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1.  Selected base sequence outside the target binding site of zinc finger protein Sp1 
Nucleic Acids Research  2001;29(24):4920-4929.
Human transcription factor Sp1 contains three contiguous repeats of the C2H2-type zinc finger motif and binds to the decanucleotide sequence 5′-(G/T)GGGCGG(G/A)(G/A)(C/T)-3′ (GC box). In order to determine whether the three-zinc finger peptide Sp1(530–623) has selectivity for sequence outside the GC box, we used a selection and amplification of binding experiment. The high affinity sequence generated from this selection is 5′-GGGTGGGCGTGGC-3′ (s-GC box), which is flanked by a novel conserved guanine triplet on the 5′-side of the core decanucleotide. Gel mobility shift assays reveal that Sp1(530–623) binds to the s-GC box with 2.3-fold higher affinity than to the wild-type GC box, 5′-GGGGCGGGGC-3′ (c-GC box). DNase I and hydroxyl radical footprinting analyses show that the area of the s-GC box protected by binding of Sp1(530–623) is wider by 1 nt than that of the c-GC box. On the other hand, alkylation interference analyses demonstrate that Sp1(530–623) forms only one special base contact at the guanine triplet. With respect to cleavage of the c-GC and s-GC boxes by the 1,10-phenanthroline–copper complex (OP-Cu), binding of Sp1(530–623) has no effect on the cleavage pattern of the s-GC box, whereas OP-Cu actually enhances cleavage of the c-GC box. Additionally, the extent of cleavage of the s-GC box by DNase I and OP-Cu is clearly different from that of the c-GC box under peptide-free conditions. The results strongly indicate that: (i) the conformation of the s-GC box is evidently distinct from that of the c-GC box; (ii) Sp1(530–623) binds to the s-GC box without induction of a conformational change in DNA detectable by cleavage with OP-Cu. The present study provides useful information for the design of multi-zinc finger proteins with various sequence specificities.
PMCID: PMC97581  PMID: 11812820
2.  Mutations of the phage lambda nutL region that prevent the action of Nun, a site-specific transcription termination factor. 
Journal of Bacteriology  1992;174(6):1983-1989.
Phage HK022 encodes a protein, Nun, that promotes transcription termination within the pL and pR operons of its relative, phage lambda. The lambda sequences required for termination had previously been shown to overlap the nut sites, which are essential for transcription antitermination during normal lambda growth. To further specify the Nun target and to determine its relation to the nut sites, we constructed deletion and base substitution mutations of the lambda nutL region and measured Nun-dependent reduction of the expression of a downstream reporter gene. The shortest construct that retained full Nun responsiveness was a 42-bp segment that included both boxA and boxB, sequences that have been implicated in lambda antitermination. Deletion of boxA reduced Nun termination, and deletion of both sequences eliminated Nun termination. Base substitutions in boxA and the proximal portion of boxB impaired Nun termination, while base substitutions between boxA and boxB, in the distal portion of boxB, and immediately downstream from boxB had no appreciable effect. The termination defect of all of the base substitution mutations was relieved by increasing the level of Nun protein; in contrast, the deletions and a multiple-base substitution did not regain full Nun responsiveness at elevated Nun concentrations. We also asked if these mutant nut regions retained their ability to interact with N, the lambda-encoded antitermination protein. A qualitative assay showed that mutations within boxA or boxB reduced interaction, while mutations outside boxA and boxB did not. These data show that (i) the recognition sites for N and Nun overlap to a very considerable extent but are probably not identical and (ii) a high concentration of Nun promotes its interaction with mutant nut sites, a behavior also reported to be characteristic of N.
PMCID: PMC205805  PMID: 1532174
3.  Functional Dissection of the Bacillus subtilis pur Operator Site 
Journal of Bacteriology  2003;185(14):4099-4109.
Bacillus subtilis PurR represses transcription of several genes involved in purine synthesis, metabolism, and transport and cofactor synthesis. PurR binds specifically to DNAs containing an inverted repeat of a 14-nucleotide “PurBox” located in the upstream control regions of genes in the PurR regulon. Further biochemical investigation of the interaction of PurR with a series of shortened upstream DNA fragments of the pur operon determined the minimum length and specificity elements of the operator. The relative affinities of the two PurBoxes differ significantly, such that upstream PurBox1 (−81 to −68 relative to the transcription start site) is designated “strong” and downstream PurBox2 (−49 to −36) is designated “weak.” Two PurBoxes are required for high-affinity PurR binding, and one of these must be strong. The shortest DNA construct with high affinity for PurR is a 74-bp perfect palindrome in which weak PurBox2 and its flanking sequences are replaced by strong PurBox1 and flanking sequences. Two PurR dimers bind to this symmetric construct. Phosphoribosylpyrophosphate (PRPP), the effector molecule that reduces affinity of PurR for DNA, requires one weak PurBox in the DNA construct to inhibit PurR binding. PRPP binds, as expected, to a PRPP-motif in PurR. A tracks outside the central conserved CGAA sequence of the PurBox may facilitate DNA bending, leading to a proposal for strong and weak designations of PurBoxes in the control regions of other genes regulated by PurR.
PMCID: PMC164870  PMID: 12837784
4.  TtsI regulates symbiotic genes in Rhizobium species NGR234 by binding to tts boxes 
Molecular microbiology  2008;68(3):736-748.
