RNA thermometers control the translation of several heat shock and virulence genes by their temperature-sensitive structural transitions. Changes in the structure and dynamics of MiniROSE RNA, which regulates translation in the temperature range of 20–45°C, were studied by site specifically replacing seven adenine residues with the fluorescent analog, 2-aminopurine (2-AP), one at a time. Dynamic fluorescence observables of 2-AP-labeled RNAs were compared in their free versus ribosome-bound states for the first time. Noticeably, position dependence of fluorescence observables, which was prominent at 20°C, was persistent even at 45ºC, suggesting the persistence of structural integrity up to 45ºC. Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant at 45°C was ablated in ribosome-bound state, when compared to those at 20°C, underscoring loss of structural integrity at 45°C, in ribosome-bound RNA. Significant increase in the value of mean lifetime for 2-AP corresponding to Shine–Dalgarno sequences, when the temperature was raised from 20 to 45°C, to values seen in the presence of urea at 45°C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45°C, only when it was ribosome bound. Taken all together, we propose a model where we invoke that ribosome binding of the RNA thermometer critically regulates temperature sensing functions in MiniROSE RNA. Although the information content of an RNA primarily resides in its sequence, the conformation and dynamics of sub-regions of mRNA are emerging as critical regulators of expression of these messages encoded in the sequence –7. TheseSensing and regulation of temperature is of vital importance in living cells. In bacteria, expression of heat-shock and cold-shock genes is regulated by temperature –12. Exprper se in modulating the structural transition and therefore the functional status of RNA thermometer, in vitro, a step closer to in vivo setting.In the present study, we explore temperature-dependent structure and dynamics of ‘MiniROSE’ RNA, a smaller segment of the thermometer sequence  by using2 and 150-mM KCl. Homogeneity of folded forms of RNA is essential for correct interpretation of fluorescence observables. 1D 1H NMR spectra , 70-mM KCl, 10-mM MgCl2]. Cells (∼50-g wet-weight) were then resuspended in 100-ml polymix buffer  containing 100-μg DNase. Cells were lysed by grinding the cell-paste with alumina. It was then centrifuged twice at 28 000 g for 30 min to clear cell debris, etc. Solid ammonium sulfate was added to the supernatant thus obtained, while stirring it to the final concentration of 210 mg/ml. The solution-pH was adjusted to 7.5 using ammonium hydroxide. Stirring was continued for another 30 min, followed by centrifugation at 28 000 g for 30 min. Clear yellow supernatant (crude ribosome preparation) was then loaded onto a column of Sephacryl S300  pre-equilibrated with buffer B (Buffer B: same as polymix buffer except NH4Cl is made 1 M). Ribosome fraction, a mixture of sub-units, eluted in the excluded volume, was primarily detectable by eluent turbidity. Absorbance was monitored at 260 nm. Peak fractions with majority of A260 material were pooled. Ribosome particles were precipitated by adding 100-mg PEG6000 per ml with stirring for 30 min at 0°C. The precipitated ribosomes were recovered by centrifuging at 16 000 g for 15 min. Pellets of ribosomes were redissolved in polymix buffer and methanol was added to the final concentration of 30% (v/v). Such final preparation of ribosome particles was dialyzed against polymix buffer containing 30% (v/v) methanol for 10 h. The absorbance of the preparation was measured at 260 and 280 nm and A260/A280 was found to be ∼1.8 and the final concentration of ribosome stock solution was 480 nM (Considering 1 A260/ml = 23 pmol/ml)  was purified from tography –30. Seph2 and 150-mM KCl were heated to 90°C for 2 min, then quench-cooled using liquid nitrogen. RNA concentrations were found to be in the range of 6–15 μM of RNA molecule.RNA samples taken in 10-mM Tris-HCl (pH 7.5), 5-mM MgClTime-resolved fluorescence intensity and anisotropy decay of 2-AP was measured by employing a passively mode-locked frequency-doubled Nd:YAG laser  driven rhodamine 6G dye laser, which generates pulses of width 1 ps at 4 MHz –23. 2-API(t), were analyzed by nonlinear least-square iterative deconvolution method based on the Levenberg–Marquardt algorithm ) and perpendicular (I⊥(t)) directions to the polarization of the excitation beam and constructing the time dependent anisotropy, r(t), with the following equation:G(λ) is the grating factor at the wavelength λ of emission. The magnitude of G-factor for the emission collection optics was determined independently using a standard solution of 2-AP for which the fluorescence lifetime was 11.3 ns using standard procedures  such that ∑iβ = 1 and ϕm is mean rotation correlation time. The ϕm, which is an operational parameter, takes note of both the local (ϕ1) and the global dynamics (ϕ2) and thus gives an indication of the overall rotational dynamics felt by 2-AP. The ϕm is proportional to the area under the anisotropy versus time curve. Furthermore, the ϕm is proportional to the steady-state anisotropy. Anisotropy decay parameters were obtained by deconvoluting the decays (Equations (r0 = 0.31 estimated in a separate experiment on 2-AP in 50% glycerol) and the intensity decay parameters (iα and iτ) estimated from Equation  with IRmentclass2pt{minimails see –23.ϕ) of a small molecule N-acetyltryptophanamide in glycerol–water mixtures with viscosity (η) in the range 1–5 cP. The values of ϕ estimated were 0.060 ± 0.027, 0.121 ± 0.034, 0.224 ± 0.029 and 0.315 ± 0.029 ns, when the solvent viscosities were 1.0, 2.1, 4.4 and 5.4 cP, respectively, as expected from the Stokes–Einstein relationship .The time resolution is limited largely by the width of the IRF, which is 40 ps. We have obtained, in several samples, fluorescence lifetimes and rotational correlation times in the range of 0.1–0.3 ns. Since these values are quite close to both the width of the IRF and the time/channel used, both of which are ∼40 ps, their reliability was checked. We measured the rotational correlation time .The solvent accessibility of 2-AP residues was monitored by collisional quenching with acrylamide. Titration stock was made with ∼5-M acrylamide in 10-mM Tris-HCl (pH 7.5), 150-mM KCl with 5-mM MgClequation  for anal1H NMR spectra of the wild-type and the loop-mutant of MiniROSE RNA was recorded in the range 288–318 K on a Bruker Avance 800-MHz NMR spectrometer equipped with a 5-mm cryogenically cooled triple-resonance probe and pulse-field gradients, with 1H carrier placed on H2O resonance (4.68 ppm) (Temperature dependence of the imino-proton region of the 1D .68 ppm) –35. The kq, estimated from dynamic quenching of fluorescence, has been of immense use in revealing the dynamic aspects, especially related to solvent accessibility of structures in biomolecules in general  at 20ºC, a temperature at which translation is inhibited where the MiniROSE RNA strand is supposed to be well structured, thus concealing the SD sequence from ribosome recognition, appropriate for initiating translation; (ii) at 45ºC, a temperature at which translation is allowed, following structural transitions in MiniROSE RNA motifs; and (iii) at 45ºC in the presence of 7-M urea, an artificial condition under which all intramolecular (tertiary and secondary) interactions in the RNA are expected to be weakened, thereby essentially providing a control that has lost all plausible structures. This strategy was expected to offer us insights into the mechanism of temperature-sensitive translation through a comparison of structure and dynamics under the above-mentioned three conditions. Further, we compared the outcome of the first two conditions in free as well as ribosome-bound states of MiniROSE RNA.Fluorescence intensity decay kinetics of all the seven 2-AP-incorporated RNA samples showed the presence of four lifetime components as seen in previous studies in DNA –23. Freeτm ( = Σiτiα), is given in Table τm in this work. Individual lifetimes and their amplitudes are given in Supplementary Table S1. τm showed significant dependence on the position of 2-AP in MiniROSE RNA at 20ºC as expected for a structured RNA. Figure τm plotted as the variation from the mean value of the parameter averaged over all the seven positions. This representation brings out the position dependence in a stark manner. It is instructive to focus our attention on the site-specific variation of the fluorescence observables. As mentioned above, the individual lifetime components (Supplementary Table S1) could have their origin on the various sub-states of the 2-APconformation. The dominant factor that controls the lifetime of 2-AP in polynucleotides is the strength of stacking interactions of 2-AP with the neighboring bases leading to electron delocalization  and global (ϕ2) motional dynamics ))ϕ1) and Noticeably, the position dependence of fluorescence observables which was prominent at 20°C was largely persistent at 45ºC also Table , suggest1H NMR spectrum , in general, were shorter at 45°C when compared to 20°C Table –23. Globkq seen under this condition compared to the values in the absence of 7-M urea (Table kq) when compared to the structured double-stranded DNA , where significant level of structural signatures (non-flat nature of observables) was still evident at 45°C, although there were indications of faster dynamics observed from the rotational correlation times  versus high temperature (45°C) lead to any biologically relevant structural changes in ribosome-bound MiniROSE RNA as compared to the same in free MiniROSE RNA? This is a biologically relevant mechanistic question that has not received sufficient attention so far. Most high-resolution structural studies involving NMR have been limited to free RNA targets, devoid of ribosomes, due largely to NMR methodology constraints. Time-domain fluorescence observables being monitored in the current study are sensitive indicators of the impact of ribosome cues on MiniROSE RNA transitions, if there are any. To address this issue, binding of the MiniROSE RNA with ribosome was assessed at 20 and 45°C by monitoring time-resolved anisotropy decay kinetics of 2-AP. The ribosome preparation used is a mixture of sub-units collected from Sephacryl S300 gel-filtration void volume peak. The admixture of ribosome particles is expected to support initial binding of RNA by 30S particles followed by 50S sub-units required for SD opening in RNA.agsA mRNA from Salmonella ). This could be rationalized based on the origin of these three fluorescence-derived parameters, as reasoned below. Fluorescence lifetime, which is the result of photophysics of the excited state of 2-AP, is largely controlled by the interaction of the aminopurine with its near neighbors as a result of structure within the RNA. Similarly, the bimolecular quenching constant kq reflects the level of structure in the RNA. Absence of structure would result in a similar level of solvent exposure of 2-AP at all the locations of the RNA. Thus when the RNA is bound to ribosome without preserving its folded structure, we expect the fluorescence lifetime and kq of 2-AP to become largely position independent as observed. In contrast, rotational dynamics of 2-AP need not be uniform across the length of ribosome-bound RNA as the strength of binding of the single-stranded RNA is likely to be discernibly position dependent, thus leading to variation in the level of segmental dynamics of the bound RNA which contributes to the mean rotational correlation time of 2-AP. Furthermore, we note that the extent of non-flatness is higher for rotational correlation time when compared to the other two observables even at 45ºC and urea  destabilization of the RNA structural motif and subsequent binding of the SD region of the RNA to ribosome . Observa5°C destaThat the folded structure of MiniROSE RNA is largely intact at 45°C is seen from site-specific dispersion (non-flatness) of fluorescence parameters associated with 2-AP Figure . Loss ofτm did not increase appreciably (indicating opening of the hairpin structure) when the temperature was increased from 20°C to 45°C especially in the presence of ribosome , etc. Our study strongly underscores the need to investigate RNA switches  in their native interaction milieu also apart from studying them as free RNA molecules.Supplementary Data are available at NAR Online.


