectrometer at 25°C. Cuvettes with 5 mm path length were used and 2 µM ADL in 50 mM Tris-HCl pH 7.0, 50 mM NaCl and 3 mM β-me was used for these studies. For tryptophan fluorescence, the protein sample was excited at 295 nm and the emission spectra recorded in the range 300–400 nm. For ANS binding studies, the corresponding values were 370 nm and 400–600 nm respectively. For ANS binding studies, a dye to protein molar ratio of 20∶1 was used and the samples were incubated with ANS for 30 minutes and gently shaken before taking measurements. The effect of urea and Guanidinium chloride (GdmCl) on ADL was observed in the concentration range 0–6 M by using tryptophan and ANS fluorescence. Fluorescence spectra with increasing concentrations of SAM and putrescine (0–200 µM) up to saturation were measured and the change in tryptophan fluorescence observed at 341 nm. As a control, titrations with buffer alone did not produce any significant change in the emission signal. The change in fluorescence can be then related to the binding of SAM and putrescine by the following equation max is the difference between the observed fluorescence intensities at zero and saturating substrate concentration, [Substrate] tot, and Kf is the apparent dissociation constant. The Kd values were determined from a non-linear least-squares regression analysis of titration data. With all samples, fluorescence spectra were corrected for the background fluorescence of the solution (buffer+substrate). Deconvolution of curves was performed using the Prism software (GraphPad software Inc) & Phase diagrams describing GdmCl and urea induced changes of fluorescence intensities were constructed.Where ΔF is the magnitude of the difference between the observed fluorescence intensities in the presence and absence of the substrate at a given concentration of substrate, ΔFhttp://www.ogic.ca/projects/k2d3/) was used to estimate the secondary structure content of the protein L donovani ADL, in apo and in complex with SAM and putrescine were performed in the same spectropolarimeter using the standard protocol between 25°C–90°C. The folded fraction of the protein at these temperature values were determined The far-UV CD measurements were made on a Jasco J810 spectropolarimeter and Chirascan™ CD spectropolarimeter (Applied Photophysics) calibrated with ammonium (+)-10- camphorsulfonate. Three spectra  from 2 µM protein samples in 50 mM Tris– HCl pH 7.0, 50 mM NaCl were taken and averaged. All measurements were taken using standard protocol http://www.sbg.bio.ic.ac.uk/~phyre/) which too takes the amino acid sequence as input and combines predicted secondary structure information in addition to PSI-BLAST generated alignment profile L. donovani ADL) and the template as input L. major, L. infantum, L. donovani and L. brazilensis were also made to confirm the interactions involved in heterodimer formation.In the absence of suitable template hits by PSI-BLAST against the PDB, the templates for homology modeling were found by searching structures with similar fold, using the PHYRE server  http://cluspro.bu.edu/login.php) and GRAMM-X (http://vakser.bioinformatics.ku.edu/resources/gramm/grammx) The protein-protein interaction was analyzed by STRING 9.0 , and interestingly, none of these residues are conserved in L. donovani ADL. In fact, a majority of them are chemically different in L. donovani ADL- Phenylalanines 7 and 223 correspond to Asp 15 and 192 in ADL, the stretch of residues corresponding to human Glu 247 is deleted in and at position 65, L. donovani ADL has a methionine instead of a leucine. Residues involved in putrescine binding, though are partially conserved in L. donovani ADL as well. In summary, the residues involved in autocatalysis and SAM positioning and binding are entirely different which suggests that the L. donovani ADL might function in a novel manner. To further explore and understand the role of L. donovani ADL, the protein was cloned, over-expressed, purified and structurally and functionally characterized.Structure-based sequence analysis of the SAM and putrescine bound crystal structure of human AD (PDB ID 1I7B) L. donovani ADL gene was cloned, over-expressed and purified using IMAC and was observed on SDS-PAGE as a single band corresponding to 33 kDa, indicating the absence of any autocatalytic cleavage reaction  in the buffer at every step of purification or keeping protein concentration below 0.2 mg/ml decreased the precipitation, suggesting the stabilizing effect of putrescine.The reaction , consistreaction . L. donoL. donovani ADL shows that it elutes at 10.4 ml on the Superdex™ 75 10/300 column, corresponding to ∼66 kDa, suggesting L. donovani ADL is a dimer  and is in agreement with the secondary structure compositions predicted by the PHD server (http://npsapbil.ibcp.fr)  .Leishmania has a digenetic lifecycle alternating between a promastigote stage in sandfly and an amastigote stage present in human macrophage between which it faces a variation in temperature (25°C in sand fly and 37°C in human macrophage), we analyzed the stability of L. donovani ADL over a range of temperature. The thermal denaturation studies show L. donovani ADL is stable up to 53°C, beyond which it begins to unfold. The Tm of protein was found to be ∼70°C and shows a sigmoidal curve of unfolding  µM. However, no digestion was seen  which had the minimum E value was used as the template for homology modeling using Modeller 9.10 Leishmania spp. and Trypanosoma spp were also generated using similar protocol. The output models were validated using standard tools and the models having minimum internal energy, with 92.7% residues in the favored region of the Ramachandran plot and an r.m.s.d value less than 2 Å was selected for further rationalization of protein ligand interaction and protein-protein interaction studiesTo understand the structural rationale of protein ligand interactions, attempts were made to crystallize the protein, both in its L. donovani ADL model is representative of the core architecture of proteins belonging to the SAM-decarboxylase superfamily, consisting of four layer αββα sandwich architecture with the β-sheets comprising of seven and eight β-strands arranged in antiparallel fashion  which is involved in interaction with the SAM in human AD. Closer examination of the L. donovani ADL docked with SAM with the crystal structure of human AD reveals the differences in the ligand binding  L. donovani ADL, due to the short length of β-strand and completely different orientation of proceeding loop as compared to the human AD. The putrescine binding site of human AD interacts with the residues from both β-sheets while in L. donovani ADL putrescine is closer to one β-sheet and interacts with the residues on this sheet only. The putrescine binding pocket of L. donovani is comprised of similar Glu and Asp residues as found in the putrescine bound crystal structure of human AD, the only difference being the presence of Leu in place of Thr . Further, ClusPro 2.0 and GRAMM-X shows positive docking result for L. donovani as well as T. brucei AD-ADL pair. From these results, it is reasonable to expect that these two proteins do interact as a heterodimer for its function, as in Trypanosoma. Analysis of the docked structures shows that the interaction is stabilized by salt bridges between Lys96, Arg124, Asp173, Lys206 and Arg216 of AD with Asp253, Glu106, Arg21, Asp25 and Asp25 of L. donovani ADL (L. donovani AD (data not shown). Preliminary interaction analysis using pull-down assays suggest that t that L. donovani AD indeed form a heterodimer complex with ADL , superimposed with structure of human AD (pink), shows that L. donovani ADL also have same structural organization αββα as in case of human AD, but ADL has one β-strand missing due to short length as shown in figure. Figure is made with the help of Chimera 1.6.1.(TIF)Click here for additional data file.Figure S2Unfolding studies of ADL in presence of urea. The changes in tertiary structure were monitored by fluorescence studies using tryptophan as intrinsic fluorophore and ANS as extrinsic fluorophore. (A–B) Effect of increasing concentration of urea on tryptophan fluorescence was monitored at tryptophan emission maxima 341 nm, shows increasing concentration of urea causes gradual red shift of ADL due to unfolding of protein with complete unfolding at 6 M urea. (C–D) ANS fluorescence emission spectra with increasing concentration of urea, monitored at 465 nm. Graph shows emission maxima increases with increase in concentration of urea up to 0.5 M urea, then gradually decreased with minima at 6 M urea, due to loss of hydrophobic patches.(TIF)Click here for additional data file.Figure S3Unfolding studies of L. donovani ADL in presence of GdmCl. The changes in tertiary structure were monitored by fluorescence studies using tryptophan as intrinsic fluorophore and ANS as extrinsic fluorophore. (A–B) Effect of GdmCl on tryptophan fluorescence of L. donovani ADL monitored at 341 nm emission maxima. Graph shows increased concentration of GdmCl causes unfolding of protein with maximum transition at 1.5 M GdmCl and protein gets fully exposed at 2 M concentration. (C–D) ANS fluorescence emission spectra with increasing concentration of GdmCl monitored at 465 nm shows emission maxima increases with increasing concentration of GdmCl up to 0.5 M GdmCl, and then gradually decreased to a minimum value at 4 M GdmCl.(TIF)Click here for additional data file.Figure S4AD-ADL interaction observed from GST pull down assay. The cell lysate containing AD-GST construct (in Tris-HCl pH 7.5 and 150 mM NaCL) was incubated with glutathione agarose for two hours and the unbound cell lysate discarded and washed with buffer containing Tris-HCl pH 7.5and 1M NaCl, before incubating with cell lysate containing 6×His-ADL for 2 hours, washed with 5 column volumes of the same buffer and eluted with reduced glutathione. A similar experiment using GST alone instead of AD-GST was also performed as control. The elution products were analyzed on 12% SDS PAGE (top panel): Lane 1 marker, Lane 2 elution of GST with ADL, Lane 3: elution of AD-GST with ADL and Lane 4: AD alone. The band at ∼60 kDa corresponds to full length AD-GST while the band at ∼35 kDa corresponds to the autocatalyzed N-terminal fragment of AD (9 kDa) fused to 26 kDa GST (Mr 35 kDa) as well as ADL (Mr 33 kDa). To resolve this, the eluted products were then probed with anti-GST (middle panel) and anti-His (bottom panel) antibodies which show the presence of the two species. The absence of a band corresponding to ADL in Lane 2 in the anti-His blot confirms specific AD: ADL interaction.(TIF)Click here for additional data file.