Infection of legumes by Rhizobium sp. NGR234 and subsequent development of nitrogen-fixing nodules are dependent on the coordinated actions of Nod factors, proteins secreted by a type III secretion system (T3SS) and modifications to surface polysaccharides. The production of these signal molecules is dependent on plant flavonoids which trigger a regulatory cascade controlled by the transcriptional activators NodD1, NodD2, SyrM2 and TtsI. TtsI is known to control the genes responsible for T3SS function and synthesis of a symbiotically important rhamnose-rich lipo-polysaccharide, most probably by binding to cis elements termed tts boxes. Eleven tts boxes were identified in the promoter regions of target genes on the symbiotic plasmid of NGR234. Expression profiles of lacZ fusions to these tts boxes showed that they are part of a TtsI-dependent regulon induced by plant-derived flavonoids. TtsI was purified and demonstrated to bind directly to two of these tts boxes. DNase I footprinting revealed that TtsI occupied not only the tts box consensus sequence, but also upstream and downstream regions in a concentration-dependent manner. Highly conserved bases of the consensus tts box were mutated and, although TtsI binding was still observed in vitro, gfp fusions were no longer transcribed in vivo. Random mutagenesis of a tts box-containing promoter revealed more nucleotides critical for transcriptional activity outside of the consensus.
PMCID: PMC2770584  PMID: 18363648
5.  The HilA Box and Sequences outside It Determine the Magnitude of HilA-Dependent Activation of PprgH from Salmonella Pathogenicity Island 1 
Journal of Bacteriology  2001;183(16):4876-4885.
Salmonella requires genes on the Salmonella pathogenicity island 1 (SPI1) for the intestinal phase of infection in several models of pathogenesis. In Salmonella enterica serovar Typhimurium, most SPI1 genes are arranged in operons that are coordinately regulated by the SPI1-encoded protein HilA. In the past, it has been shown that HilA directly activates two promoters on SPI1, PinvF-1 and PprgH. PinvF-1 contains a HilA binding site, termed a HilA box, that is necessary and sufficient for activation by HilA. The HilA box is 17 nucleotides long and contains a direct repeat comprised of two hexamers separated by 5 nucleotides, centered at −45 relative to the start site of transcription. PprgH also contains a HilA box, and here we investigate its role at PprgH. We have found that the HilA box is necessary, but not sufficient, for HilA-dependent activation of PprgH. Instead, half-site-like hexamers outside the HilA box appear to be required for HilA-dependent activation of PprgH, even though HilA binds to the HilA box in the absence of these hexamers. Thus, although HilA-dependent activation of PinvF-1 and PprgH coordinates the expression of the structural genes for a type III secretion apparatus and the effectors secreted by that apparatus, it is also possible that mechanisms not apparent under in vitro inducing conditions could separate the expression of invFGEABC-spaMNOPQRS-sicA-sipBCDA-iacP-sicP-sptP and prgHIJK-orgABC.
PMCID: PMC99542  PMID: 11466291
6.  Activin A induces ovine follicle stimulating hormone beta using -169/-58 bp of its promoter and a simple TATA box 
Activin A increases production of follicle stimulating hormone (FSH) by inducing transcription of its beta subunit (FSHB). This induction has been studied here in LbetaT2 gonadotropes using transient expression of ovine FSHBLuc (-4741 bp of ovine FSHB promoter plus exon/intron 1 linked to Luc). Several sequences between -169/-58 bp of the ovine FSHB proximal promoter are necessary for induction by activin A in LbetaT2 cells, but deletions between -4741/-752 bp decrease induction > 70% suggesting the existence of other important 5' sequences. Induction disappears if a minimal T81 thymidine kinase promoter replaces the ovine FSHB TATA box and 3' exon/intron. The study reported here was designed to determine if sequences outside -169/-58 bp are important for induction of ovine FSHB by activin A.
Progressively longer deletions of ovine FSHBLuc were created between -4741/-195 bp. Deletions internal to this region were created also, but replaced with substitute DNA. The ovine FSHB TATA box region (-40/+3 bp) was replaced by thymidine kinase and rat prolactin minimal promoters, and substitutions were made in 3' intron/exon sequences. All constructs were tested for basal and activin A-induced expression in LbetaT2 cells.
Successive 5' deletions progressively lowered fold-induction by activin A from 9.5 to zero, but progressively increased basal expression. Replacing deletions with substitute DNA showed no changes in basal expression or fold-induction. Induction by activin A was supported by the minimal rat prolactin promoter (TATA box) but not the thymidine kinase promoter (no TATA box). Replacement mutations in the 3' region did not decrease induction by activin A.
The data show that specific ovine FSHB sequences 5' to -175 bp or 3' of the transcription start site are not required for induction by activin A. A minimal TATA box promoter supports induction by activin A, but the sequence between the TATA box and transcription start site seems unimportant.
PMCID: PMC2714312  PMID: 19552818
7.  Control of Pathogenicity and Disease Specificity of a T-Lymphomagenic Gammaretrovirus by E-Box Motifs but Not by an Overlapping Glucocorticoid Response Element▿  
Journal of Virology  2008;83(1):336-346.