Flaviviridae family are representatives of viral polymerases that carry out RNA synthesis through a de novo initiation mechanism. They share a ≈ 600-residue polymerase core that displays a canonical viral RdRP architecture resembling an encircled right hand with palm, fingers, and thumb domains surrounding the active site. Polymerase catalytic motifs A–E in the palm and motifs F/G in the fingers are shared by all viral RdRPs with sequence and/or structural conservations regardless of the mechanism of initiation. Different from RdRPs carrying out primer-dependent initiation, Flaviviridae and other de novo RdRPs utilize a priming element often integrated in the thumb domain to facilitate primer-independent initiation. Upon the transition to the elongation phase, this priming element needs to undergo currently unresolved conformational rearrangements to accommodate the growth of the template-product RNA duplex. In the genera of Flavivirus and Pestivirus, the polymerase module in the C-terminal part of the RdRP protein may be regulated in cis by the N-terminal region of the same polypeptide. Either being a methyltransferase in Flavivirus or a functionally unclarified module in Pestivirus, this region could play auxiliary roles for the canonical folding and/or the catalysis of the polymerase, through defined intra-molecular interactions.RNA-dependent RNA polymerases (RdRPs) from the  Flaviviridae family include a large number of important human and animal pathogens with notable members including hepatitis C virus (HCV) from the genus Hepacivirus, dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) from the genus Flavivirus, and bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) from the genus Pestivirus. The non-segmented single-stranded RNA genome of these viruses is positive sense and has a typical length of 9.5–12.3 kilo-bases . S. SFlavivitiation . Similarectively b. Note ttemplate . In a vetivities . These drocesses . Howeverynthesis ,55. To dynthesis ,57,58. HPicornaviridae RdRP elongation complex (EC) crystal structures [The ϕ6 and HCV polymerase IC structures could represent the situation in the first and second nucleotide addition cycles of the initiation process, respectively. From these one could deduce the possible outcome as the template-product RNA duplex grows to a length of about 7–8 base pairs and occupies the entire front channel of the polymerase, a status observed in multiple ructures ,44. Presructures ,59, and pol, a representative of primer-dependent RdRP [Among other basic aspects of RdRP catalysis, the details of elongation catalytic cycle are largely clarified in PV 3Dent RdRP . In thisent RdRP . Transloent RdRP ,25. As ment RdRP ,34. Howein cis regulation of the core polymerase. This difference is reflected in the crystal structures of these RdRPs to some extent  and the structurally critical residue pair in motif G  are in-line for NTP/+1 templating nucleotide interactions and for template RNA binding, respectively  adopts the canonical fold  and motif G is RdRP specific. Whether or not the primary translocation mechanism is shared by RdRPs and RTs, it probably differs from what is taken by the Pol I family polymerases based on structural divergences. In practice, the mystery of the priming element conformational changes can be uncovered in any de novo viral RdRPs systems including those from Cystoviridae. With the low level of sequence conservation of the priming element considered, the details of the rearrangement during the transition process could be very different. However, the general strategy to complete these conformational changes is likely to be similar.As ideal systems to study the function of in cis regulation on some of the Flaviviridae RdRPs by their N-terminal domain has brought in interactions and functions beyond the fundamental processes of RdRP catalysis. In Flavivirus NS5, crystal structures of the full-length protein have shed light on how the regulation could take place, and also provide valuable clues to investigate the function of NS5 beyond polymerase catalysis. Pestivirus NS5B still awaits a high-resolution full-length structure to reveal the true face of the N-terminal domain and its relationship with the polymerase module.The


Drosophila. Guidance receptors are thought to mediate this evolutionarily conserved process. A core of transcription factors from the NK2, GATA and T-box families are also believed to orchestrate this process in both vertebrates and invertebrates. Nevertheless, whether they accomplish their function, at least in part, through direct or indirect transcriptional regulation of guidance receptors is currently unknown. In our work, we demonstrate how Tinman (Tin), the Drosophila homolog of the Nkx-2.5 transcription factor, regulates the Unc-5 receptor during DV tube morphogenesis. We use genetics, expression analysis with single cell mRNA resolution and enhancer-reporter assays in vitro or in vivo to demonstrate that Tin is required for Unc-5 receptor expression specifically in cardioblasts. We show that Tin can bind to evolutionary conserved sites within an Unc-5 DV enhancer and that these sites are required for Tin-dependent transactivation both in vitro and in vivo.During early heart morphogenesis cardiac cells migrate in two bilateral opposing rows, meet at the dorsal midline and fuse to form a hollow tube known as the primary heart field in vertebrates or dorsal vessel (DV) in  Drosophila during DV morphogenesis [Drosophila [Early stages of heart development, both in vertebrates and invertebrates, include the migration of bilaterally paired condensations of cardiac precursors and the formation of a linear tube. The tube is formed once these symmetrical groups of mesodermal cells meet, and attach to each other leaving a luminal space between them , 2. The ogenesis –8. Verteosophila , 10. Somosophila , 12 or posophila , 14, havDrosophila homolog, Tinman (Tin), play an important role in early cardiogenesis starting with the specification of cardiac precursors to remodeling and functionality of the adult heart [Cardiogenesis in both vertebrates and invertebrates also requires the key regulatory actions of a core of evolutionarily conserved families of transcription factors  . They arlt heart , 17. GivDrosophila dorsal vessel (DV). The DV develops from mesodermal cardiac precursors. After precursor division, heart cells line up bilaterally into two rows where myocardial cells or cardioblasts (CBs) are positioned dorsally and pericardial cells (PCs) ventrolaterally. Finally, they migrate together towards the dorsal midline of the embryo where CBs fuse to form the tubular heart  control heart tubulogenesis through the regulation of guidance receptors, we have studied Unc-5 receptor’s regulation in the ar heart . The CBs A and B , 19 and  A and B , 21 and  A and B . Unc-5 i A and B , 8. tin most CBs . Furtherenhancer . In thisUnc-5 as one of its targets [Drosophila is dependent on the homeobox transcription factor Tin [tin loss-of-function mutants lack a DV due to its early role in the mesoderm to specify cardiac progenitors [tin mutant animals , where tin is re-expressed in a mutant background under the control of enhancer elements (ABD) recapitulating its entire endogenous expression pattern except in the dorsal vessel. Myocardial cells are specified and the DV forms in these mutants [tin regulates Unc-5, we analyzed its expression in tin mutant DVs (Unc-5 mRNAs is significantly reduced when compared with tin heterozygous DVs (compare 1B with 1C). Thus, tin is required for Unc-5 expression in the DV at the onset of tubulogenesis.The Unc-5 receptor localizes preferentially at the luminal side in CBs at the onset of tubulogenesis and it is required to preserve the luminal space between CBs , 8. Howe targets , 24. Prector Tin , 26. As genitors . Neverth mutants . To detetant DVs . Unc-5 mtin’s sufficiency to induce Unc-5 transcription in vivo by misexpressing tin in ectodermal stripes with an engrailed-Gal4  driver. We detected Unc-5 mRNA through in situ hybridization and we confirmed that, indeed, ectopic tin expression in the ectoderm is sufficient to induce Unc-5 in the characteristic engrailed stripes  to the 5th intron within the Unc-5 locus [Unc-5 ATG sufficient to drive GFP expression in the DV at late stages of PCs and CBs migration and during tube formation Unc-5 expenhancer .tin is required for Unc-5 expression in the DV . While the wild-type reporter is ectopically expressed in en stripes our results revealed little or no activity in embryos with the R8,9,10-GH mutant reporter eyD, AlpeyD: eyD, Unc-5 GH-GFP, Unc-5 GH-Gal4 (described below), TinC-Gal4 [UAS-tau-Myc.The following stocks were used: svp-lacZ , en-Gal4inC-Gal4 , and UASUnc-5 locus dissection was carried out by PCR-amplifications using genomic DNA as template to amplify overlapping fragments of random sizes. The PCR products were cloned using TOPO TA Cloning (Invitrogen), sequenced and recombined into destination vectors: pGateway-nlsVenus-attB and/or pGateway-Gal4 and integrated into the attP2 site [pGateway-Rluc vector was used for luciferase assays. PCR amplified tin was cloned into pActC-GFP or pAct5C-FLAG plasmids to generate GFP-Tin and FLAG-Tin used in ChIP or luciferase assays, respectively. For site directed mutagenesis of Tin binding sites the most conserved nucleotides within the CACTTGA consensus motif, the “CA” dinucleotide and the first “T” [CACGGTATAGAGGCAACGG and CCGTTGCCTCTATACCGTG for R8, GTTCGTCTACAGGGCAGTCAC and GTGACTGCCCTGTAGACGAAC for R9, and TGCTGTCTAGTTTTGTGTGTTCTG and CAGAACACACAAAACTAGACAGCA for R10.tP2 site . The pGairst “T” , were muUnc-5 in situ hybridization was performed with digoxigenin-labelled probes as previously described [Embryo collection, immunohistochemistry and in situ hybridization were performed as previously described . Reporteescribed . HRP-conStacks of images were obtained using Zeiss Confocal LSM700 Microscope and 20X or 40X oil-immersion. ImageJ was used for quantification of fluorescence within regions of interest (ROI). For GFP fluorescence quantification, all controls and samples were fixed together using the same procedure, stained with GFP antibodies and imaged using the same configurations. Samples and controls were mounted in the same slide for imaging. Image analyses were done using ImageJ software. Background correction was performed individually for each embryo and the intensity for GFP ROIs was divided by the intensity of control areas and finally averaged for each genotypic group.Statistical significance of alterations in luciferase activity levels for reporters with different mutations or fluorescence intensity in different samples were calculated using one-tailed t-test for pair-wise comparisons and histograms were generated using Microsoft Excel 2013.pAct5C-GFP-Tin or pAct5C (as mock control) were fixed in 1% formaldehyde for 10 minutes at room temperature and then lysed. Following shearing the chromatin by sonication, lysates were incubated with rabbit anti-GFP  for 2 hours at 4°C followed by incubation with protein A-sepharose  for an additional 2 hours. Beads were then washed and the immunoprecipitated material were eluted at 70°C overnight. Phenole-chloroform DNA extraction was performed the next day to purify the precipitated DNA. The immunoprecipitated DNA was subsequently quantified by real-time qPCR.ChIP was performed and analyzed essentially as described previously . In summ5 cells were transfected  with the transcription factor plasmid, Rluc construct, and PolIII-Fluc . Cells were analyzed for luciferase activity 36 hours post transfection using the Dual-Glo Luciferase Kit (Promega) according to manufacturer’s instructions. Samples were assessed in triplicate.S2R+ cells were used for luciferase assays. Approximately 10S1 Figtin-ABD; tin346/tin346 mutant background a few CBs maintain reporter gene expression . LacZ co-staining, in the presence of Svp-LacZ reporter, (blue in A or magenta in A”) indicates that these are Tin-negative, Svp-positive CBs (SMCs).In (TIF)Click here for additional data file.