Mutation of the voltage-gated potassium channel KCNQ4 causes DFNA2-type nonsyndromic autosomal dominant sensorineural hearing loss. KCNQ4 is expressed predominantly in the auditory sensory outer hair cells, which are critical for sound amplification.KCNQ4 from Japanese patients with sensorineural hearing loss, and identified a novel missense mutation encoding a Tyr270His located at the N-terminus of the highly conserved pore helix sequence. As this patient was not accessible to us and information about them was limited, we used molecular modeling to investigate whether this novel mutation is hypothetically pathogenic. A careful examination of an in silico structural model of the KCNQ4 pore region revealed that the Tyr270His mutation caused an alteration in the electrostatic surface potential of the pore helix.We sequenced + through the channel by electrostatic repulsion.We propose two possible means by which the Tyr270His mutation causes hearing loss: a positively charged His270 side chain might enhance the helix dipole moment of the pore helix, thereby destabilizing the helix and/or the pore region, or it might disturb transport of K In particular, autosomal dominant nonsyndromic sensorineural deafness type 2 (DFNA2) hearing loss affects the ability of children to hear high frequencies, and results in hearing loss at all frequencies later in life [KCNQ4 is the causative gene [Deafness is a frequently inherited sensory disorder. For every 1000 newborns, more than 1% have bilateral sensorineural hearing loss (SNHL), and 50-70% of the cases are monogenic disorders . Heredit in life . KCNQ4 i+ channel) channels present in ear and epithelia [KCNQ4 mutations show progressive hearing loss, development of drugs that improve KCNQ4 function should attenuate hearing loss.KCNQ4 is a member of the five muscarinic receptor-regulated and voltage-gated potassium  induced hearing loss, we built models of the KCNQ4 and Tyr270His pore regions. By examining models that included electrostatic potentials, we were able to propose two molecular mechanisms that could have caused the patient's hearing loss.While conducting an ongoing genetic study of hereditary hearing loss, we identified a mutated KCNQ4 were reported to be associated with hearing loss [KCNQ4 exons 5, 6, and 7 (which encode the pore region) of Japanese patients with bilateral nonsyndromic hearing loss. A new heterozygotically presented KCNQ4 missense mutation was found at position 808, in which the wild-type thymine (T) was replaced by cytosine (C). This results in a Tyr270His mutation in the protein  DNA samples were not obtained from her family.The patient who carried the Tyr270His mutation was a 28-year-old female with congenital, progressive hearing loss. An audiometric examination found mild hearing loss in her left ear and moderate hearing loss in her right ear, with steeply sloping audiometric configurations in both ears  channels, KCNQ4 probably contains six transmembrane α-helices, (S1-S6), a pore helix (PH), a pore-loop (P-loop), a short N-terminal region, and a long C-terminal region  are somewhat conserved, suggesting that the pore residues, including Tyr270, are necessary for the proper functioning of the channel  for Histidine. Moreover, His270 is surrounded by the negatively charged residues Asp272, Asp266, Asp262, Glu260, and the polar residues Ser269, Ser268, and Ser265, which are capable of hydrogen bonding , the side-chain pKa of His97, which is located in the loop domain between helix3 and helix4 and surrounded by negatively charged residues of Asp94 (located contiguous to the C-terminus of helix 3) and Asp99 (N-terminus of helix 4) within 5 Å distance, is shifted to an abnormally high value (pKa = 7.8) [pK value that is greater than the standard value, and, therefore, it was predicted that more than half of the histidine side chains at position 270 carry a positive charge near physiological pH  . For theH Figure .The alteration of the electrostatic surface potential of a single helical residue in the pore region might affect the structural stability of the channel as a consequence of an alteration in the helix dipole moment. Substantial dipole moments with positive and negative unit charges at the N- and C-terminals, respectively, of α-helices are often found . Compari+ transport. The electrostatic repulsion between a positively charged His270 and K+ would be stronger than that between the electrically neutral Tyr270 and K+. In the Tyr270His model, the distance between His270 and the center pore of the channel was ~20 Å  because the electrostatic force is strong and affects charges separated by 500-1800 Å [+ might possibly impede K+ passage through the channel [+ would be smaller in the extracellular region.Alternatively, or in conjunction with the destabilization effect, His270 may impede KÅ Figure . This di0-1800 Å -28. Addi0-1800 Å . The ele channel , wherease.g., Leu274His and Trp276Ser) in the KCNQ4 PH have been correlated with hearing loss [+ transport, or both, caused hearing loss in the patient, and provide insight into how to reverse hearing loss caused by KCNQ4 mutants.Several mutations . Before sequencing the pore regions of the patients' KCNQ4, their mitochondrial DNA sequences (m.1555A > G or m.3243A > G) and GJB2 were determined to be normal . EacKCNQ4 exons 5, 6, and 7, the primer sets 5'-GAGATGGGGGACCTTTATCC-3' and 5'-AGCCCTACAAAGACCCTCAC-3'; 5'-GACCAGTCCTGCCTGTAACC-3' and 5'-AACTGAGCAGGAGGCAACTC-3'; and 5'-ACCCTTGCAGCCTCTTACTG-3' and 5'-CTGCTCCTAGGGCTTCTTCC-3; respectively, were used in conjunction with AmpliTaq Gold DNA polymerase  and a PC-818 Program Temp Control System . The PCR program was: 95°C for 4 min; 30 three-step cycles of 95°C for 30 s, 59.4°C for 30 s, and 72°C for 45 s; and then 72°C for 5 min. Primer sets for all KCNQ4 exons  as the reference. Control DNA was obtained from 96 Japanese subjects with normal hearing.Genomic DNA was extracted from blood samples using the reagents of the Gentra Puregene Blood kit . For polymerase-chain-reaction (PCR) amplification of + channel with a sequence homologous to that of KCNQ4 that could be used as the structural template for the modeling exercise, we searched the Protein Data Bank using Gapped BLAST [http://www.ebi.ac.uk/pdbsum/. The transmembrane helices and the voltage sensor and pore regions of KCNQ4 and Tyr270His were then modeled using SWISS-MODEL [3LUT, chain B) as the template [To find a voltage-gated Ked BLAST  and PDBsSS-MODEL -17, withtemplate . Both KCtemplate ,19. Finatemplate  to visuaDFNA2: Nonsyndromic autosomal dominant sensorineural deafness type 2; KCNQ4: Potassium voltage-gated channel; KQT-like subfamily: Member 4; Kv: Voltage-gated potassium channel; PCR: Polymerase chain reaction; SNHL: Sensorineural hearing loss.The authors declare that they have no competing interests.KN was involved in the design of the study, performed the experiments and data analysis, and drafted the manuscript. HM was involved in the conception of the study and polished the manuscript. HK was involved in the conception of the study and gave considerations about structural information. SH provided information about the patient with sensorineural hearing loss. TM conceived the study, and participated in its coordination, design, data analysis and production of the manuscript. All authors read and approved the final manuscript.Figure S1. The quality of the models was assessed by the Verify-3D program. The vertical axis indicates the average 3D-1D score for residues in 21-residue sliding window, the centre of which is at the sequence position indicated by the horizontal axis. Initial models of KCNQ4 exhibited better quality than those of the Kv1.2 template (Cys327 to Thr716). Nearly 100% of the scores were positive and most of these were higher than 0.1. Thus, we took the predicted KCNQ4 structure to be trustworthy.Click here for fileTable S1. The primer sets for KCNQ4. The number of each primer set indicates the target exons of KCNQ4. Each primer specific for a given genomic sequence (upper case) is conjugated with either a universal forward M13 (lower case) or a reverse M13pUC (lower case).Click here for file


Background. Colorectal cancer mostly arises from the polyps of colon. The aim of our study was to examine the association of body mass index (BMI) and serum lipids with the colorectal polyps in old Chinese people. Methods. The risk of developing colorectal polyps was studied in 244 subjects  who underwent colonoscopy for the first time from January 2008 to July 2012 at the Navy General Hospital, Beijing, China. According to the results of colonoscopy, all the subjects were divided into 112 normal control, 38 right colorectal polyps, 53 left colorectal polyps, and 41 both right and left colorectal polyps groups. The total plasma cholesterol, plasma triglyceride, plasma creatinine concentration, blood urinary nitrogen, and fasting glucose were determined using a multichannel analyzer. Results. There were significant differences among normal control, right colorectal polyps, left colorectal polyps, and both right and left polyps groups, which were the BMI, total cholesterol, triglycerides, creatinine, and urinary nitrogen. In binary logistic regression analysis, there were two risk factors associated with the occurrence of colorectal polyps, which included BMI and systolic blood pressure. Conclusions. Colorectal polyps were significantly associated with increased BMI, total cholesterol, and triglycerides levels. According to a report of the World Health Organization, cancer was the leading cause of death in 2007, accounting for 7.9 million deaths, or 13% of the total amount. The same report stated that colorectal cancer was the fourth most