Dairy cattle may experience discomfort in a myriad of ways throughout their life cycle, particularly when sustaining hock injuries or suboptimal locomotion. Lactating dairy cattle divide their lying time equally between left and right sides; however, discomfort experienced during pregnancy or following cannulation can cause a shift in the normal lying laterality. The objective of this study was to determine the effect of hock injuries and lameness on the lying behaviors of dairy cattle, particularly lying laterality. Lying laterality did not differ from the expected 50% (left side lying time) in cattle with hock injuries, lameness, or both. The current results suggest that lying laterality does not differ between varying levels of hock injury or lameness severity. Going forward, further research could determine if lying laterality shifts over the course of the animal developing a hock injury or lameness.Lactating dairy cattle divide their lying equally between their left side and their right side. However, discomfort, such as pregnancy and cannulation, can cause a cow to shift lying side preference. The objective of this study was to determine the effect of lameness and hock injuries on lying behaviors, particularly lying laterality, of lactating dairy cows. Cows from four commercial farms in eastern Croatia that had lying behavior data, health score data, and production records were used in the study. Health scores including hock injuries and locomotion were collected once per cow. Severely lame cows had greater daily lying time compared to sound cows and moderately lame cows. Overall, cows spent 51.3 ± 1.2% of their daily lying time on the left side. Maximum hock score, locomotion score, hock injury laterality, or parity did not result in lying laterality differing from 50%. Reducing the prevalence of hock injuries and lameness is key in improving cow welfare due to the discomfort associated with these conditions and the overall high prevalence. Previous research has consistently reported substantial rates of hock injuries globally with rates ranging from 57% in France, 73% in southern British Columbia  to 81% iThe relationship between hock injuries and lameness may be due to similar environmental risk factors such as housing ,8,9. FreInadequate bedding and overstocking can also alter normal lying behaviors by decreasing lying time in cows ,13. FurtThis study was a retrospective cross-sectional study. The aim was to determine if hock injuries  or lameness contributed to a shift in lying laterality from the expected 50% per side or an alteration in other lying behaviors. Therefore, the objective of this study was to determine the impact of hock injuries and lameness on lying behaviors, particularly lying laterality for lactating dairy cows with hock injuries and lameness. It was hypothesized that lying laterality in animals with hock injuries and/or lameness would differ from the expected 50%. Increased lying time has also been associated with higher parity , therefoLactating Holstein dairy cows from four farms across eastern Croatia were evaluated for this study. Farms were selected due to the Memorandum of Understanding in place between the farm and the University of Osijek and their role in the Fulbright Scholarship with the intention of measuring welfare of Croatian dairy farms. A breakdown of descriptive cow data is included in Lying behaviors , lying bout durations (min/bout), and number of lying bouts per day (n/d)) were collected using Hobo Pendant G data loggers  as previously validated  for a miCow records were obtained from each farms’ herdsman, translated from Croatian. Individual cow data included cow ID, lactation number, days in milk (DIM), pregnancy status, breeding status, days to calving, number of inseminations, days to peak milk, peak milk yield, and milk quality . Additionally, five cows that had health scores assessed twice due to being moved to a different pen were removed from the data set to keep the number of assessments consistent between cows. Power was calculated to be 0.61  due to the fact that only cows on farms with existing relationships with the university were available for data collection. To achieve a power of 0.80, a total of 306 cows would have been necessary. Unfortunately, the number of available cows was much lower and the small power may reduce the ability to detect effects. However, the intent of this study was to discover potential behavioral differences using the data and cows made available.Cows used in this study were part of a general assessment project where lying behaviors were collected from 278 cows and health scores were collected from 792 cows. For data analysis in the current study, only cows with lying behavior, health scores (locomotion and hock injury scores), and cow records (lactation number and DIM) were used  differed from 50%. Four t-tests were performed to determine differences in four variables of interest: maximum hock score, locomotion score, hock injury laterality, and parity. An additional t-test was performed to determine if lying laterality differed from 50% in the overall population of cows used in the study. In order to determine variability of lying behaviors (lying time (h/d), lying bout duration (min/bout), and bouts (n/d)) in relation to maximum hock scores, hock score laterality, locomotion scores, and parity univariate associations of SD  were analyzed using a mixed model  to determine effect of maximum hock score, hock injury laterality, locomotion score, and parity. Maximum hock scores of 0 and a hock injury laterality of “neither” were the same population of cows, therefore maximum hock score had to