In synthesizing a double-stranded DNA from viral RNA, HIV-1 reverse transcriptase (RT) generates an RNA/DNA intermediate. RT also degrades the RNA strand and synthesizes the second DNA strand. The RNase H active site of RT functions as a nuclease to cleave the RNA strand; however, the structural basis for endonucleolytic cleavage of the RNA strand remains elusive. Here we report crystal structures of RT-RNA/DNA-dATP and RT-RNA/DNA-nevirapine (NVP) ternary complexes at 2.5 and 2.9 Å resolution, respectively. The polymerase region of RT-RNA/DNA-dATP complex resembles DNA/DNA ternary complexes apart from additional interactions of 2′-OH groups of the RNA strand. The conformation and binding of RNA/DNA deviates significantly after the seventh nucleotide versus a DNA/DNA substrate. Binding of NVP slides the RNA/DNA non-uniformly over RT, and the RNA strand moves closer to the RNase H active site. Two additional structures, one containing a gapped RNA and another a bulged RNA, reveal that conformational changes of an RNA/DNA and increased interactions with the RNase H domain, including the interaction of a 2′-OH with N474, help to position the RNA nearer to the active site. The structures and existing biochemical data suggest a nucleic acid conformation-induced mechanism for guiding cleavage of the RNA strand. Lys,3, complementing the primer-binding sequence of the viral ssRNA, forms a double-stranded nucleic acid segment that binds RT to initiate RNA-dependent DNA polymerization; (ii) RT adds nucleotides complementing the (+)RNA strand to synthesize (−)DNA strand starting from the 3′-end of the annealed tRNA; (iii) RNase H activity of RT degrades RNA strand from RNA/DNA hybrid leaving short A-T rich segments, known as polypurine tracts (PPTs), attached to the (−)DNA strand and (iv) the (+)DNA strand synthesis is initiated from the 3′-end of a PPT segment. A non-PPT RNA is usually cleaved in a non-sequence-specific manner. However, the presence of specific nucleotides at positions adjacent to the RNase H site and distal sites in the nucleic acid-binding cleft of RT can enhance RNase H cleavage efficiency (HIV-1 reverse transcriptase (RT) is a central enzyme responsible for copying the viral single-stranded RNA (ssRNA) into a double-stranded DNA (dsDNA) in the cytoplasm of an infected cell . This evficiency . The ratficiency ,7. Unlikficiency , i.e. HIficiency ; see revficiency  and Beilficiency . The prificiency ,15 mightEscherichia coli RNase H is a functionally independent enzyme, whereas the HIV-1 RNase H domain requires additional elements from the polymerase domain for RNase H cleavage activity  are approved for treating HIV-1 infection. In contrast, the RNase H activity has not yet been successfully targeted for blocking viral replication. HIV-1 RNase H has a two cation-dependent nuclease activity , and theactivity . The RNaactivity ,30. TakeStructurally, RT is a heterodimer of p66 and p51 polypeptide chains. The p66 subunit contains both the polymerase and RNase H active sites. The p51 subunit is proteolytically derived from a p66 chain by HIV-1 protease cleaving off the C-terminal RNase H domain; p51 plays a structural role. RT is a highly flexible enzyme that undergoes conformational changes to carry out its activity. Several conformational and functional states of RT have been structurally characterized . While mHere we report four structures of HIV-1 RT-RNA/DNA complexes in the presence of either a dATP substrate or an NNRTI drug, nevirapine (NVP); the RNA/DNA sequences used in the current study are shown in Figure HIV-1 RT construct RT127A containing an RNase H active site mutation D498N and a thumb mutation Q258C for RT-RNA/DNA cross-linking was cloned, expressed and purified following a previously reported protocol . The RNA2. The crystals were taken out of the drop and transferred to a stabilization solution containing 1.5 μl of 50 mM bis-tris propane pH 7.0, 14% PEG 8000 (w/v), 20 mM MgCl2, 5% sucrose (w/v), 5% glycerol (v/v) and 100 mM ammonium sulfate for 10 min. The crystals were then treated for 15 min in a solution containing 5 mM dATP, 14% PEG 8000 (w/v) and 1 mM MgCl2 for forming the RT-RNA/DNA-dATP complex in crystals. The crystals were cryoprotected by dipping for 1 min into the above solution with glycerol concentration increased to 20% (v/v), and flash frozen in liquid N2.The RT-RNA/DNA binary complex was concentrated to 9 mg/ml in 75 mM NaCl and 10 mM Tris pH 8.0. Crystals were grown at 4°C by hanging-drop vapor diffusion against well solution containing 50 mM bis-tris propane-HCl pH 7.0, 10% PEG 8000 (w/v), 100 mM ammonium sulfate, 5% glycerol (v/v), 5% sucrose (w/v) and 20 mM MgCl2.The RNA/DNA, gapRNA/DNA and bulgeRNA/DNA complexes were buffer exchanged and concentrated to 12 mg/ml in 75 mM NaCl and 10 mM Tris-HCl pH 8.0. The crystals were grown at 4°C in sitting drops, by vapor diffusion against well solution containing 50 mM bis-tris propane pH 7.2 – 7.4, 9 – 11% PEG 8000 (w/v), 100 mM ammonium sulfate, 5% glycerol (v/v), 5% sucrose (w/v) and 20 mM MgCl2+ ions, then into 50 μl of solution II [solution I containing 2 mM NVP and 4% DMSO (v/v)] for 3 h 30 min and finally into 10 μl solution III [solution II with glycerol concentration increased to 20%] for 1.5 min. The RT-RNA/DNA-NVP crystals harvested from solution III were flash frozen in liquid N2. A similar procedure was followed to obtain the crystals of RT-bulgeRNA/DNA-NVP or RT-gapRNA/DNA-NVP complexes , 10% (v/v) glycerol and 12% (w/v) PEG 8000] for 30 min to remove Mgs Figure –C. Two m3 crystal of RT-RNA/DNA-dATP, a 200 × 200 × 60 μm3 crystal of RT-DNA/RNA-NVP, a 280 × 120 × 30 μm3 crystal of RT-gapRNA/DNA-NVP and a 240 × 120 × 40 μm3 crystal of RT-bulgeRNA/DNA-NVP complex. Individual datasets were processed and scaled using HKL2000 (http://www.pymol.org/) or ChemBioDraw (http://www.cambridgesoft.com/).Flash-frozen crystals were transported to the F1 beam line at the Cornell High Energy Synchrotron Source (CHESS). X-ray diffraction data were collected from a 280 × 120 × 30 μm HKL2000   compared to the other (Mol-2). Differences in subdomain arrangements are also observed from comparison of multiple crystal structures of RT/NVP binary complex; e.g. PDB IDs 3HVT  versus 1In structures of HIV-1 RT in complexes with RNase H inhibitors, the inhibitors chelate both RNase H active site cations . We aligProtein–nucleic acid interaction and crystallographic B-factor analyses of dATP and NVP complexes suggest that the binding of RNA/DNA to RT is primarily defined by its interactions with palm, thumb and finger subdomains in the polymerase domain, and with the RNase H primer grip in and nearby the RNase H domain . The parThe RNase H primer grip is important for degradation of RNA by RT, and mutations of the primer grip residues cause significant loss of RNase H cleavage activity . When coAn RNA strand of an RNA/DNA duplex is cleaved periodically. The template RNA strand does not consistently interact with RT near the RNase H domain, and thereby no structural element of RT continuously threads the RNA to the RNase H active site for the cleavage of phosphodiester bonds. It seems likely that an RNA/DNA duplex constantly undergoes conformational changes while RT slides over it ,15, and To artificially introduce conformational variations and investigate the effects on the proximity of the RNA strand with respect to the RNase H active site, we introduced (i) a single-nucleotide bulge and (ii) a single-nucleotide gap in the RNA strand of the RNA/DNA duplex Figure  and C. FThe structures of gapRNA-NVP and bulgeRNA-NVP complexes were determined at 3.2 and 3.0 Å resolution, respectively Table . As desi2+ ions are the cofactors for RNase H activity in vivo. However, structures of inhibitors chelated at the RNase H active site of RT could only be obtained in the presence of Mn2+ ions  is observed bound at position A at the RNase H active site, and no binding was observed at position B  might be preventing the formation of a catalytic complex in the current structures. Another structural constraint in nearly all crystal structures of RT is the formation of an extended β-sheet involving two RNase H domains of adjacent molecules. This extended sheet may also restrict the flexibility of RT to attain an RNase H catalytic complex in crystals.2+ (or Mn2+) ion present at the RNase H active site in bulgeRNA or gapRNA structures (with a minimum distance of ∼4 Å between the oxygen and ion)  increased by the binding of an NNRTI and (ii) decreased by the binding of a dNTP . All str) Figure  and D. C) Figure  and B. C) Figure  and D.A recently reported nicked RNA/DNA-RT-efavirenz structure shows a significant deviation in the binding of the RNA/DNA compared to the current structures  and all RT-nucleic acid complexes previously described in the literature; the authors proposed that enhanced interaction between the p51 C-terminus and RNA strand might be critical for RNase H degradation . Our anaThe current series of structures provide insights into how the non-uniform sliding of RNA/DNA, and conformational changes of the duplex near to and distal from the RNase H active site assist the RNA strand in accessing the active site for cleavage ,52,54. FThe atomic coordinates and structure factors for the structures of RT-RNA/DNA-dATP, RT-RNA/DNA-NVP, RT-gapRNA/DNA-NVP and RT-bulgeRNA/DNA-NVP complexes are deposited in Protein Data Bank (PDB) under accession numbers 4PQU, 4PUO, 4Q0B and 4PWD, respectively.Supplementary Data are available at NAR Online.