Although transcription factors of the basic helix-loop-helix family have been shown to regulate enhancers of lymphomagenic gammaretroviruses through E-box motifs, the overlap of an E-box motif (Egre) with the glucocorticoid response element (GRE) has obscured their function in vivo. We report here that Egre, but not the GRE, affects disease induction by the murine T-lymphomagenic SL3-3 virus. Mutating all three copies of Egre prolonged the tumor latency period from 60 to 109 days. Further mutating an E-box motif (Ea/s) outside the enhancer prolonged the latency period to 180 days, suggesting that Ea/s works as a backup site for Egre. While wild-type SL3-3 and GRE and Ea/s mutants exclusively induced T-cell lymphomas with wild-type latencies mainly of the CD4+ CD8− phenotype, Egre as well as the Egre and Ea/s mutants induced B-cell lymphomas and myeloid leukemia in addition to T-cell lymphomas. T-cell lymphomas induced by the two Egre mutants had the same phenotype as those induced by wild-type SL3-3, indicating the incomplete disruption of T-cell lymphomagenesis, which is in contrast to previous findings for a Runx site mutant of SL3-3. Mutating the Egre site or Egre and Ea/s triggered several tumor phenotype-associated secondary enhancer changes encompassing neighboring sites, none of which led to the regeneration of an E-box motif. Taken together, our results demonstrate a role for the E-box but not the GRE in T lymphomagenesis by SL3-3, unveil an inherent broader disease specificity of the virus, and strengthen the notion of selection for more potent enhancer variants of mutated viruses during tumor development.
PMCID: PMC2612299  PMID: 18945767
8.  Variable sequences outside the SAM-binding core critically influence the conformational dynamics of the SAM-III/SMK box riboswitch 
Journal of molecular biology  2011;409(5):786-799.
The SMK box (SAM-III) translational riboswitches were identified in S-adenosyl-L-methionine (SAM) synthetase metK genes in members of the Lactobacillales. This riboswitch switches between two alternative conformations in response to the intracellular SAM concentration and controls metK expression at the level of translation initiation. We previously reported the crystal structure of the SAM-bound SMK box riboswitch. In this study we combined SHAPE chemical probing with mutagenesis to probe the ligand-induced conformational switching mechanism. We revealed that while the majority of the apo SMK box RNA molecules exist in an alternatively base paired (ON) conformation, a subset of them pre-organize into a SAM-bound-like (READY) conformation, which upon SAM exposure is selectively stabilized into the SAM-bound (OFF) conformation through an induced-fit mechanism. Mutagenesis showed that the ON state is only slightly more stable than the READY state, as several single-nucleotide substitutions in a hypervariable region outside the SAM-binding core can alter the folding landscape to favor the READY state. Such SMK variants display a “constitutively-OFF” behavior both in vitro and in vivo. Time-resolved and temperature-dependent SHAPE analyses revealed adaptation of the SMK box RNA to its mesothermal working environment. The latter analysis revealed that the SAM-bound SMK box RNA follows a two-step folding/unfolding process.
PMCID: PMC3479645  PMID: 21549712
9.  Sequence, structure, and stacking 
RNA Biology  2013;10(12):1761-1764.
The term riboswitch usually refers to small molecule sensing regulatory modules in the 5′ untranslated regions of a mRNA. They are typically comprised of separate ligand binding and regulatory domains. The T box riboswitch is unique from other identified riboswitches because its effector is an essential macromolecule, tRNA. It senses the aminoacylation state of tRNA to regulate genes involved in a variety of functions relating to amino acid metabolism and tRNA aminoacylation. T box riboswitches performs an intuitively simple process using a complex structured RNA element and, until recently, the underlying mechanisms were poorly understood. Only two sequence-specific contacts had been previously identified: (1) between the specifier sequence (codon) and the tRNA anticodon and (2) between an anti-terminator stem loop and the tRNA acceptor arm CCA tail. tRNA aminoacylation blocks the latter interaction and therefore serves as the switch between termination and anti-termination. Outside of these two contacts, the structure and functions of T box riboswitches have come to light in some recent studies. We recently described the X-ray crystal structure of the highly conserved T box riboswitch distal Stem I region and demonstrated that this region interacts with the tRNA elbow to anchor it to the riboswitch. Independently, Lehmann et al. used sequence homology search to arrive at a similar model for Stem I-tRNA interactions. The model was further supported by two recent structures of the Stem I-tRNA complex, determined independently by our group and by Zhang and Ferré-D’Amaré. This article highlights some of these contributions to synthesize an updated model for tRNA recognition by the T box riboswitch.
PMCID: PMC3917978  PMID: 24356646
RNA; structure; T-loop; interlocking T-loop; RNA-RNA complex
10.  The effects of moderate fatigue on dynamic balance control and attentional demands 
During daily activities, the active control of balance often is a task per se (for example, when standing in a moving bus). Other constraints like fatigue can add to the complexity of this balance task. In the present experiment, we examined how moderate fatigue induced by fast walking on a treadmill challenged dynamic balance control. We also examined if the attentional demands for performing the balance task varied with fatigue.
Subjects (n = 10) performed simultaneously a dynamic balance control task and a probe reaction time task (RT) (serving as an indicator of attentional demands) before and after three periods of moderate fatigue (fast walking on a treadmill). For the balance control task, the real-time displacement of the centre of pressure (CP) was provided on a monitor placed in front of the subject, at eye level. Subjects were asked to keep their CP within a target (moving box) moving upward and downward on the monitor. The tracking performance was measured (time spent outside the moving box) and the CP behavior analyzed (mean CP speed and mean frequency of the CP velocity).