common fatal cancer, after lung, stomach, and liver cancers –7 and ch NSAIDs ; past history of cancer, coronary artery disease, diabetes mellitus, inflammatory bowel disease, familial polyposis or thyroid disease; major gastrointestinal surgery, including partial or total gastrectomy or colectomy; colon cancer diagnosed during sigmoidoscopic examination; vegetarian patient; dieting; liver cirrhosis or SGPT levels three times higher than the normal limit; nephrotic syndrome or serum creatinine levels higher than 115 umol/L; The CEA levels higher than 10 ng/mL; incomplete examination and missing data.The subjects comprised 244 individuals who underwent a routine screening colonoscopy at the navy general hospital, Beijing, china, between January 2008 and July 2012. There were 212 men  and 32 women . For the analysis of the occurrence of colorectal polyps, we included patients with the following conditions: complete, available medical records; normal blood pressure (diastolic blood pressure of less than 90 mmHg and systolic blood pressure of less than 150 mmHg) or well-controlled hypertension (diastolic blood pressure of less than 90 mmHg and systolic blood pressure of less than 150 mmHg). The hypertensive patients were taking antihypertensive medication, and all the observed patients had taken acetylsalicylic acid (100 mg/day). Exclusion criteria were as follows: using medication for hyperlipidemia and otherHypertension was defined as a systolic blood pressure (SBP) ≧ 140 mmHg or diastolic blood pressure (DBP) ≧ 90 mmHg according to the Seventh Report of the Joint National Committee [The diagnostic criteria for coronary artery disease included the previous onset of myocardial infarction or more than 75% narrowing of the coronary artery, as determined by radiography.The protocol of the present study was reviewed and approved by the Ethics and Research Committee of the Navy General Hospital . The study population gave informed consent before blood sampling.Endoscopists were physicians who had been trained for 6 months or longer at our hospital and had carried out 500 or more colonoscopies. Each endoscopist carried out six or more colonoscopies per day. Insertion up to the cecum was successful in 96.8% of examinations. Digital examination of the rectum was routine prior to the insertion of the endoscope. In preparation for colonoscopy, subjects took a laxative  on the night before the examination and on the day of colonoscopy at 08:30 h (colonic lavage using approximately 2000 mL of solution was carried out). Small-caliber electronic colonoscopies  were used for colonoscopy. All visualized lesions were biopsied and histologically assessed by experienced pathologists.For the purpose of our analysis, colorectal polyps which arise proximal (right) or distal (left) to the splenic flexure were divided into three groups , which aThe blood samples were drawn from participating patients after an overnight fast of more than 12 h, and also they stopped smoking, drinking alcohol, tea, or coffee. The serum levels of plasma creatinine concentration, blood urinary nitrogen, total plasma cholesterol, plasma triglyceride were measured using a multichannel analyzer .The CEA and CA19-9 levels were measured by means of immunometric chemiluminescent assay kit .Anthropometric measurements were made by well-trained examiners on individuals wearing light clothing and without shoes. Height was measured to the nearest 0.1 cm and weight to the nearest 0.1 kg; body mass index (BMI) was calculated for these patients as weight (in kilograms) divided by the square of height (in meters).P < 0.05. Tests for several independent samples were used to explore the association between different categories of colorectal polyps groups and sex, hypertension, hyperplastic polyp, tubular adenoma, and tubulovillous adenoma. The one-way ANOVA was used for others appropriate factors between different categories of colorectal polyps. Binary logistic regression analysis was used to determine the factors associated with colorectal polyps in the entire population. Data were expressed as mean ± SD or counts. Statistical analysis was performed using SPSS version 16.0 , and the level of statistical significance was defined as All the patients were divided into four groups based on their colorectal polyps: normal, right colorectal polyps, left colorectal polyps, and both right and left colorectal polyps. The 244 patients were evaluated at baseline and found to be eligible for the present analysis. The demographic characteristics of these people are summarized in P < 0.05), total cholesterol (P < 0.01), triglycerides (P < 0.01), creatinine (P < 0.01) and urinary nitrogen (P < 0.05) were the significant differences among normal control, right colorectal polyps, left colorectal polyps, and both right and left polyps groups (P < 0.01); the total cholesterol of both right and left polyps group (4.92 ± 1.25) was significantly different from the normal control groups (4.01 ± 0.80) (P < 0.01), right colorectal polyps group (4.04 ± 1.02) (P < 0.01), and left colorectal polyps group (4.31 ± 0.90) (P < 0.01); the total cholesterol of left colorectal polyps group was significantly different from the normal control group (P < 0.05). The triglycerides of both right and left polyps group (1.65 ± 1.15) were significantly difference from the normal control groups (1.06 ± 0.49) (P < 0.01) and right colorectal polyps group (1.14 ± 0.59) (P < 0.05); the triglycerides of left colorectal polyps group (1.65 ± 1.48) were significantly different from the normal control groups (P < 0.01), right colorectal polyps group (P < 0.05). The fasting glucose of both right and left polyps group (6.07 ± 2.47) was significantly different from the right colorectal polyps group (5.40 ± 0.96) (P < 0.05). The creatinine of both right and left polyps group (103.15 ± 26.48) was significantly different from the right colorectal polyps group (118.68 ± 45.27) (P < 0.05); the creatinine of left colorectal polyps group (94.88 ± 21.36) was significantly different from the right colorectal polyps group (118.68 ± 45.27) (P < 0.01); the creatinine of right colorectal polyps group (118.68 ± 45.27) was significantly different from the normal control group (99.68 ± 29.38) (P < 0.01). The urinary nitrogen of left colorectal polyps group (5.69 ± 2.16) was significantly different from the right colorectal polyps group (6.98 ± 2.98); the urinary nitrogen of right colorectal polyps group was significantly different from the normal control group (6.14 ± 1.37) (P < 0.05).One-way ANOVA post hoc tests showed that the BMI (s groups . The BMIP > 0.05).The comparison of female, hyperplastic polyp, tubular adenoma, tubulovillous adenoma, and hypertension among the four groups was not a significant difference, which was analysed by tests for several independent samples (P < 0.05) and systolic blood pressure   . A number of reports have demonstrated that obesity is a risk factor for colorectal polyps (both adenomatous and hyperplastic polyps) and cancer in men –20. Our The mechanisms underlying the association between BMI and colorectal polyps and its role in predicting colorectal polyps in obesity patients are unclear. There are two possible mechanisms that might explain our findings. First, recently, insulin and insulin-like growth factors (IGF) have been suggested to play a role in colorectal carcinogenesis as the underlying mechanism for the increased incidence in obese subjects , 22. It In our study, patients with colorectal polyps were more likely to have high total cholesterol, triglycerides, fasting glucose, and urinary nitrogen levels; total cholesterol and triglycerides were associated with the site and number of colorectal polyps. One study by Tabuchi et al. performed a large scale retrospective study with 4,887 patients to analyze the correlation between the incidence of colorectal adenoma and serum levels of total cholesterol and triglyceride. Multiple logistic regression analysis with adjustment for age and gender revealed that triglyceride was an independent correlation factor in males with tubular adenoma but not with villous adenoma . Most stThe possible mechanisms of relationship between colorectal polyps and serum lipids might explain our findings. First, hypertriglyceridemia is associated with hyperinsulinemia and insulin resistance , 31, andAbout the relationship of colorectal polyps and fasting glucose levels, one possible explanation is that fasting glucose levels were related to obesity or serum lipids, so that fasting glucose levels were increased as obesity or serum lipids. This study had several limitations. There may have been a selection bias in that subjects in this study were recruited from individuals who visited the hospital for regular health examination and underwent colonoscopies; thus, they were more concerned about their health status and were of a higher socioeconomic status than the general population. Approximately 54% of these individuals had colorectal polyps, an incidence higher than that in other studies , 34. In The current study provides new evidence linking the obesity and serum lipids to colorectal polyps' etiology and shows a positive association between obesity and colorectal polyps risk as well as between total cholesterol, triglycerides, and colorectal polyps risk in old Chinese people. To consider the role of dietary fat as well as serum lipids in carcinogenesis, more detailed studies are needed to clarify the effects of both systemic and local effect of lipid constituents on gastrointestinal cancer development. Further investigations in this subject should also take into account other metabolic determinants including insulin and gut microbiota.