Dose-dependent differential gene expression provides critical information required for regulatory decision-making. The lower costs associated with RNA-Seq have made it the preferred technology for transcriptomic analysis. However, concordance between RNA-Seq and microarray analyses in dose response studies has not been adequately vetted.t) cut-off while microarray results varied more based on the fold change criteria, although responses strongly correlated. Verification by WaferGen SmartChip QRTPCR revealed that RNA-Seq had a false discovery rate of 24% compared to 54% for microarray analysis. Dose–response modeling of RNA-Seq and microarray data demonstrated similar point of departure (POD) and ED50 estimates for common DEGs.We compared the hepatic transcriptome of C57BL/6 mice following gavage with sesame oil vehicle, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 μg/kg TCDD every 4 days for 28 days using Illumina HiSeq RNA-Sequencing (RNA-Seq) and Agilent 4×44 K microarrays using the same normalization and analysis approach. RNA-Seq and microarray analysis identified a total of 18,063 and 16,403 genes, respectively, that were expressed in the liver. RNA-Seq analysis for differentially expressed genes (DEGs) varied dramatically depending on the P1 contains supplementary material, which is available to authorized users. Toxicogenomic evaluations by microarrays have been invaluable in elucidating underlying mechanisms of toxicity ,2, invesAnalysis approaches have been developed specifically for RNA-Seq -21, yet While microarray data submissions are encouraged by regulatory agencies and widely accepted in toxicogenomic research ,13, the p-dioxin (TCDD) is a persistent environmental contaminant that binds to the aryl hydrocarbon receptor (AhR) which then translocates to the nucleus and dimerizes with the aryl hydrocarbon receptor translocator (ARNT) [2,3,7,8-Tetrachlorodibenzo-r (ARNT) ,27. The r (ARNT) ,27, and r (ARNT) -31. Doser (ARNT) ,32,33. I50.Overall, RNA-Seq and Agilent generated comparable results. Quantitatively, RNA-Seq detected more genes expressed in the liver, and identified more differentially expressed genes (DEGs) compared to Agilent. Qualitatively, direct sequencing by RNA-Seq provided more accurate transcript identification. Verification by WaferGen SmartChip QRTPCR indicates RNA-Seq had fewer false-positives and false-negatives compared to Agilent. Dose response modeling was also consistent between both platforms, but the ability of RNA-Seq to detect low abundance transcripts and its larger dynamic range provided superior qualitative and quantitative data for DEG identification, and estimates of point of departure (POD) and EDGenome-wide hepatic gene expression was examined using NGS Sequencing and Agilent 4×44 K oligonucleotide microarrays using the same RNA samples. RNA-Seq was performed at an average read depth of 30 M resulting in ~21 M high quality reads per sample Figure . Reads wComparison of detected genes using three biological replicates identified 12,165 genes which were detected either by RNA-Seq or microarray and annotated with corresponding Entrez and Ensembl Gene IDs Figure . Among tt)) cut-offs to investigate the effects of filtering criteria on DEG detection without violating parametric hypothesis testing assumptions.Contrary to previous comparative studies, we used the same semi-parametric normalization and empirical Bayes analysis to identify DEGs in both RNA-Seq and microarray datasets. Empirical Bayes analysis allows the varying of fold change and posterior probability (P1(t) value correlation  filtering criteria. Increasing the P1(t) cut-off from ≥0.8 to ≥0.9999 while maintaining a |fold change| cut-off of ≥1.5 decreased the number of DEGs from 3,546 to 460 whereas increasing the |fold change| cut-off from >1.5 to >2.0 decreased the number of DEGs from 3,546 to 1,249  values  ≥ 0.8 were used for RNA-Seq while a |fold change| ≥ 1.5 and P1(t) ≥ 0.999 were used for microarray in all subsequent comparisons in this study.These differences in sensitivity to filtering criteria may be attributed to the larger dynamic range of RNA-Seq which exhibits a wider range of fold changes Figure  and a mos Figure . ConsequCyp1a1, Cyp1a2, Nqo1, and Tiparp  value correlations, indicating a strong correlation between RNA-Seq and Agilent. As seen with sample size, relaxing the cut-off criteria for the union of the RNA-Seq and microarray data sets increased the overlap suggesting differences were most likely due to genes approaching but not satisfying hard fold change and/or P1(t) cut-offs, despite evidence of differential expression.Within the 12,165 common RNA-Seq and microarray genes, 901 and 1,270 DEGs were identified, respectively Figure . ComparaDespite the overlap of only 449 genes, comparison of functional enrichment analyses identified DEGs associated with lipid binding, processing, and metabolism, oxidative stress, immune responses, cell adhesion and movement, and extracellular matrix remodeling within RNA-Seq and Agilent data sets , 13 positive controls (changed in both datasets), 35 RNA-Seq specific genes, and 34 Agilent specific genes  estimates for the benchmark dose  and the benchmark dose limit (BMD(L); lower limit of a one-sided 95% confidence interval on the BMD) were determined using BMDExpress . BMDExpr50 estimates, 709 of 1,249 RNA-Seq DEGs exhibited a sigmoidal dose response as determined by the ToxResponse Modeler [50s , found Agilent ED50s to be higher compared to RNA-Seq at low estimates. In contrast, RNA-Seq ED50s were higher at higher estimates  values on a per gene and dose basis [t) values do not test hypotheses, and can be used to rank and prioritize DEGs based on their probability of differential expression. This allows cut-off criteria to be varied in order to include well characterized responsive genes and mechanistic significance in the context of observed phenotypes, without violating any assumptions. In our study, varying the cut-off criteria demonstrated that RNA-Seq was particularly sensitive to P1(t) changes likely due to its much larger dynamic range compared to microarrays  cut-off. Comparing RNA-Seq and Agilent datasets for DEGs identified a 35% – 50% overlap  identified two principal contributing factors. First, some Agilent probes were misannotated and did not target the expected gene possibly due to outdated annotation provided by manufacturer, but nevertheless, requiring re-evaluation and re-annotation. Second, multiple probes for the same gene also showed divergent responses indicating either non-specific binding (cross-hybridization) or the presence of variant transcripts that targeted individual probes. RNA-Seq analysis mitigates these disadvantages by using the most recent genome build available and its associated annotation, while providing the opportunity to identify treatment-specific transcript variants.50 values and point of departure (POD) estimates such as BMD and BMD(L). Despite the challenges of incorporating omic data into risk assessment, the goal is to provide data that complements existing testing guidelines and requirements to support a more quantitative, mechanistically-based risk assessment.Risk assessments typically involve four components: hazard identification, dose response characterization, exposure assessment and risk characterization. Omic technologies are expected to improve risk assessment by providing more qualitative and quantitative data. This includes the use of gene expression profile or fingerprint classifiers that not only identify comparable modes/mechanisms of action in hazard identification, but could also justify the use of refined uncertainty factors for extrapolation between species. In addition, RNA-Seq provides a greater number of responsive genes that can be functionally annotated, associated with a key event, and modeled for ED50 values were considered to be more accurate due to its ability to detect lower abundance genes and greater dynamic range  and had free access to deionized water. On PND 28 and every following 4th day animals (N = 5) randomly assigned to treatment groups were orally gavaged with 0.1 mL sesame oil vehicle control or 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 μg/kg TCDD  for a total of 28 days. On day 28 mice were sacrificed and livers were frozen in liquid nitrogen, and stored at-80°C. We assumed that randomization and consistent conditions across all treatment groups negated any gene expression effects due to differences in estrous stage. All procedures were carried out with the approval of the Michigan State University All-University Committee on Animal Use and Care.Female C57BL/6 mice from Charles Rivers Laboratories  were received on postnatal day 25 (PND25), housed in polycarbonate cages with cellulose fiber chips  at 30–40% humidity, and acclimatized for 4 days using a 12 h light/dark cycle. Mice were fed 260/A280 ratio and by visual inspection on a denaturing gel. All sample processing and analysis was performed blinded to treatment group when possible.Frozen liver samples (~100 mg) were transferred to 1.3 mL of TRIzol  and homogenized using a Mixer Mill 300 tissue homogenizer . Total RNA was isolated according to manufacturer’s protocol with an additional phenol:chloroform extraction . Isolated RNA was resuspended in RNA storage solution (Life Technologies). Total RNA was quantified and assessed for purity by nanodrop , Qubit (Life Technologies), and Bioanalyzer . Total RNA quality was also assessed using the ATotal RNA from treated animals were hybridized with vehicle control samples to 4×44 K Agilent microarrays . Microarrays were performed with three biological replicates (N = 3), commonly used in microarray assessments ,32,33, ut)) values were calculated using an empirical Bayes method based on a per gene and dose basis using model-based t values [Data were normalized using a semi-parametric approach  in SAS vt values . Priors t values  and depohttps://rtsf.natsci.msu.edu/genomics). In summary, libraries from five independent biological replicates (N = 5) were prepared using the Illumina TrueSeq RNA Sample Preparation Kit  according to manufacturer’s instructions. Library sizes were confirmed using Caliper GX , and quantified by qPCR using the Kapa Biosystems quantification kit . Sequencing of libraries was performed by pooling 10 random samples at equimolar ratios, quantified again by qPCR (Kapa Biosystems), and distributed across two lanes of the flow cells to maintain library complexity, and loaded onto an Illumina HiSeq 2500 and clustered onboard.RNA-Sequencing was performed at the Michigan State University Research Technology Support Facility Genomics Core  was used for read processing and analysis. Reads, 1×50 bp with a seven-base index, were demultiplexed and quality was determined using FASTQC v0.11.2 (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adaptor sequences were removed using Cutadapt v1.4.1 [http://hannonlab.cshl.edu/fastx_toolkit/index.html). Reads were mapped to the mouse reference genome (GRCm38 release 74) using Bowtie 1.0.0 and TopHat v1.4.1 [samtools.sourceforge.net/). Gene counts were determined using HTSeq v0.6.1 [The Michigan State High Performance Computer  according to the DESeq reference manual. Data was normalized using a semi-parametric approach [t) values were calculated using an empirical Bayes method based on a per gene and dose basis using model-based t values [Counts were transformed through variance stabilizing transformation (VST) using the DESeq package  in R  [Functional enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery   filtered-ΔΔCt method standardized to the geometric mean of reference genes ActB, B2m, Gapdh, Hmbs, Hprt, Rn18s, and Rps13. Primer sequences are available in Additional file post-hoc test, which contrasts to vehicle control, using SAS 9.3.Total RNA (2 μg) was reversed transcribed using SuperScript II (Invitrogen) and oligo-dT primers. The WaferGen SmartChip  was prepared according to manufacturer’s instructions at a final cDNA concentration of 1.25 ng/μl, primer concentration of 250 nM, and 1X SYBR Green mastermix  dispensed using the WaferGen SmartChip Multisample Nanodispenser. Amplification  and detection was performed using the WaferGen Real-Time PCR Cycler at RTSF. Expression was determined using the 250s was performed using the ToxResponse Modeler [50 modeling, gene responses were split into groups of 50 and run in parallel on the MSU HPCC. Only genes with sigmoidal dose-responses  estimates benchmark dose (BMD) and lower 95% confidence limit BMD (BMDL) were estimated as previously described using BMDExpress [Dose–response modeling for the estimation of ED Modeler . For RNAl., 2008  for examDExpress ,44. Brie