be adjusted. Maximum hock score was analyzed as maximum hock score within hock laterality. Only maximum hock scores of 1 had both bilateral and unilaterally injured groups. All cows with a maximum hock score of 2 were injured unilaterally. The experimental unit was cow and the random effect was farm. Degrees of freedom were estimated using Kenward-Roger option. One sample ns of SD  were detn = 7) or right injury (n = 8) were combined into the unilateral category due to the limited number of cows with only left or right hock injuries. Dependent variables included lying time, lying bouts, and lying bout duration. Independent variables included in the model were maximum hock score, hock injury laterality, locomotion score, and parity.Maximum hock scores were determined by using the maximum hock score from each cow. Hock scores of 2 and 3 were combined into one score (score = 2) due to the limited number of cows with a hock score of 3. Hock injury laterality was categorized into three categories: unilateral (cow has only one hock that is scored at 1 or above), bilateral (cow has both hocks scored at 1 or above), and neither (both hocks were scored as 0). Cows with only a left injury (p = 0.003) and moderately lame cows (p = 0.002). No differences in lying time were found between hock injury laterality (p = 0.52), max hock score (p = 0.79), or parity (p = 0.27).Severely lame cows had greater daily lying time than sound (p = 0.004) and severely lame cows (p = 0.01). Lying bout duration did not differ between hock injury laterality (p = 0.29), max hock score (p = 0.97) or parity (p = 0.07).Sound cows had shorter lying bout duration compared to moderately lame cows (p = 0.01) but not severely lame cows (p = 0.54). Lying bouts did not differ between hock injury laterality (p = 0.35), max hock score (p = 0.85) or parity (p = 0.31).Sound cows had greater daily lying bouts than moderately lame cows (p = 0.28). The lying laterality did not differ from 50% for cows with varying maximum hock scores (p ≥ 0.20), locomotion scores (p ≥ 0.21), side of hock injury (p ≥ 0.20), or parity (p ≥ 0.14).Overall, cows spent 51.3 ± 1.1% daily lying time on their left side which did not differ from 50%  compared to sound cows (10.5 ± 2.7 h/d) [n = 122) than primiparous (n = 73). Both heifers and cows prioritize resting [Parity did not influence lying time contrary to previous research, which reports increased lying time with increased parity ,25. Howe resting ,27 and a resting .Overall, mean lying bout duration for multiparous (80.6 ± 30.8 min/bout), primiparous (71.5 ± 19.8 min/bout), uninjured (75.5 ± 30.9 min/bout), bilaterally injured (76.6 ± 28.4 min/bout), and unilaterally injured cows (87.2 ± 30.9 min/bout) did not exceed the 90 min/bout threshold that was found to be associated with severely lame cows . This suSound cows had a greater number of lying bouts per day compared to moderately lame cows. This is contrary to previous data that reports no difference in lying bouts between moderately lame and sound cows ,18. PrevLying laterality did not differ between maximum hock scores, locomotion scores, parity, or hock injury laterality. Previous research reports increased right side laterality with age but not specifically with increased parity and no laterality preference in pregnant heifers . In thatEvaluating SD of lying behaviors can indicate variability of lying behaviors among individual cows . VariatiThe current results suggest that lying laterality does not differ between varying levels of hock injury or lameness severity. However, further research could determine if a shift in lying side occurs on an individual cow level as hock injuries or lameness progress. The current study focused on a short period of time.


Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event.Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as  Escherichia coli SSB (EcoSSB) is a homotetramer, with each subunit consisting of a single OB domain for ssDNA binding, in conjunction with a flexible C-terminal extension involved in protein–protein interactions  are essential for the genome maintenance of all known cellular organisms (Mushegian and Koonin Sulfolobus solfataricus SSB (SsoSSB) demonstrated its close structural relationship with the ssDNA-binding domains of human RPA70  and a ten times molar excess of fluorescent dye with the addition of urea to a final concentration of 8 M. The labelling reaction was left at room temperature for 3 h, and then overnight at 4 °C. The labelling mixture was then diluted with labelling buffer to half the concentration of urea. A pure sample of labelled proteins was obtained using an affinity column to remove the unlabelled proteins and any remaining free dye, as described previously , and Cy3 and Cy5 dyes are indicated.SsoSSB samples were dialysed extensively against 20 mM MES buffer, pH 6.5, 100 mM potassium glutamate, and 1 mM MgCl2 (ITC buffer). Oligonucleotides (DNA or RNA) were also dissolved in ITC buffer. Binding experiments were performed in triplicate at 50 °C. A 370 µL syringe with stirring at 300 rpm was used to titrate the oligonucleotide (40 µM) into the sample cell containing approximately 1.4 mL of SsoSSB (10 µM). Each titration consisted of a preliminary 1 µL injection followed by up to 25 subsequent 10 µL injections. Heats of dilution (ΔH) were measured in corresponding blank titrations by adding oligonucleotide to ITC buffer and/or ITC buffer to protein and were found to be similar to heats observed at the end of protein-DNA titrations. ITC-binding isotherms were analysed using a Single Set of Identical Sites model built-in to ITC Data Analysis in ORIGIN provided by