We investigated auditory perception and cognitive processing in individuals with chronic tinnitus or hearing loss using functional magnetic resonance imaging (fMRI). Our participants belonged to one of three groups: bilateral hearing loss and tinnitus (TIN), bilateral hearing loss without tinnitus (HL), and normal hearing without tinnitus (NH). We employed pure tones and frequency-modulated sweeps as stimuli in two tasks: passive listening and active discrimination. All subjects had normal hearing through 2 kHz and all stimuli were low-pass filtered at 2 kHz so that all participants could hear them equally well. Performance was similar among all three groups for the discrimination task. In all participants, a distributed set of brain regions including the primary and non-primary auditory cortices showed greater response for both tasks compared to rest. Comparing the groups directly, we found decreased activation in the parietal and frontal lobes in the participants with tinnitus compared to the HL group and decreased response in the frontal lobes relative to the NH group. Additionally, the HL subjects exhibited increased response in the anterior cingulate relative to the NH group. Our results suggest that a differential engagement of a putative auditory attention and short-term memory network, comprising regions in the frontal, parietal and temporal cortices and the anterior cingulate, may represent a key difference in the neural bases of chronic tinnitus accompanied by hearing loss relative to hearing loss alone. Subjective tinnitus is the phantom perception of sound in the absence of an external source. The annoyance and distress associated with tinnitus range from mild to severe, with the latter type having a major impact on a person's life, making sleep difficult and intellectual work challenging We used two tasks to investigate the neural bases of short-term memory and auditory processing in individuals with hearing loss without tinnitus, participants with hearing loss accompanied by tinnitus and normal hearing control subjects without tinnitus. Both tasks used identical non-speech stimuli; they differed only in the tasks: passive listening or active discrimination. We had previously used similar tasks and stimuli to investigate short-term memory and auditory processing in young normal-hearing adults We wanted to test differences in behavior and neural response of those with hearing loss with and without tinnitus, for sounds they could hear and discriminate well. Both of these groups would be compared to a control group of normal hearing participants without tinnitus. We ensured that all participants could hear the sounds equally well by (a) recruiting only those with either normal hearing for octave frequencies 0.25–8 kHz (control group) or normal hearing through 2 kHz  and (b) creating stimuli that included frequencies only up to 2 kHz. Our prediction was that the response of a distributed set of regions in the frontal, parietal and temporal cortices would be enhanced for those with hearing loss and would be increased further for those with hearing loss and tinnitus compared to normal hearing controls during these tasks. Such increased response would be more apparent for the discrimination task relative to the passive listening task because of the greater engagement of a distributed cortical network in the former, possibly due to attentional and short-term memory processing. The prediction was based on our previous fMRI study of young normal hearing adults using similar stimuli and a discrimination task. However, because all participants could hear the sounds and if their behavior did not differ significantly, the null hypothesis would be that there would be no appreciable difference in the response of the auditory processing network between the normal hearing and hearing impaired group without tinnitus; the group with tinnitus would differ due to the additional distracting factor of chronic tinnitus.Three groups of participants were recruited in the study from the greater Washington, D.C. metropolitan area. All participants gave written informed consent. The National Institutes of Health/National Institute of Neurological Disorders and Stroke-National Institute on Deafness and Other Communication Disorders Institutional Review Board approved the study (protocol 06-DC-0218) and all participants were suitably compensated.The tinnitus group (TIN) consisted of 8 male volunteers  with bilateral, mild to moderately-severe high-frequency sensorineural hearing loss and chronic subjective tinnitus that had persisted for between 3–38 years at the time of their scan . The tinThe second group (HL) (n = 7) was matched in age , gender and hearing loss and had bilateral, mild to moderately-severe hearing loss but did not have tinnitus. The third group (NH) (n = 11) was age  and gender-matched and had normal hearing with no tinnitus. All subjects scored in the minimal depression range on the Beck Depression Inventory (BDI-II) http://www.asha.org/public/hearing/disorders/types.htm) for 3–8 kHz. There were no statistically significant differences in the pure-tone average hearing loss  (p = 0.89 using Wilcoxon Rank Sum test) or at the higher frequencies  (p = 0.69 using Wilcoxon Rank Sum test) for the TIN and HL groups. Using the method described in All participants underwent full audiologic evaluation before and after the scanning session at the NIH Clinical Center. The audiologic examination, including speech recognition and pure-tone air- and bone-conduction thresholds (0.25–8 kHz), was conducted in a double-walled audiometric test suite using ER-3A transducers in accordance with American National Standards Institute standards , New York, NY: American National Standards Institute, 2003, and S3.1-1996 American National Standard Specification for Audiometers (Standard S3.6). New York, NY: American National Standards Institute; 2004). Additional audiometric measures, including distortion product otoacoustic emissions, tympanometry, and acoustic reflex thresholds and decay, were conducted to ensure that there were no audiometric signs of conductive or retrocochlear pathology. Loudness tolerance evaluation using recorded samples of scanner noise was also conducted to ensure that each participant's loudness discomfort levels were sufficiently high to permit scanning without loudness discomfort. We excluded potential participants who exhibited symptoms of hyperacusis, either via loudness tolerance evaluation or subjective questionnaire. All participants in the NH group had pure-tone thresholds of 25 dB HL or less for all of the test frequencies. Participants in the TIN and HL groups had pure-tone thresholds of 25 dB HL or less for 0.25–2 kHz, and sensorineural hearing loss in the mild to moderately-severe range  and 3 high frequency tones . There were two types of frequency modulated sweep stimuli: “down-up” and “up-down”. The up-down stimuli consisted of a 200 ms up sweep, a 100 ms pure tone and a 200 ms down sweep. The three segments were concatenated such that each complete stimulus was continuous and the total duration was 500 ms. For both the up-down and the down-up stimuli, the 100 ms pure tone frequency was 1 kHz. There were 3 up-down stimuli with varying frequencies. The starting frequencies of the up-down stimuli were 0.65, 0.55, 0.45 kHz. The initial 200 ms up sweep always ended at 1 kHz (the frequency of the pure tone) regardless of the starting frequency. The down sweep then started at a frequency of 1 kHz and dropped in frequency to 0.65, 0.55 and 0.45 kHz to match each beginning frequency. The down-up stimuli were identical to the up-down in duration and segmentation but consisted of a concatenated down sweep, pure tone (with frequency at 1 kHz) and up sweep. There were 3 down-up stimuli with starting and ending frequencies of 2.0, 1.8, 1.6 kHz. Each stimulus-pair (either ‘same’ or ‘different’) was presented 5 times in pseudo-random order with 10 silent (rest) trials mixed amongst the listening trials. Thus, there were 30 trials with pure tones and 30 trials with the sweeps, with equal distribution of same and different trials. The tones and sweeps were generated using Audition 2.0 . None of the stimuli overlapped the hearing loss range of the listeners who had normal hearing at frequencies less than 2 kHz. The sounds were further low-pass filtered with a cut-off point at 2 kHz and were normalized to have the same root-mean-square amplitude. Sounds were played at most comfortable level for the participants, during the ‘silent’ portion of the sparse sampling acquisition. Post-hoc measurements revealed that this was between 70–80 dB SPL.Subjects performed (a) a passive listening task (PL) where they listened to pairs of stimuli without responding and (b) a discrimination task (DT) in which they responded whether a pair of tones or a pair of sweeps was ‘same’ or ‘different’. Responses were collected via button-presses. Subjects performed a brief training session for 5–10 minutes to familiarize them with the tasks and stimuli. The training sounds were similar to but not identical to the stimuli used in the experiment. Subjects began the actual experiment once they achieved a threshold of 85% accuracy on the task.Participants were scanned in a 3 Tesla GE Excite scanner using an eight-channel receive-only coil. Subjects were scanned using an Echo Planar Imaging (EPI) sparse sampling technique shown in  so that http://www.fil.ion.ucl.ac.uk/spm/software/spm5/) was used to analyze the data. The fMRI data were pre-processed: images were realigned, co-registered to a high resolution template, normalized to MNI space, and finally smoothed with an 8-mm full-width-half-maximum filter. The smoothed data from individual subjects was entered into a fixed-effects analysis for purposes of statistical analyses at group level. A design matrix of all three groups was defined