HIV viral load (VL) testing is the gold standard for antiretroviral treatment monitoring, but many barriers exist to VL testing in resource-limited settings, including storage and transport limitations for whole blood and plasma. Data from various studies indicate that HIV RNA is stable beyond current recommendations. We conducted a systematic review to assess stability data of HIV RNA in whole blood and plasma across times and temperatures.Using a pre-defined protocol, five databases were searched for studies where blood samples from HIV patients were stored at time and temperature points that exceeded manufacturer recommendations. RNA stability, the primary outcome, was measured by the difference in means compared to samples stored within established thresholds. RNA stability was defined as ≤0.5 log degradation. The search identified 10,716 titles, of which nine full-text articles were included for review. HIV RNA maintained stability in EDTA whole blood and plasma at all measured time points up to 168 hours when stored at 4°C, while stability was detected at 72 hours (95% confidence) in whole blood at 25°C, with data points before and beyond 72 hours suggesting stability but not reaching statistical significance. For EDTA plasma stored at 30°C, stability was maintained up to 48 hours (95% confidence), with OLS linear regression estimates up to 127 hours, suggesting stability. Overall, quality of studies was moderate. Limitations included small sample sizes, few studies meeting inclusion criteria, and no studies examining RNA stability in low viremia  environments.Whole blood and plasma samples in EDTA may remain stable under conditions exceeding current manufacturer recommendations for HIV VL testing. However, given the limited number of studies addressing this question, especially at low levels of viremia, additional evaluations on HIV RNA stability in EDTA tubes and PPT in field conditions are needed. The 2013 WHO HIV treatment guidelines strongly recommend routine viral load (VL) testing six and twelve months after treatment initiation and then at least every year thereafter  Despite the benefits of VL testing, HIV RNA sample transport constraints limit testing access in resource-limited settings. It is estimated that only 20% of people on antiretroviral treatment (ART) in Africa access routine VL testing For VL testing, samples may be shipped to the laboratory as whole blood, plasma, or dried blood or plasma spots (DBS/DPS), depending on the resources available. Optimizing sample collection options depends on the trade-offs between test accuracy and logistical capacity.DBS/DPS remain stable over extended time and temperature periods, often making this the only practical transport option in remote areas. However, the smaller sample volume reduces sensitivity and the cell-associated nucleic acid can reduce specificity Plasma transport with ethylenediaminetetraacetic acid (EDTA) tubes or plasma preparation tubes (PPT) is the gold standard for VL testing. However, generating plasma samples requires trained phlebotomists and electricity in health posts to power the centrifuges. Current recommendations for plasma storage and transport require that plasma is transported within 24 hours at 25°C in EDTA tubes, or within 5 days at 4°C for EDTA tubes or PPT, after centrifugation  [6]. If The limited sample transport time and temperature thresholds effectively limit the radius of whole blood transport to 6 hours at 25°C or to 24 hours at 4°C. The implications of such cautious labelling would not significantly affect care in developed countries, since maintaining the cold chain is less difficult, and there is widespread access to centrifuges and efficient specimen transport services. However, in resource-poor environments, such restrictive guidance on whole blood and plasma transport greatly limits access to VL testing to only those in close proximity to national or regional laboratories. This systematic review seeks to measure HIV RNA stability in EDTA tubes and PPT at extended time and temperature thresholds, compared to the established thresholds, in all published studies.th, 2013 or October 28th, 2013, depending on database.Using a pre-defined protocol, five databases  were searched according to pre-specified search terms . No restAfter an initial title search, selected abstracts were reviewed in duplicate using piloted forms. Any disagreements were resolved with a third reviewer. The same procedure was followed for inclusion of full text articles and data extraction. Articles were included if they measured HIV RNA stability beyond the established time and temperature thresholds for whole blood or plasma with either EDTA tubes or PPT. Extracted information included characteristics of the study, population, intervention, outcome and risk of bias. Recommended time and temperature thresholds include: <6 hours at room temperature (defined as 25°C) for whole blood in EDTA tubes or PPT and <24 hours in whole blood in EDTA at 4°C. Plasma can be stored for 24 hours at room temperature (defined as 25°C) in EDTA tubes or 5 days at 4°C for EDTA tubes or PPT.Risk of bias was estimated for each study according to a modified Jadad scale adapted for basic science The primary outcome measured was mean log declines in RNA stability beyond recommended time and temperature thresholds, with significant declines in RNA stability reported as a secondary outcome. Data was analyzed by mean VL differences at different temperature thresholds. Mean and standard errors were calculated from median and range when not otherwise provided A viral degradation cutoff of > 0.5 log copies/mL was used to define clinical significance, consistent with the literature The database search yielded 10,716 unique titles ; 60 artiStudies measured RNA degradation in EDTA whole blood (n = 6)With regards to risk of bias, according to a modified Jadad scale , all stuFor EDTA whole blood, six studies assessed RNA stability at 4°C and room temperature (25°C), with time points at zero, six, 18, 24, 48, 72, and 168 hours . HIV RNAThree studies assessed the stability of RNA in EDTA plasma, at 4°C, 22°C, 30°C and 37°C, with time points at 24, 48, 168, and 336 hours . There wAt 30°C, HIV RNA maintained stability in EDTA plasma until 48 hours (0.12 log reduction). At 168 hours there was a significant decline of 0.74 log copies/mL, with continued declines at 336 hours (1.6 log copies/mL decline) Only one study assessed the stability of PPT whole blood. It found a no significant degradation 30 hours post collection at 25°C The studies were evaluated under a GRADE framework and found to be of moderate quality, owing to the small sample sizes and failure to exclude publication bias .Our analysis indicates that HIV RNA is stable in EDTA tubes beyond the current manufacturer recommendations for time and temperature and highlights the need for more definitive studies at higher temperatures that may be encountered in countries with high HIV burdens. For EDTA whole blood transport, RNA remains stable until 72 hours, with data points at times beyond 72 hours suggesting stability, but with 95% confidence intervals that spanned the stability threshold. For EDTA plasma at 30°C, measures of RNA were within the stability threshold at 48 hours. While a meta-regression suggested stability beyond 48 hours (estimated stability until 127 hours), no data points were available between 48 and 168 hours and one cannot assume linear RNA degradation under these conditions. More data is needed to confirm the true stability of EDTA plasma at 30°C beyond 48 hours. These studies indicate that transport for both EDTA whole blood and plasma can potentially be greatly expanded, thus increasing the feasibility of transporting blood tubes to laboratories in resource limited settings.Acceptable RNA degradation over the course of sample transport needs to be assessed through the lens of clinical relevance. A VL in whole blood or plasma of 1,000 copies/mL is recommended by the 2013 consolidated WHO HIV treatment guidelines as the threshold by which to define virological failure The precedent for expanded sample transport thresholds have already been set by the British HIV Association. In their 2011 guidelines for routine monitoring, EDTA whole blood storage for 2–3 days at room temperature is recommended, with evidence cited from at least one well-designed quasi-experimental study Adapting sample transport regulations to match the demonstrated HIV RNA stabilities would enable VL sample collection to expand and reach more people living with HIV. A one-week sample transport window would allow for bi-weekly or weekly sample collection, provided samples were kept at 25°C, as might be possible with passive cooling devices, such as a cooler box.Countries implementing VL testing in resource–limited settings will need guidance on acceptable time and temperature thresholds for sample transport, particularly in hot climates. Given that the studies included in this systematic review indicate that stability at extended time and temperature thresholds is greater than current recommendations indicate, we recommend that suppliers of EDTA tubes and PPT, as well as viral load tests, urgently undertake the necessary studies to allow for more flexible sample transport recommendations.Some of these studies are not sufficiently powered to assess significance at 95% confidence at particular timepoints. Much of this variability may be attributed to variation across viral loads in patient samples, rather than over time. As only aggregate data was available, a matched pair comparison of differences in HIV RNA stability over time was not possible, thus confidence intervals around the point estimates are conservative estimates.The majority of included studies were published prior to 2000 and the Roche Amplicor HIV-1 Monitor was the most utilized VL assay. While the age of these studies would not impact the RNA stability, newer VL technologies may better detect RNA and could narrow the RNA degradation gap. Our study did not incorporate the variability in precision between VL assays, although these have been documented These studies were laboratory-based, rather than examining the field impacts of sample transport beyond time and temperature thresholds, with only one study examining PPT transport. Given the logistical constraint of centrifugation, the added value of PPT may be limited if whole blood remains stable in EDTA tubes for extended periods. Additionally the inconsistent assessments of time and temperature thresholds limited comparability across studies.The impact of sample transport conditions on low and undetectable VLs need to be further examined. The majority of people in these studies had VLs over 3.5 log copies/mL with some not receiving ART. Populations accessing VL monitoring today are generally receiving ART, and VLs in these populations are lower than those not on ART This systematic review examined RNA stability over time and temperature, but it did not examine increases in HIV RNA past time and temperature thresholds. Extended storage time may erroneously detect intracellular viral particles after cell lysis, which is of unknown clinical significance All published studies show that HIV RNA has an extended time and temperature stability compared to current product labelling. Further studies are needed to determine the precise threshold of expansion and to correlate possible time and temperature-induced increases on HIV RNA at low VLs, especially with the newest VL technologies. This information is urgently needed in order to revise sample transport guidelines and facilitate the expansion of HIV VL monitoring in low- and middle-income countries.Appendix S1Standard thresholds by manufacturer.(DOCX)Click here for additional data file.Appendix S2Review Protocol.(DOC)Click here for additional data file.Appendix S3Risk of bias adapted Jadad Scale.(DOCX)Click here for additional data file.Appendix S4GRADE Analysis.(DOCX)Click here for additional data file.Appendix S5Stata meta-regression command and output.(DOCX)Click here for additional data file.Checklist S1PRISMA 2009 Checklist.(DOC)Click here for additional data file.