Moderate fatigue led to an immediate decrement of the performance on the balance control task; increase of the percentage of time spent outside the box and increase of the mean CP speed. Across the three fatigue periods, subjects improved their tracking performance and reduced their mean CP speed. This was achieved by increasing their frequency of actions; mean frequency of the CP velocity were higher for the fatigue periods than for the no fatigue periods. Fatigue also induced an increase in the attentional demands suggesting that more cognitive resources had to be allocated to the balance task with than without fatigue.
Fatigue induced by fast walking had an initial negative impact on the control of balance. Nonetheless, subjects were able to compensate the effect of the moderate fatigue by increasing the frequency of actions. This adaptation, however, required that a greater proportion of the cognitive resources be allocated to the active control of the balance task.
PMCID: PMC1592501  PMID: 17007646
11.  MyoD and myogenin act on the chicken myosin light-chain 1 gene as distinct transcriptional factors. 
Molecular and Cellular Biology  1993;13(11):7153-7162.
Expression of MyoD, myogenin, MRF4, and Myf-5 converts nonmuscle cells to muscle cells. In an attempt to analyze the roles of these factors, we have investigated their effects on transcription driven by the promoter of the chicken myosin alkaline light-chain (MLC1) gene. The activation by CMD1 or c-myogenin (chicken MyoD or myogenin, respectively) was dependent on the existence of a muscle-specific regulatory region located from positions -2096 to -1743. Its distal half, containing a pair of E boxes (CANNTG), had been previously characterized as an enhancer responsive to CMD1 but not to c-myogenin. In this study, we report the identification of another enhancer in the muscle-specific regulatory region which is preferentially responsive to c-myogenin. Deletion and mutation analyses indicated that this enhancer requires a single E box and its flanking sequences. Furthermore, analysis of chimeric proteins of CMD1 and c-myogenin indicated that regions outside the basic helix-loop-helix domain of c-myogenin are involved in the specificity of the enhancer. These results show that CMD1 and c-myogenin act on the MLC1 gene by recognizing different upstream DNA sequences and that direct or indirect interactions between the regions outside the basic helix-loop-helix domain and flanking sequences of E boxes are involved in the target sequence specificity.
PMCID: PMC364776  PMID: 8413304
12.  Endoscopic knot tying: In vitro assessment in a porcine stomach model 
AIM: To determine if surgical knotting performed via endoscopy is an effective closure method for natural orifice translumenal endoscopic surgery.
METHODS: The proposed method was tested on an in vitro pig stomach model using standard endoscopy suite materials. A single use laparoscopy trocar (Versaport Plus manufactured by Tyco Healthcare) was fixed onto a plastic rectangular box in a horizontal position. A fresh pig stomach was tightly attached via its esophageal end to the trocar opening on the inner side of the box. The stomach cavity was closed at the duodenal end with Kocher forceps. A standard upper gastrointestinal endoscope fitted at its tip with a transparent plastic cap was introduced into the stomach through the outer trocar opening, so that the passage of the surgical trocar would mimic the passage of an esophagus. The stomach was subsequently inflated, followed by irrigation and washing. A neutral electrode of an electrocautery unit was placed inside the plastic box, underneath the pig stomach. The stomach’s outer surface was kept moist using normal saline in order to maintain the natural elasticity and to ensure good contact with the electrode.
RESULTS: The submucosal space on the anterior face of the stomach was accessed using the technique of endoscopic submucosal dissection. First, a site on the anterior face of the stomach was chosen, near the angle. Then, saline was injected into the submucosa with a standard endoscopic needle, so as to create a 20 mm diameter elevation. A linear 15 mm vertical incision was created at its center using a Dual Knife (KD650U manufactured by Olympus). This incision was used to access the submucosal space, and about 10 mm was dissected on both sides of the incision. The endoscope was then pushed through to the outside of the stomach after dilating a small puncture made by the Dual Knife in the muscularis propria, which simulated the peritoneoscopy procedure. Then, a 0.025” guidewire (Jagwire/450 cm manufactured by Boston Scientific) was inserted into the puncture, followed by a dilating balloon (Quantum TT manufactured by Cook Medical) that was used to enlarge the aperture orifice. After withdrawing the scope back into the stomach, the procedure continued with guidewires being passed from the submucosal space into the gastric lumen through small orifices on the left and right sides of the mucosal opening. These orifices were made with the Dual Knife, and the guidewires were inserted via a guiding catheter (HGC-6 manufactured by Cook Medical). As the guidewires were pulled outside of the stomach, they were replaced with a single surgical suture that had been initially attached to their tip and was now untied. Finally, one loop of this surgical suture was formed on the exterior. One loop end was fixed while the opposite suture end was pulled by biopsy forceps through the endoscope channel as the scope was inserted into the stomach. The loop was advanced until it approached and fixed the two mucosal incision margins. Three alternating loops were made in this manner to create a genuine tight surgical knot.
CONCLUSION: Endoscopic knotting of the gastric wall is feasible, but an in vitro survival study is necessary to validate clinical significance.
PMCID: PMC3547117  PMID: 23330051
Endoscopy; Endoscopic submucosal dissection; Natural orifice translumenal endoscopic surgery; Suture; in vitro
13.  Genetic Association Studies of Cleft Lip and/or Palate With Hypodontia Outside the Cleft Region 
The purpose of this study was to determine whether the candidate genes previously studied in subjects with cleft lip, cleft palate, or both are associated with hypodontia outside the region of the cleft.