comprising contrasts testing for significant effects of various task components from each group. The contrasts were Task (either DT or PL)>Rest or Rest>Task. Because the PL and DT tasks were in separate EPI runs, they were analyzed separately. To analyze commonalities across the two hearing loss groups , we performed a conjunction analysis Statistical parametric software , hearing loss , tinnitus .As shown in All three groups, on average, showed greater response of the bilateral superior and middle temporal cortex when discriminating sounds compared to rest see . In addiConjunction analysis, which was used to identify commonalities between HL and TIN groups, revealed widespread activations in the bilateral superior temporal cortex and in the central regions of anterior cingulate and in the medial frontal gyri . There were no regions of significant difference for the passive listening task between the three groups, except participants in the NH group activated the left inferior/middle frontal gyrus to a greater extent relative to those in the hearing loss groups with and without tinnitus (TIN and HL). The patterns of response across the three groups varied more for the discrimination task compared to the passive listening task. In the discrimination task compared to rest, we found an elevated response in the frontal and parietal cortices in addition to the temporal cortex for the normal hearing and hearing impaired without tinnitus participants. This is not surprising because the discrimination task is a short-term memory task. The activation patterns seen in the normal hearing control group are similar to those seen in our previous study of young normal hearing adults One of the surprising results of our study was that high-frequency hearing loss affected perception and discrimination of low-frequency sounds, not in terms of behavior, but in terms of the response of the auditory, frontal and parietal cortices. Because the sounds were low-pass filtered at 2 kHz to be within the normal hearing range of all participants, the null hypothesis was that there would be no difference in the response of brain for the participants regardless of their hearing status (disregarding tinnitus). This was mostly true for the passive listening task; however, we observed differential involvement of a distributed set of brain regions in the three groups for the discrimination task, varying both on hearing and tinnitus status.The distributed set of regions  highlighted in our results have been proposed previously to play an important role in auditory attention and working memory. Although our study did not employ tasks that require attention explicitly, the short-term memory task uses attention implicitly. Studies investigating attention in the auditory modality have reported on the involvement of the following cortical and subcortical structures: prefrontal cortex, parietal cortex, superior temporal gyrus, temporoparietal junction, anterior cingulate gyrus, basal ganglia, thalamus and inferior colliculus  have also implicated the involvement of the attentional network in tinnitus perception; however, for the most part they have not taken into account the effect of hearing loss and have reported disparate findings. Jacobson and colleagues Although we found brain activation pattern differences between the groups, we did not find any statistically significant behavioral differences. Behavioral studies have noted attentional deficits in selective and divided attention in chronic tinnitus sufferers, specifically in the form of slower response times. In one such study We compared TIN and HL groups in order to understand better the neural correlates of tinnitus, however, the interaction between hearing loss and tinnitus may not be linear. The TIN and HL comparison gave us greater understanding of the brain regions most affected by hearing loss and those that may be most influenced by tinnitus, within the context of simple listening tasks. The relation between hearing loss and tinnitus is complex and includes other cortical and subcortical networks such as those subserving emotion or somatosensory processing We collected structural MR and diffusion tensor imaging (DTI) data on the same group of participants in the current study We chose to perform a fixed-effects analysis because of the limited number of participants in our study and additionally, the patients belonged to a subset  of a complex heterogeneous population. Fixed-effects analysis also lends itself to conjunction analysis In conclusion, our study suggests the differential involvement of a putative AASM network in hearing loss with and without tinnitus. This network consisted of regions in the frontal, parietal and temporal cortices and the anterior cingulate. In participants with hearing loss without tinnitus, the attentional network response was enhanced relative to normal hearing controls. In individuals with tinnitus and hearing loss, the response of some nodes of the attentional network was diminished with respect to hearing loss only group, whereas response of other nodes was enhanced. This suggests a complex role for the attentional network in those with chronic tinnitus and studies are needed that will elaborate on the functioning of this network.


Recent studies have shown that plant genomes have potentially undergone rampant horizontal gene transfer (HGT). In plant parasitic systems HGT appears to be facilitated by the intimate physical association between the parasite and its host. HGT in these systems has been invoked when a DNA sequence obtained from a parasite is placed phylogenetically very near to its host rather than with its closest relatives. Studies of HGT in parasitic plants have relied largely on the fortuitous discovery of gene phylogenies that indicate HGT, and no broad systematic search for HGT has been undertaken in parasitic systems where it is most expected to occur.Rafflesia cantleyi Solms-Laubach and its obligate host Tetrastigma rafflesiae Miq. using phylogenomic approaches. Our analyses show that several dozen actively transcribed genes, most of which appear to be encoded in the nuclear genome, are likely of host origin. We also find that hundreds of vertically inherited genes (VGT) in this parasitic plant exhibit codon usage properties that are more similar to its host than to its closest relatives.We analyzed the transcriptomes of the holoparasite Rafflesia for maintaining these genes. Finally, a similar convergence in codon usage of VGTs has been shown in microbes with high HGT rates, which may help to explain the increase of HGTs in these parasitic plants.Our results establish for the first time a substantive number of HGTs in a plant host-parasite system. The elevated rate of unidirectional host-to- parasite gene transfer raises the possibility that HGTs may provide a fitness benefit to  If the transcript was closer to Malpighiales, then it was assigned to be Mapighiales-like; otherwise, it was assigned to be Vitis-like.The VGT transcripts were first converted into four nucleotide frequencies , 16 dinucleotide frequencies, and 61 codon frequencies (excluding stop codons), and normalized by the length of each transcript. Next, χ cluster . Finallybp: Base pair; BS: Bootstrap support; cDNA: Complementary DNA; gDNA: Genomic DNA; HGT: Horizontal gene transfer; RPKM: Reads per kilobase per million reads; VGT: Vertical gene transfer.The authors declare that they have no competing interests.CCD, JSR, RKB, and KJW designed all analyses; ZX, KB, KMW, and MS collected the data and conducted the analyses; CCD, JSR, and ZX wrote the initial draft of the paper; all other authors contributed to subsequent revisions to the final draft. All authors read and approved the final manuscript.Figure S1. Histogram of the assembled cDNA transcript lengths from Illumina Genome Analyzer II sequencing of  Rafflesia cantleyi .Click here for fileFigure S2. Histogram of the assembled cDNA transcript lengths from GS-FLX 454-sequencing of  Tetrastigma rafflesiae .Click here for fileTable S1. Data sources of protein coding DNA sequences from whole genome sequencing used in the comparative phylogenomic analyses.Click here for fileFigure S3. Bar chart showing the distribution of bootstrap support for putative VGT (dark grey) and HGT (light grey) transcripts identified for  Rafflesia cantleyi .Click here for fileFigure S4. Elevated inference of HGT genes in  Rafflesia cantleyi  is not sensitive to our bootstrap cutoff thresholds. The percentage of HGT genes in Malpighiales  was the number of genes sister to Vitis vinifera, in proportion to the total number of genes with resolved relationships, at the specified bootstrap thresholds. For Tetrastigma rafflesiae, HGT percentage is the number of genes that are sister to Malpighiales in proportion to the total number of genes with resolved relationships, at the specified bootstrap thresholds.Click here for fileFigure S5. (A) Summary of nucleotide substitution rates in Rafflesia cantleyi HGT transcripts versus homologous Vitis vinifera transcripts; (B) Summary of nucleotide substitution rates in Rafflesia cantleyi VGT transcripts versus homologous Manihot esculenta and Ricinus communis transcripts. The boxplot was truncated so that the median can be better visualized. The number of transcripts used to construct each boxplot is shown in parentheses.Click here for fileTable S2. Putative gene location and function of Rafflesia cantleyi HGT transcripts based on NCBI BLAST search results.Click here for fileFigure S6. Percentages of Rafflesia cantleyi HGTs (dark grey) and VGTs (light grey) for which gDNA Illumina reads can be positively mapped onto the assembled transcripts, as a function of bootstrap support for the VGT and HGT inference.Click here for fileFigure S7. Percentages of Rafflesia cantleyi HGTs (dark grey) and VGTs (light grey) for which gDNA Illumina reads can be positively mapped onto the assembled transcripts, as a function of transcript length for the VGT and HGT inference.Click here for fileFigure S8. Nucleotide sequence alignment for a typical intron-bearing HGT transcript identified from Rafflesia cantleyi genomic DNA sequencing. Nucleotides are denoted as dots when identical to the consensus sequence. The Rafflesia HGT transcript is printed in red, and sequences from genomic DNA are marked with asterisks. The intron is highlighted in grey for Rafflesia.Click here for fileFigure S9. Summary of the number of Rafflesia cantleyi cDNA Illumina reads re-mapped onto the assembled VGT and HGT transcripts. The boxplot was truncated so that the median can be better visualized. The number of transcripts used to construct each boxplot is shown in parentheses. (RPKM = reads per kilobase per million reads).Click here for file