Through these mechanisms, lncRNAs form RNA-dsDNA triplex. CCAT1, GAPLINC, GAS5, H19, MEG3, and TUSC7 play oncogenic or tumor suppressor roles by correlated with tumor suppressor P53 or onco-protein c-Myc, respectively. In conclusion, interaction with DNA, RNA and proteins is involved in lncRNAs’ participation in gastric tumorigenesis and development.Long noncoding RNAs (lncRNAs) are non-protein coding transcripts longer than 200 nucleotides. Aberrant expression of lncRNAs has been found associated with gastric cancer, one of the most malignant tumors. By complementary base pairing with mRNAs or forming complexes with RNA binding proteins (RBPs), some lncRNAs including GHET1, MALAT1, and TINCR may mediate mRNA stability and splicing. Other lncRNAs, such as BC032469, GAPLINC, and HOTAIR, participate in the competing endogenous RNA (ceRNA) network. Under certain circumstances, ANRIL, GACAT3, H19, MEG3, and TUSC7 exhibit their biological roles by associating with microRNAs (miRNAs). By recruiting histone-modifying complexes, ANRIL, FENDRR, H19, HOTAIR, MALAT1, and PVT1 may inhibit the transcription of target genes  Generally defined as transcripts longer than 200 nucleotides, long noncoding RNAs (lncRNAs) are lack of significant open reading frames . AccordiAmong the malignant tumors, gastric cancer remains the fourth most prevalent one and the second leading mortality . Over thIn this article, we summarized the molecular mechanisms of lncRNAs on gastric cancer. By interacting with DNA, RNA, and proteins, lncRNAs play crucial roles in gastric tumorigenesis and development.FOXF1) adjacent non-coding developmental regulatory RNA (FENDRR) anchors to targeted promoter fragments [DHFR) gene combines with the key regulatory region of its host gene in a triple helical model [LncRNAs may combine with histone-modifying complexes and then target on DNA . For exaragments . HOX traragments . Additioal model . These tal model .The base paring is formed between protein-coding transcript and the complementary lncRNA, the natural antisense transcript (NAT). Interestingly, the RNA duplex may be created by incompletely base pairing between Alu elements in a targeted mRNA and the complementary sequence harbored in an lncRNA. Staufen 1 (STAU1) protein recognizes the dsRNA binding sites and then results in mRNA degradation. This type of lncRNAs is called 1/2-STAU1-binding site RNAs . And theIrrespective of the direct interaction, the indirect mode between lncRNAs and their targeted mRNAs is illustrated by metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) Figure  and gastGHET1 potentiates the combination between insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and c-Myc mRNA Figure . Albeit Sry gene enable the formation of a circular RNA [About a quarter of lncRNAs are consist of one or more Alu elements . Inverteular RNA . Apart fBy assembly of RNA induced silencing complex (RISC), miRNAs lead mRNA degradation . StudiesmiR-675, processed from its precursor , has the properties of promoting proliferation and epithelial-mesenchymal transition (EMT) Figure . H19 is Besides, lncRNAs’ expressions may be repressed by miRNAs -49. The MIR-99A and MIR-449A  and thus induces the overexpression of MEG3 . By recrA Figure .vice versa, can also be found in other parts of this article.The examples about miRNAs’ regulating lncRNAs via epigenetic modification, Emerging evidences suggest that lncRNAs may participate in competitive endogenous RNA (ceRNA) network , in whicPlasma HOTAIR levels in gastric cancer patients are higher than health controls . ThroughBased on miRNA and lncRNA microarray data of gastric cancer, our group constructed a ceRNA network interlaced by gastric cancer-associated lncRNAs, miRNAs, and mRNAs . For insAs many as 38% lincRNAs cooperate with at least one of multiple histone-modifying complexes . This suMIR-34A loci  to target genes  promoter -specific methyltransferase 2A (MLL) . FENDRR c cancer . Regardlr Figure . The ful4a locus . PRC2 isp53 mutations, DNA hypermethylation, as well as activated E2F, Myc and Ras oncogenic pathways [Among the transcription factors (TFs) activating carcinogenesis, P53 and c-Myc represent potent inducers. The proliferative subtype is a well-defined subtype of gastric cancer characterized by pathways . H19 is pathways  and oncopathways .Recent study identified several lincRNA loci enriching consensus P53 responsive elements (PREs) . Meanwhic-Myc enhancer region [Myc protein contains a basic DNA binding domain that binds to E-box DNA recognition fragment (CACGTG) . CpG islr region .With the exception of the direct interaction with proteins, how do lncRNAs regulate the levels of proteins is arresting. GAS5 positively influences YBX1 protein stability without increasing its transcription . The putH19 is found bound with Isthmin 1 (ISM1)  and posiper se matches the potential of HOTAIR in metastasis in gastric cancer.There are also inhibitory effects of lncRNAs on the binding proteins, such as nc886 on double stranded RNA-dependent protein kinase (PKR) Figure  and HOTALncRNAs are characterized by the complexity of their mechanisms. We summarized lncRNAs’ interaction with DNA, RNA, and proteins in gastric cancer occurrences.It is tempting to speculate that a multitude of lncRNAs may interrupt definitive steps in numerous tumor suppressive and oncogenic pathways. The uncovering of the underlying mechanisms of lncRNAs may benefit our understanding of gastric cancer's pathogenesis.


Craniofacial reconstruction is to estimate an individual's face model from its skull. It has a widespread application in forensic medicine, archeology, medical cosmetic surgery, and so forth. However, little attention is paid to the evaluation of craniofacial reconstruction. This paper proposes an objective method to evaluate globally and locally the reconstructed craniofacial faces based on the geodesic network. Firstly, the geodesic networks of the reconstructed craniofacial face and the original face are built, respectively, by geodesics and isogeodesics, whose intersections are network vertices. Then, the absolute value of the correlation coefficient of the features of all corresponding geodesic network vertices between two models is taken as the holistic similarity, where the weighted average of the shape index values in a neighborhood is defined as the feature of each network vertex. Moreover, the geodesic network vertices of each model are divided into six subareas, that is, forehead, eyes, nose, mouth, cheeks, and chin, and the local similarity is measured for each subarea. Experiments using 100 pairs of reconstructed craniofacial faces and their corresponding original faces show that the evaluation by our method is roughly consistent with the subjective evaluation derived from thirty-five persons in five groups. Craniofacial reconstruction  aims to The craniofacial face is one of the most complex geometric objects in the natural world. How to evaluate the results of the craniofacial reconstruction is still a challenging problem. Existing evaluation methods of the craniofacial reconstruction can be divided into three types: subjective qualitative evaluation method, objective quantitative evaluation method, and the combination method of subjective and objective evaluation. Subjective evaluation methods evaluate the craniofacial reconstruction results subjectively by designing different evaluation strategies. Quatrehomme et al.  invited Some scholars made a preliminary exploration on evaluating craniofacial reconstruction results by objective method. Feng et al.  used a rSeveral researchers combined subjective and objective evaluations. For example, VaneZis  invited In this paper, we propose a new global and local evaluation method of craniofacial reconstruction based on the geodesic network. We define the weighted average of the shape index value in a neighborhood as the feature of one vertex. The absolute value of the correlation coefficient of the feature of all corresponding geodesic network vertices between two models is taken as the similarity. It lays a foundation for qualitative and quantitative analysis of craniofacial reconstruction results and provides guidance for improvement of the craniofacial reconstruction methods.The rest of this paper is organized as follows. The materials and methods are presented in This study was approved by the Institutional Review Board (IRB) of Image Center for Brain Research, National Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University. The study was carried out on a database of 208 whole head CT scans on voluntary persons that mostly come from Han ethnic group in North of China, aged from 19 to 75 years. The CT scans were obtained by a clinical multislice CT scanner system (Siemens Sensation16) in Xianyang Hospital located in western China. As The craniofacial reconstruction evaluation is to assess the similarity between the reconstructed craniofacial face and the original face. We propose an objective method of the craniofacial reconstruction evaluation based on the shape index of the geodesic network vertices. The method evaluates the similarity both globally and locally. The geodesic network of the reconstructed craniofacial face and the original face are built, respectively, by geodesics and isogeodesics, whose intersections are geodesic network vertices. The weighted average of the shape index values within a neighborhood of each geodesic network vertex is computed for each craniofacial face model. The weighted average is considered as the feature of the vertex. The absolute value of the correlation coefficient of the features of all corresponding geodesic network vertices is taken as the holistic similarity between two models. To evaluate the reconstruction locally, these geodesic network vertices of each model are divided into six subareas, forehead, eyes, nose, mouth, cheeks, and chin. The absolute value of the correlation coefficient of the features of corresponding network vertices in each subarea between two craniofacial models is taken as the local similarity.Geodesic is the curve with geodesic curvatures equal to zero. It is the shortest path between two points on a surface. Due to its intrinsic invariance, geodesic can be applied to face recognition that is insensitive to expression. According to geodesic, a geodesic polar coordinate system can be constructed by exponential mapping , 16. InsO is the highest point of the whole model, that is, the point which has the biggest y value  in Frankfurt coordinate. Secondly, n isogeodesics are extracted according to the geodesic distance to the source point. The extracted n isogeodesics are well-distributed with the same interval between two of them. The interval is one out of n of the shortest geodesic distance from the nose tip point to the boundary point set B. So the isogeodesics set IG can be obtained by the following formula:O, dXj) represents the isogeodesic that the geodesic distance from the source point O to the point on the curve is equal to dXj. Geodesic distance can be solved by the existing geodesic algorithms, such as MMP [m geodesics are computed from the tip nose according to an equal angular interval. The equal angle can be found as follows: (1) the direction of the nose tip and the middle point of eyebrows is selected as the initial direction; (2) the outermost isogeodesic is projected into the tangent plane of the nose tip, and the cross points of the equal angular line from the nose tip with the projection of the outermost isogeodesic on the tangent plane are computed; (3) project the cross point into original outermost isogeodesic, and these equal division points Pi are taken as the target points of geodesics; (4) geodesic Gi from the nose tip to the target points Pi can be computed as follows:Qij of the geodesics Gi and isogeodesics IGj are calculated, and all intersection points constitute the intersection point set Q, that is, geodesic network point set:The detailed steps for constructing the geodesic network are described as follows. Firstly, we find the nose tip of the craniofacial face model and make it as the source point of geodesics. Because the craniofacial models are frontal, the nose tip point h as MMP , ICH [18h as MMP , and SVGh as MMP . Here, wh as MMP . ThirdlyQ1 and Q2 be the geodesic network point sets of the craniofacial models M1 and M2, respectively. The geodesic network point Qij1 of M1 and Qij2 of M2 are the corresponding points of the two craniofacial face models since they have the same initial directions and the same source (the nose tip).SI) is the feature generated by the principal curvatures κ1 and κ2. It can capture the “local” shape of a surface. Thus, we evaluate the craniofacial reconstruction based on the correlation coefficient of the shape index value of the corresponding geodesic network vertices between the original and reconstructed craniofacial faces. Shape index SI is defined by the curvedness-orientation-shape map on sphere (COSMOS) representation [P. Every distinct surface shape corresponds to a unique value of SI. At a point P, the shape index SI is defined asDue to the intrinsic invariance of geodesic, the features of the geodesic network vertices can be used to evaluate the results of craniofacial reconstruction. Shape index  and SR(P′), respectively. To avoid the interference of the noise and obtain the robust shape index value, we define the weighted average of the shape index value in a neighborhood as the feature of one vertex. Owing to the important role of the geodesic network vertex in evaluating the two craniofacial models, the weight of the geodesic network vertex itself should be the highest, and the weights of the 1-ring neighborhood of the vertex should be higher than those of the 2-ring neighborhood. In this paper, we choose the weights of 100, 10, and 1 for the network vertex, 1-ring neighborhood, and 2-ring neighborhood, respectively. The features of the corresponding network vertex P and P′ of the two craniofacial models are SO =  and SR =  where N is equal to n × m, that is, the product of the number of isogeodesics and the number of geodesics.For the corresponding network vertexes Generally, the correlation coefficient is used to measure how two sets of variables are linearly related. Here, we use the correlation coefficient to measure the similarity of two craniofacial models. For evaluating the craniofacial reconstruction result globally, we compute the correlation coefficient of the features of all corresponding geodesic network vertices between the original and the reconstructed craniofacial models by the following formula:The global evaluation reflects only the whole similarity between the reconstructed and the original craniofacial models and cannot discern whether the region is well reconstructed or not. In order to evaluate the reconstructed craniofacial model locally, we divide the geodesic network vertices into six parts, that is, forehead, eyes, nose, mouth, cheeks, and chin  method  to reconIn order to evaluate the proposed objective method, we invited 35 subjects to evaluate the 100 reconstructed craniofacial face models. 