One hundred twenty subjects from the Iowa Craniofacial Anomalies Research Center were selected based on the availability of both dental records and genotype information.
The type of orofacial clefting and type and location of dental anomalies (missing teeth, supernumerary teeth, or peg laterals) were assessed by dental chart review and radiographic examination. Genotype analysis of candidate genes was performed using polymerase chain reaction/single-strand conformation polymorphism analysis.
The prevalence of hypodontia in this sample was 47.5%, with 30.0% of subjects having missing teeth outside the cleft. There was a positive association between subjects with cleft lip or cleft lip and palate who had hypodontia outside the cleft region (compared with noncleft controls) and both muscle segment homeo box homolog 1 (MSX1) (p = .029) and transforming growth factor beta 3 (TGFB3) (p = .024). It was not possible in this analysis to determine whether this association was specifically associated with orofacial clefting combined with hypodontia or whether it was due primarily to the clefting phenotype.
In this sample, there was a significantly greater incidence of hypodontia outside the cleft region in subjects with cleft lip and palate, compared with cleft lip only or cleft palate only. Cleft lip and/or palate with hypodontia outside the cleft region was positively associated with both TGFB3 and MSX1, compared with noncleft controls.
PMCID: PMC2752356  PMID: 12733956
cleft lip and palate; genetics; hypodontia
14.  Novel conserved sequence motifs in plant G-box binding proteins and implications for interactive domains. 
Nucleic Acids Research  1994;22(3):470-478.
The G-box is a cis-acting DNA sequence present in several plant promoters that are regulated by diverse signals such as UV irradiation, anaerobiosis, abscissic acid and light. Several basic/leucine zipper (bZIP) proteins from different plant species have been identified as high affinity G-box binding proteins. Although their capability to enhance transcription has been demonstrated, their precise function in transcriptional activation is still unknown. We have isolated three cDNAs from young tomato fruit that encode bZIP G-box binding proteins (GBF4, GBF9 and GBF12). They bind to the G-box sequence in the tomato rbcS1, rbcS2 and rbcS3A promoters. GBF9 binding resulted in a DNase I footprint identical to that obtained with tomato nuclear extract and different from the DNase I protection obtained with GBF4 and GBF12. The mRNAs of all three GBFs were most abundant in tomato fruit and seeds, moderately abundant in root and least abundant in leaves. Protein sequences outside of the bZIP domains were compared with the known GBFs from other plants and seven conserved motifs of seven to 35 amino acids length have been identified. Based on the presence of these motifs, three classes of GBFs can be defined that are conserved among plant species. GBF9, the predominantly expressed tomato GBF, is the first member of its class isolated from dicot plants. Three conserved motifs from two of the classes are highly hydrophilic and are predicted to be exposed on the surface of the proteins. These motifs likely define novel interactive domains in the different classes of GBFs that could provide a new tool to determine how distinct regulatory signals are transmitted through GBFs to activate transcription.
PMCID: PMC523606  PMID: 8127687
15.  Stimulatory Effect of Morning Bright Light on Reproductive Hormones and Ovulation: Results of a Controlled Crossover Trial 
PLoS Clinical Trials  2007;2(2):e7.
Studies have shown a shortening of the menstrual cycle following light exposure in women with abnormally long menstrual cycles or with winter depression, suggesting that artificial light can influence reproductive hormones and ovulation. The study was designed to investigate this possibility.
Placebo-controlled, crossover, counterbalanced order.
Medical centres and participants' homes in Novosibirsk (55°N), Russia.
Twenty-two women, aged 19–37 years, with baseline menstrual cycle length 28.1–37.8 d and no clinically evident endocrine abnormalities completed the study. The study lasted for two menstrual cycles separated by at least one off-protocol cycle.
During one experimental cycle, bright light was administered at home for 1 wk with a light box emitting white light at 4,300 lux at 41 cm for 45 min shortly after awakening. During the other experimental cycle, dim light was <100 lux at 41 cm with a one-tube fluorescent source.
Outcome Measures:
Blood samples and ultrasound scans were obtained in the afternoon before and after the week of light exposure, on day ∼7 and 14 after menstruation onset. Further ultrasound scans after day 14 documented ovulation. Serum was assayed for thyroid-stimulating hormone (TSH), prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol (E2).
Concentrations of PRL, LH, and FSH were significantly increased with bright versus dim light exposure, as was follicle size (ANOVA, intervention × day, p = 0.0043, 0.014, 0.049, and 0.042, respectively). The number of ovulatory cycles increased after exposure to bright compared to dim light (12 versus 6 cycles, Wilcoxon tied p = 0.034).
Morning exposure to bright light in the follicular phase of the menstrual cycle stimulates the secretion of hypophyseal reproductive hormones, promotes ovary follicle growth, and increases ovulation rates in women with slightly lengthened menstrual cycles. This might be a promising method to overcome infertility.