The chief objective of the current study was to investigate the auditory status in a group of adults with AIDS before commencing antiretroviral therapy (ART) in a hospital outpatient clinic in Gauteng, South Africa. A total sample of 150 participants, aged between 20 and 46 years, was assessed following a prospective qualitative research design. All participants underwent case history interviews and medical record reviews, otoscopy and tympanometry, as well as conventional pure tone audiometry testing. Descriptive statistics was used to analyse data obtained. Prevalence, type, degree, configuration, and symmetry of the auditory manifestations; as well as type of onset of hearing loss and the possible causes of the auditory manifestations were analysed. Findings indicated that auditory manifestations in adults with AIDS are varied in nature and are possibly due to a number of causes. Manifestations including hearing loss, tinnitus and vertigo (in varied combinations) were found; with the types of hearing loss being mainly sensorineural in nature. The severity of hearing loss ranged from mild to severe, occurred either unilaterally or bilaterally; with the type of onset being mainly gradual and progressive in nature. The varied causes of hearing loss included HIV infection or AIDS illness as a primary cause, opportunistic infections, and various ototoxic therapies that the patients had undergone. Implications for future research as well as future assessment and management of patients with AIDS are raised. The HIV pandemic in South Africa, now in its third decade, remains one of the biggest challenges this country has to confront. This occurs together with poverty, joblessness and other socio-economic challenges that the departments of health and social development strive to eradicate . The HIVGlobally, research has shown numerous associations between HIV/AIDS and auditory performance in adults, including sensorineural hearing loss possibly linked with antiretroviral therapy (ART). However, the acquired evidence is predominantly from the developed world and research from developing countries remains limited. The known effects of HIV/AIDS on the auditory system are based mainly on cross-sectional studies and case reports conducted internationally in industrialised countries, with very scant information emanating from third-world countries where the presentation of the virus and its treatments may be different. Although much research has been conducted internationally, replication of such studies in the developing world is still required. The current study investigates specifically the auditory manifestations of AIDS in adults.There are suggestions that hearing changes may be one of the presentations at any stage of HIV disease . The docConsistent with the reports that state minor auditory manifestations of HIV/ AIDS, are claims that this small incidence rate is exclusive to adults, but may be vastly different in children. These studies have resolved that there is an extremely low incidence (or no incidence) of otologic disease in adults with HIV infection, with possibly much higher incidence in paediatric patients . The higWith regard to comparing the occurrence of auditory manifestations to other head and neck effects of HIV/AIDS, there is significant variation in the reports. For instance, A review of the literature on auditory findings implies variability as far as nature, degree, and configuration of the hearing losses diagnosed in patients with HIV/ AIDS. The documented hearing impairment may be conductive, sensorineural, or central; the severity of the loss may range from mild to severe; the onset of the loss may be sudden or gradual; and the hearing loss may be stable or may fluctuate . Khoza aVery limited published literature exists with regards the degree of hearing loss in patients with HIV/AIDS. The description of hearing impairment in HIV/ AIDS varies even when one considers type of onset and symmetry of hearing loss . SeveralNumerous clinical and medically oriented studies have exhibited the occurrence of hearing loss and other auditory manifestations in HIV/AIDS. According to the literature, auditory abnormalities associated with HIV/AIDS and its treatments have been reported in persons with varying degrees of HIV infection, among both symptomatic and asymptomatic patients, as well as in patients on ART . IndicatA review of the literature with regard to causes of hearing loss in cases of HIV/ AIDS confirms the substantial role that opportunistic infections play in the development of auditory manifestations. Literature states that conductive hearing loss in this population is commonly caused by otitis media, which may be due to eustachian tube dysfunction . VariousA large body of evidence exists in the literature regarding causes of SNHL in HIV/AIDS . Larsen Because of all the diseases and infections that the population with HIV/ AIDS presents with, it is not startling to find patients with hearing loss attributable to ototoxicity, as this population undergoes treatment that often involves potentially ototoxic medications . BankaitThe current study which aimed at determining the auditory manifestations of AIDS was therefore conducted in order to increase the evidence base that is more contextually relevant.To investigate the auditory status in a group of adults with AIDS prior to commencement of antiretroviral therapy in a hospital outpatient clinic in Gauteng, South Africa.To estimate the prevalence of hearing loss and the presence of other otologic effects over and above hearing impairment To assess the type, degree and configuration of the hearing lossTo explore the type of hearing symptom onset To document case history data such as signs and symptoms of each participating participant and to identify any associations between obtained signs and symptoms and hearing lossAs an extensive literature search yielded a paucity of both South African and internationally published data on this topic, the study was exploratory. The design utilized was prospective and qualitative in nature . The aim3 were assessed and counselled for ART at this clinic - and the current study targeted this group before they enrolled in the treatment.A total sample of 150 participants comprised the study. The patients selected for this study were recruited from the Hospital's Adult HIV/AIDS clinic. Patients who attend this clinic have already been diagnosed with HIV/AIDS and are seen there for general medical management as well as antiretroviral treatment and monitoring. At the time of the study all patients with CD4+ counts below 200 cells/mmDue to the exploratory nature of the current study; and given the fact that little, if any, published research has been conducted on this aspect of HIV/AIDS in South Africa, the researcher believed that it was crucial to have a sample that was as closely representative of the general AIDS population as possible; while closely recording medical history that could have had an influence on the results. This medical history was obtained from both case history interviews and from medical records. To this end, criteria stipulated in A nonprobability convenience sampling technique was utilized in the study since the sample was restricted to a part of the population that was readily available , and truParticipants underwent case history interviews and medical record reviews, otoscopy and tympanometry, as well as conventional pure tone audiometry testing. Data were collected from assessing participants' dependant variables before administration of ART. Following infection control measures proposed by Case History:A case history form that targeted the signs and symptoms of auditory manifestations was utilized in order to gather all the important case history information, audiological data and some medical variables that could have exerted an impact on the results of the study.Otoscopy:The researcher evaluated the participants' ears for the presence of impacted wax; otitis externa; possible otitis media; perforated tympanic membranes; collapsed ear canals; presence of any growths and any other ear disorders . These oTympanometry:Tympanometry (through the use of the Inter-Acoustic AZ26 audiotympanometer) was utilized to assess the status and integrity of middle ear functioning. Standard single frequency tympanometry using an 85dB SPL tone set at 226Hz was done.Pure tone audiometry:Conventional (250Hz–8000Hz) pure tone audiometry was performed on all participants through the use of the Inter-Acoustic AC 40 diagnostic audiometer. The criteria used to define normal hearing, was that of pure tone thresholds of 25dBHL or lower across all frequencies, with the absence of an air-bone gap . Where pTest reliability was controlled and maintained at a high level by standardizing test administration, ensuring proper equipment calibration, and controlling patient variables. For all audiological assessments precautionary measures advocated by Participants' results were descriptively analysed and classified as either