100 pairs of craniofacial face models were divided into five groups and each group had 20 pairs. 35 subjects were divided into five groups, and each group had seven subjects to evaluate twenty pairs of reconstructed craniofacial faces and corresponding original craniofacial faces. Each subject was asked to observe every pair of faces on the screen and choose the overall similarity degree from the following five degrees as The global evaluation is to compare the features of all geodesic network vertices to get the objective similarity score between the reconstructed craniofacial face and the corresponding original face. Local evaluation is the evaluation of the similarity in six subareas: forehead, nose, eyes, mouth, cheeks, and chin between the reconstructed craniofacial face and the corresponding original craniofacial face. We find the most and the least similar areas and compare them with the subjective assessments. We take three pairs of craniofacial faces in We evaluate all of the 100 reconstructed craniofacial faces locally and compare the local evaluation of six subareas with the global evaluation. We compute the mean similarity score of each subarea of the 100 pairs of faces. The mean similarity scores are shown in Craniofacial reconstruction has a widespread application in forensic medicine, archeology, medical cosmetic surgery, and so forth. However, nearly all researches on craniofacial reconstruction focus on the reconstruction method itself, and little attention is paid to the evaluation methods of the reconstruction. This paper proposes an objective method to evaluate globally and locally the reconstructed craniofacial faces based on the shape index of geodesic network vertices. The geodesic networks of the craniofacial faces are built by geodesics and isogeodesics. For each geodesic network vertex, we define the weighted average of the shape index value in a neighborhood as the feature of the network vertex. The absolute value of the correlation coefficient of the feature of all corresponding geodesic network vertices between two models is taken as the similarity. We used 100 pairs of reconstructed craniofacial faces and their corresponding original faces to evaluate our method. To compare with the subjective evaluation, we also invited 35 subjects to evaluate visually the reconstructed craniofacial faces. Experimental results show that the evaluation by our method is roughly consistent with the subjective evaluation. By evaluating the craniofacial reconstruction effects both globally and locally, we can provide guidance for improvement of the craniofacial reconstruction methods. In addition, since small face expression can be regarded as the isometric deformation, under which the geodesic distance is invariant, the proposed method is also fit for the craniofacial faces of small expression variation.


H replacement refers to RAG-mediated secondary recombination of the IgH genes, which renews almost the entire VH gene coding region but retains a short stretch of nucleotides as a VH replacement footprint at the newly generated VH–DH junction. To explore the biological significance of VH replacement to the antibody repertoire, we developed a Java-based VH replacement footprint analyzer program and analyzed the distribution of VH replacement products in 61,851 human IgH gene sequences downloaded from the NCBI database. The initial assignment of the VH, DH, and JH gene segments provided a comprehensive view of the human IgH repertoire. To our interest, the overall frequency of VH replacement products is 12.1%; the frequencies of VH replacement products in IgH genes using different VH germline genes vary significantly. Importantly, the frequencies of VH replacement products are significantly elevated in IgH genes derived from different autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and allergic rhinitis, and in IgH genes encoding various autoantibodies or anti-viral antibodies. The identified VH replacement footprints preferentially encoded charged amino acids to elongate IgH CDR3 regions, which may contribute to their autoreactivities or anti-viral functions. Analyses of the mutation status of the identified VH replacement products suggested that they had been actively involved in immune responses. These results provide a global view of the distribution of VH replacement products in human IgH genes, especially in IgH genes derived from autoimmune diseases and anti-viral immune responses.V H genes, 27 DH genes, and 6 JH genes within the human IgH locus. The diversified IgH repertoire is generated at different levels, including the random recombination of V, D, and J genes segments, imprecise processing of the coding-ends, addition of non-template nucleotides by terminal deoxynucleotidyl transferase (TdT), random pairing of IgH with Igκ or Igλ light chains, and later through somatic hypermutation and class switch recombination during antigen dependent germinal center reaction  specificities through somatic rearrangement of previously separated variable (V), diversity (D) (for heavy chain only), and joining (J) gene segments to form the variable domain exons of immunoglobulin genes –3. V(D)Jreaction , 11. Forin genes , 13. AnaJ recombination is essential for generating a diverse IgH repertoire, however, it also produces non-functional IgH genes or IgH genes encoding autoreactive antigen receptors , 20. Eargκ locus , 24.H replacement process using the cryptic recombination signal sequences (cRSSs) embedded within the framework-3 regions of previously rearranged VH genes  and by amplification of different VH replacement excision circles  program was developed using the NetBeans 7.01 IDE with Java development kit (JDK) and tested under Windows, Mac OS X, and Ubuntu Linux   regions and DH–JH junction (N2) regions. The frequency of VH replacement products was calculated by dividing the number of IgH genes with VH replacement footprints in the N1 regions with the total number of unique IgH gene sequences. IgH genes with 7, 6, 4, and 3-mer VH replacement footprint motifs within their N1 regions were also analyzed and discussed. The positive prediction value with 95% confidence interval using the 6, 5, 4, and 3-mer VH replacement footprint motifs to assign VH replacement products are 68, 59, 54, and 52%, respectively. In the following comparison, the VH replacement products mainly refer to IgH genes with 5-mer VH replacement footprint within their N1 regions.The IgH gene sequence files from NCBI database were first converted into FASTA files and uploaded to the Vtextes/)  and the H replacement products in IgH genes derived from different keyword sub-categories were analyzed based on the information linked to each sequence in the NCBI GenBank files. The frequencies of VH replacement products with pentameric footprints were used for all these comparisons. For mutational analysis the IgH gene sequences had a minimum of ≥80% nucleotide similarity to the assigned germline VH gene sequences.The distribution of Vt-test. p < 0.05 is considered statistically significant and p < 0.0001 is considered extremely statistically significant.Statistical significance was determined by using either the two-tailed Chi square test with Yates’ correction or the unpaired HRFA computer program to analyze large number of IgH gene sequences and to identify potential VH replacement products . The VH4–28 gene was used less frequently, which was only found in 13 IgH rearrangements (0.03%). The differential usages of individual VH germline genes did not seem to correlate with their relative location within the IgH locus . Then, the VHRFA program uses these libraries to search for the presence of VH replacement footprint motifs with specified lengths at the VH–DH junction (N1) regions or the DH–JH junction (N2) regions of IgH genes. As an initial test of the newly developed VHRFA program, we reanalyzed the 417 human IgH gene sequences that had been to manually identify potential VH replacement products analyzed in a previous study , indicating that the presence of such motifs at the N1 region is likely contributed by VH replacement rather than random nucleotide addition. Among these IgH gene sequences, 12.1% of them contain the 5-mer VH replacement footprint motifs and can be assigned as potential VH replacement products  is extremely statistically significantly higher than that in the overall functional IgH genes . Identification of VH replacement products in non-functional IgH genes fulfills the prediction that VH replacement is a random process that can generate both functional and non-functional IgH rearrangement products. Taken together, these results uncovered a previously unrealized contribution of VH replacement products to the diversification of human IgH repertoire.Within the large number of IgH genes, there are 3818 non-functional IgH gene sequences and 687 of them contain the 5-mer VHRFA program, we further analyzed the distribution of VH replacement products in IgH genes using different VH genes. The frequencies of VH replacement products in IgH genes using different VH germline genes are different  .Using the Vt Figure . For exaH replacement products in the 39,438 unique IgH genes from the NCBI database (12.1%) is much higher than what was estimated in the 417 IgH genes obtained from healthy donors. We reasoned that the majority of IgH gene sequences deposited at the NCBI database was derived from diseased subjects, which may have higher frequencies of VH replacement products. Next, we investigated the distribution of VH replacement products in IgH genes derived from different disease sub-categories. Using the keyword analysis function within the VHRFA program, we can correlate the frequencies of VH replacement products with different sub-categories of IgH gene sequences from the NCBI database. For examples, the frequency of VH replacement products in 558 IgH genes derived from healthy donors is 8.6% . The frequencies of VH replacement products are further enriched in IgH genes derived from RA synovium and in IgH genes encoding rheumatoid factors, suggesting that B-cells expressing VH replacement products are positively selected in the RA synovium to encode rheumatoid factors . Similarly, VH replacement products are highly enriched in IgH genes derived from SLE plasmablasts , suggesting that these enriched VH replacement products contribute to the production of autoAbs in SLE.The overall frequency of VH replacement in IgH genes derived from patients with different autoimmune diseases suggested that VH replacement products may contribute to the production of autoAbs. Indeed, further analyses showed that VH replacement products are statistically significantly enriched in IgH genes encoding rheumatoid factors, anti-Rh (D) Abs, and anti-acetylcholine receptor Abs .The accumulation of VH replacement products are significantly elevated in IgH genes derived from different viral infections. For examples, the frequencies of VH replacement products in IgH genes derived from HIV and HCV infected patients are statistically significantly higher than that in healthy donors . Further analyses showed that the VH replacement products contribute to about 30% of IgH genes encoding anti-HCV glycoprotein E2 Abs or