Editorial Commentary
Background: It is not clear whether light plays any role in determining menstrual cycles or ovulation in women. However, light is used as a treatment for some medical conditions related to rhythm, most notably seasonal affective disorder. In this study, the researchers wanted to examine the role of light in influencing the menstrual cycle. In the trial reported here, 22 women with lengthened menstrual cycles were provided with a light box that emitted either bright light or dim light, and the women were asked to use the light box for a defined period each day for one menstrual cycle. The study had a crossover design, where women would receive either bright or dim light for one cycle, then another cycle without the light box, and finally a cycle with either bright or dim light, whichever they did not receive first. Outcomes assessed in the trial included measurements of the blood levels of certain hormones. Specifically, the researchers looked at three hormones that determine the reproductive cycle (lutenizing hormone [LH], follicle stimulating hormone [FSH], and prolactin); these are produced by a region of the brain that is controlled by the hypothalamus, a gland that is directly responsive to light. Additionally, ultrasound scans were used to check for ovulation during each cycle. The trial was carried out in winter in Novosibirsk, a Russian city.
What the trial shows: When the researchers compared hormone levels between the “bright light” and “dim light” phases of the study, they found statistically significant increases in levels of LH, FSH, and prolactin in the blood. Ovulation was more likely in the “bright light” phase of the study as compared to the “dim light” phase. However, levels of two other hormones, thyroid stimulating hormone, and estradiol, were not significantly different when comparing the “bright” and “dim” cycles.
Strengths and limitations: In this trial the use of a crossover design enabled each woman to act as her own control, thereby reducing the number of participants that were needed in the trial. A further strength in this study was the use of ultrasound scans to allow the researchers to pinpoint ovulation in the participants; similar studies have not tried to examine the effects of light both on reproductive hormones and ovulation. A weakness of the design is that participants were assigned to receive either dim light first or bright light first using alternation, not true randomization. Therefore blinding was not possible and the researchers recruiting participants would have known in advance what intervention each participant would receive first. Other weaknesses include the limited number of participants in this study, and the nature of the participant population, all of whom had lengthened menstrual cycles and were living at fairly northerly latitude.
Contribution to the evidence: This study adds data suggesting that in women with lengthened menstrual cycles, ovulation can be stimulated by bright light. However, this finding would need to be replicated in a larger sample of women and it is not yet clear whether bright light will have the same effect on ovulation in women outside northerly latitudes and with average-length menstrual cycles.
PMCID: PMC1851732  PMID: 17290302
16.  A gene that is related to SRY and is expressed in the testes encodes a leucine zipper-containing protein. 
Molecular and Cellular Biology  1995;15(7):3759-3766.
SRY-related cDNA encoding a protein with a high-mobility-group (HMG) box and a leucine zipper motif, which was designated SOX-LZ, was isolated from a rainbow trout testis cDNA library. Comparison of this cDNA with the mouse homologous cDNA isolated from a testis cDNA library exhibits an overall amino acid sequence identity of 77%, which is in striking contrast to the abrupt loss of amino acid sequence homology outside the HMG box found among mammalian SRY genes. In both rainbow trout and mice, Northern (RNA) blot analyses have revealed the presence of a testis-specific 3-kb-long SOX-LZ mRNA, and this transcript appeared coincidentally with the protamine mRNA, suggesting its expression in the germ line. A recombinant HMG box region protein encoded by SOX-LZ could bind strongly with an oligonucleotide containing an AACAAT sequence, which is also recognized by mouse Sry and Sox-5. Upon cotransfection into CHO cells, SOX-LZ transactivated transcription through its binding motif when the region including the leucine zipper motif was deleted [SOX-LZ (D105-356)]; however, the intact SOX-LZ failed to transactivate. The intact SOX-LZ could form homodimers through the leucine zipper, which resulted in inhibition of DNA binding by the HMG box, while SOX-LZ (D105-356), which was incapable of dimerization, showed specific binding with the AACAAT sequence. Thus, the repressed transactivation of the intact SOX-LZ in CHO cells was primarily attributable to the low level of DNA binding of SOX-LZ homodimers.
PMCID: PMC230614  PMID: 7791783
17.  Structural determinants of DNA recognition by plant MADS-domain transcription factors 
Nucleic Acids Research  2013;42(4):2138-2146.
Plant MADS-domain transcription factors act as key regulators of many developmental processes. Despite the wealth of information that exists about these factors, the mechanisms by which they recognize their cognate DNA-binding site, called CArG-box (consensus CCW6GG), and how different MADS-domain proteins achieve DNA-binding specificity, are still largely unknown. We used information from in vivo ChIP-seq experiments, in vitro DNA-binding data and evolutionary conservation to address these important questions. We found that structural characteristics of the DNA play an important role in the DNA binding of plant MADS-domain proteins. The central region of the CArG-box largely resembles a structural motif called ‘A-tract’, which is characterized by a narrow minor groove and may assist bending of the DNA by MADS-domain proteins. Periodically spaced A-tracts outside the CArG-box suggest additional roles for this structure in the process of DNA binding of these transcription factors. Structural characteristics of the CArG-box not only play an important role in DNA-binding site recognition of MADS-domain proteins, but also partly explain differences in DNA-binding specificity of different members of this transcription factor family and their heteromeric complexes.
PMCID: PMC3936718  PMID: 24275492
18.  A simple modification of the Farnsworth-Munsell 100-Hue test for much faster assessment of color vision 
Indian Journal of Ophthalmology  2014;62(6):721-723.
The Farnsworth-Munsell (FM) 100-hue test is well known but is also time consuming, especially its analytical component. To reduce this needless time-waste during precious working hours, a simple modification was devised.
Prospective, comparative, observational study.