normal or abnormal. Normal hearing was taken as normal tympanometric results in the presence of normal otoscopic findings with pure tone responses at or better than 25 dB HL, with hearing loss being thresholds greater than 25dB HL with air-bone gaps greater than 10dB . This stThe hearing losses were classified into the three well-documented types of hearing losses  .Configuration of hearing loss was established by describing the pure tone results as depicting flat, irregular, rising (low frequency), sloping (high frequency) configuration . SymmetrPrior to commencement of the study, permission to conduct the research project was sought from the University of the Witwatersrand Human Research Ethics Committee  which gave unconditional ethical clearance in the form of protocol number M041131. The researcher ensured that permission to conduct the study was obtained from the Hospital management and from the Heads of the Audiology and HIV/AIDS clinics at the research site. Written informed consent to participate in the study was obtained from all participants before the study was conducted with an assurance that confidentiality of all records would be maintained. Furthermore, to ensure anonymity, the researcher ensured that no personal or identifying information was included in the research report and research coding numbers instead of identifying information were used. The current study also reduced risks to the participants to a minimum by conforming to the ethical principles  and obse3 which was consistent with the CD4+ requirement that persons need to have before they can be enrolled in an ART programme in South Africa (i.e. CD4+ has to be below 200 cells/mm3).As depicted in Close inspection of the demographic profile of the participants in the current study reveals similarities between the sample evaluated and the general South African population infected with HIV/AIDS with regard to age, gender and race . This thOf the total sample of 150 participants evaluated, 135 (90%) had normal hearing, and 15 (10%) presented with a clinical hearing loss . Over anThese findings are consistent with previous reports on auditory manifestations of HIV/AIDS. The 10% that presented with hearing loss in the current sample is considered to be high since this sample only consisted of participants in the AIDS stage and excluded the earlier stages of the disease. This sample also did not include the effects of medications on hearing function since participants had not enrolled in ART at the time of data collection. This high occurrence of hearing loss in the sample assessed underscores the need for more research into this population to enhance generalizability of results so that vital decisions can be made regarding the anticipated burden of disease. Given the prevalence and disease burden of undetected hearing impairment and the availability of effective treatments, it is important for audiologists to engage early in the assessment and management of this population before quality of life is severely compromised.Of the 15 participants with clinical hearing loss, 11 (73%) had sensorineural hearing loss (SNHL); 4 (27%) had conductive hearing loss (CHL); and none had mixed hearing loss (MHL) .In reviewing the literature, the current findings provide support for the reports that claim the hearing loss seen in HIV/AIDS to be of any type . The limFollowing the classification of degree of hearing loss previously reported; it became evident that the hearing loss could occur in any degree of severity as depicted in These results are consistent with those reported in the literature where severity of hearing loss has been described as ranging from mild to severe with little mention of profound hearing loss - as was found in the current study. Despite the absence of the profound degree of hearing impairment in this sample, the implications for the patients' quality of life are still significant; and the role of the audiologist with regard to early and prompt effective services to this population is highlighted. Unaided severe hearing loss can have an influence not only on the patient's everyday listening environment; but in this case, may also have an influence on the patient's ability to follow treatment protocols; and subsequently adherence to ART.No typical pattern of configuration of hearing loss could be established before antiretroviral treatment was instituted. In the total sub-sample (n = 15) of patients with hearing loss, 5 (33%) participants presented with a sloping/high frequency hearing loss while the remaining 67% presented with flat and/or irregular audiograms with an equal chance of involvement of all frequencies. These results are consistent with findings documented by Of the total sub-sample of 15 participants with abnormal hearing, 7 (47%) had unilateral hearing impairment while 8 (53%) presented with bilateral hearing loss . AlthougOf the entire sample with hearing loss, all the participants (100%) presented with gradual/progressive hearing loss with no participant presenting with sudden onset of symptoms. These results are inconsistent with earlier reports by Patients who presented with SNHL had documented medical histories of meningitis; infections (syphilis and otosyphilis); and histories of ototoxic medication used in the treatment of TB and other opportunistic infections. Four participants had unknown causes of hearing loss.Patients who presented with CHL had a history of chronic suppurative otitis media, and otitis media with effusion.Detailed analysis of the audiologic evaluation results together with the documented case history information where medical diagnoses had been confirmed by Ear, Nose and Throat Specialists  and 5 rePossible causes of hearing loss found in the current study are consistent with literature reviewed. These findings were not surprising as these conditions are commonly seen in persons living with HIV/AIDS where immunological status has been severely compromised , and in Limitations to the current study were present, and they included the fact that there was limited control over confounding variables such as previous exposure to ototoxic drugs. Furthermore, due to the sample size and the fact that the data were collected in one hospital in Gauteng, South Africa, the researcher's ability to generalize the results from the sample studied to the total population of adults with AIDS in South Africa is limited. From the findings of the current study it is clear, however, that there is great heterogeneity in presentation of the auditory manifestations of AIDS. With the exception of Audiological research into HIV/ AIDS may clearly demonstrate the potential role of the audiologist in both the assessment and treatment of patients AIDS. Most importantly, research conducted in specific contexts where the disease has varied effects as well as varied treatments may yield findings that are more relevant and evidence-based. Research conducted within the South African context; a context considered to be the epicentre of the African HIV pandemic; and a context that is acknowledged to present unique challenges of which political, social, economic, health, equipment, and personnel problems are deemed non-exhaustive; is crucial. Such research can directly inform the assessment and management protocols of this population, thereby impacting positively on their quality of life. These protocols can only be implemented if the audiological manifestations of AIDS and its treatments within a specific context have been clearly characterised.Characterisation of the burden of disease has significant implications for developing countries where financial constraints guide the prioritisation of resources. For example, if the correct prevalence of audiologic manifestations of AIDS could be established, appropriate budget planning could be employed, which would have a direct influence on resource management for this population. Furthermore, when budgets are allocated for comprehensive HIV/ AIDS management, audiological services could be included in the programmes. Recommendations may need to be made for audiological services in terms of the provision of sensitive and objective equipment (such as measures of otoacoustic emissions [OAEs]) as well as the appointment of audiologists at HIV/AIDS clinics in state institutions.While there are some clear auditory manifestations that were found in the current study, there is still a small evidence base. Findings regarding the prevalence as well as nature of auditory dysfunction in adults with HIV/ AIDS need to be replicated in other communities and populations in Africa, especially since sub-Saharan Africa has such high HIV incidence. Because of the vast variance in contexts, the differences in the patient populations studied, the wide range of methodological approaches employed, and so on — findings from international studies cannot easily be generalised to the African context. Furthermore, certain issues such as time of ART initiation, use of complimentary or alternative medicine in the African context, and how these may impact auditory function have not received attention. Hence, more context-relevant, current and updated research which utilises standardised and sensitive research instruments for ease of comparability of the findings is clearly needed to address this situation. Such research should also include more controlled studies to establish definitive links between HIV/ AIDS and its drug treatments with auditory function. Research should also include continuous reviews of prevalence and incidence data, as the nature of the disease and its manifestations may be changing. These research efforts would greatly enhance the development of a more consolidated body of evidence that can then be used to contribute to policy-making and programme design and implementation, while at the same time improving the efficacy of clinical practice.