anti-HBVsAg Abs. Such frequencies are statistically significantly higher than that in healthy donors . Taken together, these results showed that VH replacement products are highly enriched in IgH genes derived from patients with different autoimmune diseases and viral infections.To our surprise, the frequencies of VH replacement renews almost the entire VH coding region. Due to the location of the cRSS site, a short stretch of nucleotides is remained as a VH replacement footprint at the newly formed VH–DH junction after the VH replacement process (H replacement footprints can contribute up to two amino acids into the IgH CDR3 to elongate the CDR3. The average CDR3 length of the identified VH replacement products is 18.2 ± 5.0 aa (n = 4417), which is extremely statistically significantly longer than that of the non-VH replacement products   . Detailed analyses showed that the frequencies of K, R, D, and E residues encoded by the VH replacement footprints are statistically significantly higher than their usage in the N1 regions of non-VH replacement products . These results confirmed our previous prediction that VH replacement footprints preferentially contribute charged amino acids to the IgH CDR3 regions.Our previous analysis showed that the V regions , 45. ThiH replacement products derived from different autoimmune diseases or viral infections showed that the identified VH replacement footprints predominantly encode charged amino acids .Charged amino acids within IgH CDR3 are not well tolerated during Ab repertoire development, they are frequently found within the IgH CDR3 regions of autoreactive or anti-viral Abs, which may play important roles in binding charged self or viral antigens, respectively. Further analyses of Vs Figure A. DetailH replacement products have similar mutation rate when compared with the non-VH replacement product derived from healthy donors, patients with autoimmune diseases or viral infections (Figure H replacement products or non-VH replacement products in neonatal IgH gene sequences have much lower mutation rates (Figure H replacement products indicates that these enriched VH replacement products in autoimmune diseases or viral infections had been positively selected.The identified Vs Figure C. As negs Figure C. The acH replacement products in these IgH genes and explore the biological significance of VH replacement products in human antibody diversification and diseases, we developed a Java based computer program VHRFA to analyze large number of IgH gene sequences and to identify potential VH replacement products (H, DH, and JH gene usages in the 61,851 human IgH gene sequences provides a comprehensive view of the human IgH repertoire. In this dataset, the usage of every functional VH germline gene was confirmed, although their usages differ dramatically.In order to determine the distribution of Vproducts . PreviouHRFA program, we identified VH replacement products and analyzed their distributions in the 39,438 unique IgH sequences. Based on the identification of pentameric VH replacement footprint motifs within the VH–DH junctions, 12.1% of the IgH genes can be assigned as potential VH replacement products. These results confirmed our previous estimation that VH replacement products contribute to the diversification of the human IgH repertoire. Interestingly, the frequencies of VH replacement products in IgH genes using the VH2–5, VH3–30, VH3-30-3, VH3–49, VH1–69, and VH3–34 are statistically significantly higher than that in the overall IgH genes. In contrast, the frequency of VH replacement products in IgH genes using the VH6-1 gene is statistically significantly lower than that in the overall IgH genes. Among the non-functional IgH genes, 18% of them contain the pentameric VH replacement footprints and can be assigned as potential VH replacement products. These results confirmed the prediction that VH replacement is a random process that can generate both functional and non-functional IgH rearrangements. Moreover, the high frequency of VH replacement products in non-functional IgH genes suggested that VH replacement products were negatively selected during B-cell development. Based on this reasoning, the frequency of VH replacement products in the non-functional IgH genes may represent the true frequency of VH replacement during early stages of B-cell development, because these non-functional IgH rearrangements cannot encode BCRs and had not been selected during B-cell development.Using the VH replacement footprint at the VH–DH junctions following the VH replacement process (H replacement footprints will elongate the IgH CDR3 regions (H replacement products showed that the average CDR3 length of the identified VH replacement products is 2.8 aa longer than that of non-VH replacement products. Previously, it surprised us that the identified VH replacement footprints preferentially encode charged amino acids within the IgH CDR3 regions (H replacement footprints encoded charged amino acids in the IgH CDR3 regions. Normally, charged amino acids within IgH CDR3 are not well tolerated during antibody repertoire development, probably due to charged residues may generate autoAbs. Indeed, our analysis revealed that VH replacement products are significantly enriched in IgH genes derived from patients with different autoimmune diseases, including RA, allergic rhinitis, and SLE or in IgH genes encoding different autoAbs such as rheumatoid factor, anti-rhesus D antigen, and anti-acetylcholine receptor Abs. Our recent analyses of large number of mouse IgH genes also showed that the frequencies of VH replacement products are enriched in IgH genes derived from autoimmune prone mice (H replacement products contribute to the generation of autoantibodies in both human and mouse.Due to the location of the cRSS site, a short stretch of nucleotides has the potential to remain as a V process , 37, 46. regions , 37, 46. regions , 37, 46. regions . In the one mice . These rH replacement products are significantly elevated in IgH genes derived from various viral infections, including HIV, HCV, and in IgH genes encoding Abs against HCV glycoprotein E2 or HBV surface antigens. Our recent studies showed that VH replacement products are highly enriched in IgH genes encoding different subgroups of anti-HIV antibodies, especially in CD4i and PGT antibodies (H replacement products may contribute to the generation of anti-viral Abs. The majority of the VH replacement footprints identified from anti-viral Abs also encode charged amino acids, which may be important for binding charged viral antigens. Moreover, the accumulation of mutations in these VH replacement products indicated that these enriched VH replacement products in patients with viral infections are positively selected during anti-viral responses. The identification of VH replacement products in autoimmune diseases and anti-viral responses suggested a potential link between viral infections and the pathogenesis of autoimmune diseases. It has long been postulated that chronic viral infections contribute to autoimmunity. However, clear examples that Abs against viral antigens cross-react with self-antigens have only been found in a few cases (H replacement products in IgH genes derived from autoimmune diseases and anti-viral responses.Another important and interesting finding from this analysis of large number of IgH gene sequences is that the frequencies of Vtibodies . These rew cases , 51. HerH replacement was originally proposed as a receptor editing mechanism to change unwanted IgH genes that are either non-functional or encoding autoreactive Abs. The enrichment of VH replacement products in IgH genes derived from autoimmune diseases or encoding autoAbs is particular puzzling. There are several possible mechanisms to explain this finding. First, we have recently shown that crosslinking cell surface BCRs induces VH replacement in human immature B-cells (H replacement recombination might be induced in the immature B-cells during either the anti-viral immune response or autoimmune disease due to persistent antigen stimulation or chronic inflammation. In supporting of this assumption, the number of newly emigrated immature B-cells in the peripheral blood is increased during inflammatory response; and these mobilized immature B-cells may continue to undergo VH replacement recombination ectopically. Second, the intrinsic feature of VH replacement is elongating the IgH CDR3 with charged amino acid. VH replacement products may frequently encode autoAbs and they are efficiently deleted during normal B-cell development. The observed elevated frequencies of VH replacement products in different autoimmune diseases may reflect the defective negative selection in these diseased subjects. Moreover, ectopically occurred VH replacement may bypass the stringent negative selection in the bone marrow and release VH replacement products in the periphery. Last, due to the special features of VH replacement products in generating IgH genes with long and charged CDR3, it is possible that VH replacement products are positively selected by viral antigens during anti-viral responses to produce specific anti-viral Abs. In supporting of this notion, the identified potential VH replacement products encoding anti-HIV antibodies all have very long CDR3 regions with multiple charged amino acid residues (H genes of the identified VH replacement products in the current study also indicated the positive selection. However, the leftover VH replacement products generated during a chronic viral infection may encode Abs that cross-react with self-antigens and later contribute to autoimmunity. In fact, many cell surface antigens and viral antigens are negatively charged, which may be a reason for the selection of VH replacement products with long and charged CDR3 regions.V B-cells . Thus, tresidues . The accH replacement is dependent on the identification of VH replacement footprints within the VH–DH junctions. Any deletion at the 3′ of VH genes or the 5′ of VH replacement footprint motifs during the primary or secondary IgH gene recombination, respectively, may destroy the pentameric VH replacement footprints. Therefore, it is possible that the sequence analysis based study still under-estimates the frequency of VH replacement products. Using the VHRFA program, we extended our analysis our VH replacement products to include potential VH replacement footprint motifs with different lengths. For examples, 33.9% of the IgH genes contain the tetrameric VH replacement footprint motifs and 58.8% of IgH genes contain the trimeric VH replacement footprint motifs. These results revealed a significant contribution of VH replacement products to the IgH repertoire. Recent studies in mice carrying non-functional IgH genes on both IgH alleles demonstrated that VH replacement occurs efficiently to generate almost normal numbers of B-cells with diversified IgH repertoires (H replacement footprints. Therefore, the majority IgH genes generated through VH replacement recombination have no leftover VH replacement footprints. Theoretically, 66% of IgH rearrangements will be out of reading frame and 44% of developing B-cells may carry non-functional IgH rearrangements on both alleles. If all of these B-cells are rescued by VH replacement, a minimum of 44% of the IgH genes might be generated through VH replacement recombination. Under this assumption, IgH genes containing the tetrameric or the trimeric VH replacement footprint motifs at their N1 regions should also be considered as potential VH replacement products.In our sequence based analysis, the assignment of Vertoires . HoweverHRFA program also has its limitation. Although sequence motifs assemble the VH gene 3′ ending sequences can be identified in the N1 regions, such motifs can also be identified within the N2 regions at relative lower frequencies. Theoretically, VH replacement can only leave footprint within the N1 region; the existence of VH replacement footprint like motifs within the N2 regions can only be generated by random nucleotide addition. For IgH genes using the VH6-1 gene, which is the first VH germline gene 5′ to the DH locus, there should have no VH replacement footprint like motifs within the VH–DH junctions, but the VHRFA program still identifies 7.56% of the sequences contains VH replacement footprint like motifs within the VH–DH junctions. We can only refer such motifs as the contribution of random nucleotide addition.Like any sequence based analysis program, the VH replacement products to human Ab repertoire, especially in IgH genes derived from autoimmune diseases or anti-viral responses. Understanding how VH replacement is regulated and how VH replacement products are positively or negatively selected during normal or diseased conditions will be the focus of future studies, because modulation of the level of VH replacement may offer unique approaches to treat different human diseases.In summary, analyses of a large number of human IgH gene sequences from the NCBI database uncovered a significant contribution of VThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fimmu.2014.00345/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.