Materials and Methods:
A transparent clear plastic carrier box replaced the opaque one, allowing ready digital photodocumentation of top and bottom without even opening the box, or handling/inverting the caps -200 reportedly normals and 50 known color vision defectives could be easily tested on this modified-FM and results stored, allowing rapid turnover. The captured scores with patient ID were analyzed, at leisure, outside hospital time, saving 45-60 minutes/patient. After recording, the box was promptly handed over to the next subject for rearrangement. Times taken for test/patient were recorded.
Running time was reduced from 60-75 min to ~15 min/patient with no waste of invaluable lab hours. Turnover time is limited to capturing two photographs (~60 sec). The box is relatively cheap and easy to maintain.
Our simplified FM 100-hue test allowed rapid assessment of color visions with easy data storage of both top and bottom.
PMCID: PMC4131328  PMID: 25005203
Color vision; color vision testing; FM 100-Hue; Ishihara
19.  Missense mutation outside the forkhead domain of FOXL2 causes a severe form of BPES type II 
Molecular Vision  2012;18:211-218.
Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a developmental disease characterized by a complex eyelid malformation associated or not with premature ovarian failure (POF). BPES is essentially an autosomal dominant disease, due to mutations in the forkhead box L2 (FOXL2) gene, encoding a forkhead transcription factor. More than one hundred unique FOXL2 mutations have been described in BPES in different populations, many of which are missense mutations in the forkhead domain. Here, we report on a very severe form of BPES resulting from a missense mutation outside the forkhead domain.
A clinical and molecular genetic investigation was performed in affected and unaffected members of an Iranian family with BPES. The FOXL2 coding region was sequenced in an index case. Targeted mutation testing was performed in 8 family members.
We have identified a heterozygous FOXL2 missense mutation c.650C→G (p.Ser217Cys) co-segregating with disease in members of a three-generation family with BPES type II. Only few missense mutations have been reported outside the forkhead domain so far. They were all found in mild BPES, in line with in vitro studies demonstrating mostly normal localization and normal or increased transactivation properties of the mutant proteins. Unlike previous studies, affected members of the family studied here showed a severe BPES phenotype, with bilateral amblyopia due to uncorrected ptosis.
This is the first study demonstrating a severe BPES phenotype resulting from a FOXL2 missense mutation outside the forkhead domain, expanding our knowledge about the phenotypic consequences of missense mutations outside the forkhead domain in BPES.
PMCID: PMC3272052  PMID: 22312189
20.  Organisational reporting and learning systems: Innovating inside and outside of the box 
Clinical Risk  2015;21(1):7-12.
Reporting and learning systems are key organisational tools for the management and prevention of clinical risk. However, current approaches, such as incident reporting, are struggling to meet expectations of turning health systems like the UK National Health Service (NHS) into learning organisations. This article aims to open up debate on the potential for novel reporting and learning systems in healthcare, by reflecting on experiences from two recent projects: Proactive Risk Monitoring in Healthcare (PRIMO) and Errordiary in Healthcare. These two approaches demonstrate how paying attention to ordinary, everyday clinical work can derive useful learning and active discussion about clinical risk. We argue that innovations in reporting and learning systems might come from both inside and outside of the box. ‘Inside’ being along traditional paths of controlled organisational innovation. ‘Outside’ in the sense that inspiration comes outside of the healthcare domain, or more extremely, outside official channels through external websites and social media (e.g. patient forums, public review sites, whistleblower blogs and Twitter streams). Reporting routes that bypass official channels could empower staff and patient activism, and turn out to be a driver to challenge organisational processes, assumptions and priorities where the organisation is failing and has become unresponsive.
PMCID: PMC4436279  PMID: 25999777
Patient safety; incident reporting; organisational learning; risk management; resilience
21.  Unusual Centrosome Cycle in Dictyostelium: Correlation of Dynamic Behavior and Structural Changes 
Molecular Biology of the Cell  1999;10(1):151-160.
Centrosome duplication and separation are of central importance for cell division. Here we provide a detailed account of this dynamic process in Dictyostelium. Centrosome behavior was monitored in living cells using a γ-tubulin–green fluorescent protein construct and correlated with morphological changes at the ultrastructural level. All aspects of the duplication and separation process of this centrosome are unusual when compared with, e.g., vertebrate cells. In interphase the Dictyostelium centrosome is a box-shaped structure comprised of three major layers, surrounded by an amorphous corona from which microtubules emerge. Structural duplication takes place during prophase, as opposed to G1/S in vertebrate cells. The three layers of the box-shaped core structure increase in size. The surrounding corona is lost, an event accompanied by a decrease in signal intensity of γ-tubulin–green fluorescent protein at the centrosome and the breakdown of the interphase microtubule system. At the prophase/prometaphase transition the separation into two mitotic centrosomes takes place via an intriguing lengthwise splitting process where the two outer layers of the prophase centrosome peel away from each other and become the mitotic centrosomes. Spindle microtubules are now nucleated from surfaces that previously were buried inside the interphase centrosome. Finally, at the end of telophase, the mitotic centrosomes fold in such a way that the microtubule-nucleating surface remains on the outside of the organelle. Thus in each cell cycle the centrosome undergoes an apparent inside-out/outside-in reversal of its layered structure.
PMCID: PMC25160  PMID: 9880333
22.  Juvenile Survival in a Neotropical Migratory Songbird Is Lower than Expected 
PLoS ONE  2013;8(2):e56059.