GJB2 and GJB6. Sensorineural hearing loss and male infertility  is a contiguous gene deletion syndrome resulting from homozygous deletion of the CATSPER2 and STRC genes on chromosome 15q15.3. Females with DIS have only hearing loss and are fertile. Until recently this syndrome has only been described in three consanguineous families and 2 nonconsanguineous families.Hearing loss is the most common birth defect and the most prevalent sensorineural disorder in developed countries. More than 50% of prelingual deafness is genetic, most often autosomal recessive and nonsyndromic, of which 50% can be attributed to the disorder DFNB1, caused by mutations in We recently indentified a patient with hearing loss and macrocephaly who was found to be homozygous for this deletion. Her nonconsanguineous parents are both carriers. We examined our database of patients tested by array CGH and determined that just over 1% of our patients are heterozygous for this deletion. If this number is representative of the general population, this implies a 1% carrier frequency and prevalence of DIS of 1 in 40,000 individuals.We propose that DIS is a greatly under-diagnosed cause of deafness and should be considered in children with hearing loss. Likewise, current molecular genetic testing panels for hearing loss in the United States should be expanded to include deletion/duplication analysis of this region. GJB2 (which encodes the protein connexin 26) and GJB6 (which encodes the protein connexin 30) [GJB2 mutation is approximately one in 33 [Hearing loss is the most common birth defect and the most prevalent sensorineural disorder in developed countries. One out of every 500 newborns has permanent bilateral sensorineural hearing loss ≥40 dB; by adolescence, the prevalence increases to 3.5 per 1000 [CATSPER2  and STRC (responsible for hearing loss) genes on chromosome 15q15.3. Females with DIS have only hearing loss and are fertile [Sensorineural hearing loss and male infertility  is a contiguous gene deletion syndrome resulting from homozygous deletion of the  fertile  and two  fertile . HoweverThe proband is a 13-month-old girl who presented to the Medical Genetics Department for evaluation regarding congenital hearing loss. Her past medical history was unremarkable. She was the second child born to a 35-year-old mother. Except for morning nausea the pregnancy was not complicated. The mother did not take any medications during gestation. Delivery was at term (40 weeks), birth weight was 3856 grams (75th centile), and no perinatal complications were reported. Her psychomotor development has been normal. The patient was crawling at age 7 months, standing up at age 8 months, and walked unassisted at age 10 months. Her language at age 13 months was appropriately limited to babbling and a few words.At age 13 months, her physical examination was normal except for the presence of macrocephaly. Her height was 75 cm (70th centile), and her weight was 12.5 kg (94th centile). Her head circumference was 49.7 cm (>99th centile). In addition to macrocephaly, anterior fontanel was still open (2–2 cm) and there was mild brachycephaly, but the face was non-dysmorphic. A review of her growth chart revealed that she had normal head circumference at birth , but her head size exceeded the 99th centile at age 4 months. Since that time, her head circumference has consistently tracked above the 99th centile , and auditory brainstem response (ABR) suggested normal middle ear function, good 8th nerve synchrony, but a mild hearing loss at least for the 2000–4000 Hz range in both ears. The hearing loss was most likely sensorineural in nature given the normal middle ear function (evidenced by tympanometry) with absent otoacoustic emissions. Behavioral audiological testing via visual reinforcement audiometry in the sound-field was first completed at 8 months of age and has continued at 6 months intervals. Behavioral testing verified the hearing loss predicted by ABR and has suggested some progression to date. The patient’s most recent test results suggested a mild sloping to moderate hearing loss  involving the 500–6000 Hz range for at least the better ear but involving both ears Figure B. FamilyGJB2 and deletion/duplication testing of GJB6 were also negative. However, array comparative genomic hybridization (array CGH) detected an approximately 62 kilobase homozygous deletion at 15q15.3  by array CGH. Of those, 57 patients were found to be heterozygous for similar deletions including the We present here the third reported case of a patient with DIS whose parents were not consanguineous Table . In thisSTRC on the non-deleted allele; however such mutations are likely not as common as deletions [GJB2 mutation [One important limitation to point out is that our study cohort is comprised of individuals referred to clinics for a variety of reasons, including but not limited to developmental delay, dysmorphism, congenital anomalies, etc. Because our cohort does not represent a “normal” population, we cannot rule out the possibility that heterozygosity for this deletion is enriched in our patient population for a different reason, such as predisposing to phenotypic abnormalities. However, this is unlikely given the fact that heterozygous individuals are asymptomatic and that homozygous individuals have only hearing loss and, in males, infertility, as the only clinical manifestations. We also cannot rule out the possibility of a point mutation in eletions , and arreletions . Anothermutation . Taken tGenomic DNA extraction for molecular cytogenetic studies was performed using the PureGene method  on peripheral blood lymphocytes. DNA was resuspended in 1X Tris-EDTA  and the concentration was measured with a Nanodrop ND-100 Spectrophotometer . To perform array CGH, 1 μg of patient genomic DNA was labeled with Cy5 and an equal amount of gender-matched control DNA was labeled with Cy3 using the Genomic DNA Labeling Kit  according to the manufacturer’s protocol. Each sample was purified using a Multiscreen PCR 96-well plate and eluted in TE. Equal volumes (19.5 μl) of both patient and control DNA were mixed along with Cot-1 DNA and 2X hybe buffer . The mixtures were denatured at 95°C for 3 minutes and then incubated at 37°C for 30 minutes. Each mixture was then applied to an Agilent 4 × 180 K oligonucleotide array CGH slide and incubated while rotating at 65°C for 24 hours. Slides were washed and scanned on a G2565CA Microarray Scanner System . Data was analyzed using DNA Analytics version 4.0 . This research was approved by the Mayo Clinic Institutional Review Board (IRB).The authors declare that they have no competing interests.NLH analyzed the laboratory data, drafted, revised, and finalized the manuscript. UA analyzed the laboratory data and involved in manuscript preparation. PB collected and analyzed the laboratory data. NB, JW, and DB clinically examined the patient and collected the clinical data. All authors read and approved the final manuscript.


We describe a case of a 67-year-old woman with severe disabling right-sided tinnitus, mild hyperacusis, and headache. The tinnitus was associated with sudden right hearing loss and vertigo, which occurred about 18 months before. Magnetic resonance imaging (MRI) resulted in normal anatomical structures of the cochlea and of the cranial nerves showing a partial empty sella syndrome with suprasellar cistern hernia. Angio-MR revealed a bilateral contact between the anterior-inferior cerebellar artery (AICA) and the acoustic-facial nerve with a potential neurovascular conflict. Surgery was considered unnecessary after further evaluations. The right ear was successfully treated with a combination device (hearing aid plus sound generator). Shortly after a standard fitting procedure, the patient reported a reduction of tinnitus, hyperacusis, and headache which completely disappeared at the follow-up evaluation after 3, 6, and 12 months. This paper demonstrates that the combination device resulted in a complete tinnitus and hyperacusis suppression in a patient with unilateral sensorineural sudden hearing loss. Our paper further supports the restoration of peripheral sensory input for the treatment of tinnitus associated with hearing loss in selected patients. Sudden sensorineural hearing loss (SSHL) is defined as an acute deafness with abrupt onset, generally within 3 days of more than 30 dB hearing loss at three consecutive frequencies , 2. The A 67-year-old Caucasian woman came to our attention with complaints of severe disabling right-sided tinnitus, mild hyperacusis, and headache. Insomnia was also linked with the presence of tinnitus during the night. The tinnitus was associated with sudden right-sided hearing loss and vertigo, which occurred about 18 months before. Her hemogram, blood coagulation, blood sugar levels, lipid profile, and renal and liver functions were normal. Autoimmune markers and urine homocysteine tests were negative. In spite of different therapies  previously prescribed, there was no hearing improvement. Meanwhile the tinnitus worsened in the right ear (pitch: narrow band noise at 6 kHz). Tinnitus severity has been assessed with Tinnitus Handicap Inventory (THI) and visual analogue scales (VAS). Using a ten-point VAS, the tinnitus was rated by the patient according to loudness and annoyance. Insomnia and concentration problems due to tinnitus lead to severe disability and a poor quality of life . Echo-Doppler of neck vessels was normal. Pure tone audiometry testing revealed right-sided ski-slope hearing loss on high frequencies and mild hearing impairment on the high frequencies in the contralateral ear . After tEven if the lipidic and glycemic blood profile of the patient resulted in normal values we suggested a low-carbohydrate diet, which improved her quality of life with the result of a reduction of headache and tinnitus. The patient reduced her weight loosing 12 Kg in two months without adverse effects. The insomnia was successfully improved with melatonin (3 mg/die).The right ear was treated with a combination device, which provides both hearing instrument features as well as a masking sound generator, used at least four hours per day. Initially it was activated, only the open fit hearing of the device and the process of monoaural fitting were effective with mild Noise Tracker, fixed directionality, and dual stabilizer DFS on. After 1 month the sound generator was activated and set to the mixing point so that the tinnitus and the sound generator stimulus blended together. The default noise setting for the generator was set to a broadband filtering setting with the flexibility of low and high cut controls to provide more individualized comfort. The low cut filter was 1 kHz and the high cut filter was 6 kHz, on the basis of audiological tests . The ampShortly after a standard fitting procedure, the patient reported a reduction of her tinnitus, hyperacusis, and headache. The THI was significantly reduced at the follow-up evaluation after 6 months  and 12 months . After 6 months of sound therapy, the ULL test was normal and the hyperacusis disappeared as well as the discomfort related to the environmental noise. The decrease of afferent input caused by SSHL could induce tinnitus connected to central neural changes. Hyperacusis has received much less attention than tinnitus but it is a common and disabling symptom. This paper demonstrates that the combination device (hearing aid + sound generator) resulted in a complete tinnitus and hyperacusis suppression in a patient with unilateral SSHL. Our report further supports the restoration of peripheral sensory input for the treatment of tinnitus associated with hearing loss , 10 and