Attempts to estimate and identify factors influencing first-year survival in passerines, survival between fledging and the first reproductive attempt (i.e. juvenile survival), have largely been confounded by natal dispersal, particularly in long-distance migratory passerines. We studied Prothonotary Warblers (Protonotaria citrea) breeding in nest boxes to estimate first-year survival while accounting for biases related to dispersal that are common in mark-recapture studies. The natal dispersal distribution (median = 1420 m; n = 429) and a distance-dependent recruitment rate, which controls for effects of study site configuration, both indicated a pattern of short-distance natal dispersal. This pattern was consistent with results of a systematic survey for birds returning outside the nest box study sites (up to 30 km in all directions) within a majority (81%) of total available bottomland forest habitat, further suggesting that permanent emigration outside of the study system was rare. We used multistate mark-recapture modeling to estimate first-year survival and incorporated factors thought to influence survival while accounting for the potential confounding effects of dispersal on recapture probabilities for warblers that fledged during 2004–2009 (n = 6093). Overall, the average first-year survival for warblers reared without cowbird nestmates was 0.11 (95% CI = 0.09–0.13), decreased with fledging date (0.22 early to 0.03 late) and averaged 40% lower for warblers reared with a brood parasite nestmate. First-year survival was less than half of the rate thought to represent population replacement in migratory passerines (∼0.30). This very low rate suggests that surviving the first year of life for many Neotropical migratory species is even more difficult than previously thought, forcing us to rethink estimates used in population models.
PMCID: PMC3568049  PMID: 23409122
23.  Consensus Higher Order Repeats and Frequency of String Distributions in Human Genome 
Current Genomics  2007;8(2):93-111.
Key string algorithm (KSA) could be viewed as robust computational generalization of restriction enzyme method. KSA enables robust and effective identification and structural analyzes of any given genomic sequences, like in the case of NCBI assembly for human genome. We have developed a method, using total frequency distribution of all r-bp key strings in dependence on the fragment length l, to determine the exact size of all repeats within the given genomic sequence, both of monomeric and HOR type. Subsequently, for particular fragment lengths equal to each of these repeat sizes we compute the partial frequency distribution of r-bp key strings; the key string with highest frequency is a dominant key string, optimal for segmentation of a given genomic sequence into repeat units. We illustrate how a wide class of 3-bp key strings leads to a key-string-dependent periodic cell which enables a simple identification and consensus length determinations of HORs, or any other highly convergent repeat of monomeric or HOR type, both tandem or dispersed. We illustrated KSA application for HORs in human genome and determined consensus HORs in the Build 35.1 assembly. In the next step we compute suprachromosomal family classification and CENP-B box / pJα distributions for HORs. In the case of less convergent repeats, like for example monomeric alpha satellite (20-40% divergence), we searched for optimal compact key string using frequency method and developed a concept of composite key string (GAAAC--CTTTG) or flexible relaxation (28 bp key string) which provides both monomeric alpha satellites as well as alpha monomer segmentation of internal HOR structure. This method is convenient also for study of R-strand (direct) / S-strand (reverse complement) alpha monomer alternations. Using KSA we identified 16 alternating regions of R-strand and S-strand monomers in one contig in choromosome 7. Use of CENP-B box and/or pJα motif as key string is suitable both for identification of HORs and monomeric pattern as well as for studies of CENP-B box / pJα distribution. As an example of application of KSA to sequences outside of HOR regions we present our finding of a tandem with highly convergent 3434-bp Long monomer in chromosome 5 (divergence less then 0.3%).
PMCID: PMC2435359  PMID: 18660848
Human genome; alpha satellite; alphoid; higher order repeat (HOR); key string algorithm - KSA; frequency distribution of strings; consensus higher order repeat; suprachromosomal families; CENP-B box; pJα motif
24.  The USF Proteins Regulate Transcription of the Follicle-Stimulating Hormone Receptor but Are Insufficient for Cell-Specific Expression 
Expression of the FSH receptor (FSHR) is limited to granulosa cells of the ovary and Sertoli cells of the testis. Previous studies showed that an E box in the proximal promoter of the FSHR gene is required for transcription and that the predominant E box binding proteins are the ubiquitous transcription factors, upstream stimulatory factor 1 (USF1) and USF2. Through cotransfection analysis, we have shown that both wild-type and dominant negative forms of the USF proteins regulate the rat FSHR promoter and that transcriptional activation of FSHR required several domains within the amino-terminal portion of the USF proteins. Analysis of the FSHR promoter region using in vivo genomic footprinting indicated that the E box is occupied by proteins in Sertoli cells but not in cells that fail to express the receptor, despite the presence of the USF proteins. To help delineate the regions of the rat FSHR gene required for correct spatial and temporal expression, transgenic mice harboring two constructs containing variable amounts of 5′-flanking sequence (5,000 bp and 100 bp) were generated. Examination of 16 different transgenic lines revealed varied transgene expression profiles with multiple lines having different amounts of ectopic expression and two lines failing to express the transgene. In addition, little or no expression was observed in Sertoli cells. These studies indicate that additional regulatory sequences outside the region from −5,000 to + 123 bp are needed for proper expression in Sertoli cells. (Molecular Endocrinology 14: 1836–1848, 2000)
PMCID: PMC1496886  PMID: 11075816
25.  Structure and Substrate Recruitment of the Human Spindle Checkpoint Kinase Bub1 
Molecular cell  2008;32(3):394-405.
In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister-chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN-box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.
PMCID: PMC2644263  PMID: 18995837

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