the manufacturer. Non-linear least-squares fitting of the data to this model was performed using the ITC Data Analysis software. This fit does not consider any positive cooperativity and the KD values obtained are thus reported as “apparent KD’s”. This does not affect the main observation which is that RNA and DNA are bound similarly.Calorimetric experiments were carried out using a VP-ITC instrument . All solutions were degassed prior to use. SsoSSB in the same buffer. Emission intensity at each concentration of SsoSSB was corrected for dilution and the emission titration was fitted, as previously described  experiments were carried out in triplicate using a Varian Cary Eclipse fluorimeter, exciting the Cy3 dye at 550 nm. Oligonucleotides C12ssDNA Cy3 and C12ssRNA Cy3 (10 nM) were solubilized in 50 mM Tris–HCl pH 8.0, 10 mM KCl, and titrated with IDA and ID represent the intensity of the donor in the presence and absence of acceptor, respectively. Control experiments to determine the variation in the emission of Cy3 due to PIFE at each SsoSSB were also carried out.Melting experiments were carried out using and intra-molecular FRET assay using Cy3 and Cy5 as FRET pair and the energy transfer efficiency was calculated using Eq. SsoSSB with increasing concentrations of unlabelled oligonucleotide. The area under the emission spectrum was taken at each data point. All ensemble data shown represent the average of three replicates.Stoichiometric tryptophan quenching experiments were carried out as previously described ), where IA and ID represent the intensities of the acceptor and donor, respectively. Single-molecule FRET histograms were generated using the first 15 frames of each trajectory as previously reported   and mixed thoroughly, then spun at 13,000 rpm for 1 min. 5 µl from the resulting supernatant was added to 5 µl formamide (Promega) and loaded on to a denaturing gel , and 30 µl of TEMED, 5 ml TBE, 5 ml water, total volume 50 ml) run at 85 W with a temperature threshold of 50 °C for 2.5 h. The gel was scanned using Fuji FLA5000 phosphorimager and analysed using the ImageJ software.Escherichia coli SSB also indicated a much weaker affinity to ribopolymers than to their deoxy-counterparts  as that seen for a 21T DNA oligonucleotide (apparent KD = 95 ± 0.6 nM) . PIFE assays are based on the increase in the fluorescence emission of dyes due to the binding of proteins in close proximity and it has been extensively used as a molecular ruler to measure binding dynamics and distances shorter than those available by Förster resonance energy transfer (FRET)  and the Hill coefficient (1.7 ± 0.2) were obtained when the amount of titrated ssRNA was decreased to sub-nanomolar levels (~0.7 nM).To investigate this unexpected property of SsoSSB monomer was further investigated using the intrinsic fluorescence of tryptophan as a reporter of binding  are important for ssDNA binding (Wadsworth and White SsoSSB (460 nM) with increasing concentrations of a 12 C ssRNA sequence induced a 75% quenching of the tryptophan emission and yielded a value of ~6–7 ribonucleotides interacting with each bound SsoSSB   and that this melting ability is enhanced when the duplex structure contains single mismatches and lesions, such as cyclobutane pyrimidine dimers (CPD) and extra-helical adducts  showed a significant emission from the Cy5 acceptor dye (λem ~ 670 nm), indicative, as expected, of a high degree of energy transfer from the Cy3 to the Cy5 for the intact hairpin , where Iacc represents the intensity of the acceptor and Idon the intensity of the donor. The single-molecule FRET trajectories displayed occasional bursts in FRET efficiency from a value near zero to a very high efficiency value (Eapp ~ 0.9–1). These bursts represent binding events where the association of the labelled SsoSSB brings the acceptor in close proximity to the donor resulting in a high FRET efficiency. The average dwell time of these binding events is similar between ssDNA  to ensure only a single SsoSSB associates to the nucleic acid and thus allow a direct comparison of the monomer-binding dynamics to ssDNA and ssRNA. Single-molecule dwell-time histograms quantifying the association and dissociation dynamics of SsoSSB to ssDNA and ssRNA are shown in Fig. SsoSSB monomers exhibited, at this concentration, dissociation rate values of 3.8 ± 0.8 s− 1 for ssDNA and 6 ± 1 s− 1 for ssRNA  confirmed that ssDNA and RNA recognise essentially the same binding interface on the protein  with three ssDNA bases (PDB ID 2MNA) (Gamsjaeger et al. In the literature, there are examples of proteins that discriminate between ssDNA and ssRNA (Dickey et al. ded Fig. a. These ein Fig. b–e. We hIn vivo, RNA in S. solfataricus is turned over by the exosome, which functions like the eukaryotic exosome by degrading RNA in a 3′–5′ direction (Evguenieva-Hackenberg et al. SsoSSB on the efficiency of RNA degradation by the exosome in vitro (Fig. S. solfataricus exosome in the presence of increasing amounts of SsoSSB. At higher concentrations of SsoSSB, the activity of the exosome was progressively diminished, demonstrating that SsoSSB has the ability to bind and protect RNA against degradative enzymes in vitro. Partial protection of RNA by SsoSSB against digestion by benzonase was reported previously (Shi et al. SsoSSB is clearly the major ssDNA-binding protein present in Sulfolobus cell extracts, and is estimated to constitute 0.1% of total soluble protein (Wadsworth and White SsoSSB has the potential to associate with and stabilise unstructured RNA molecules, such as mRNA, and thus increase its half-life at the elevated temperatures characteristic of hyperthermophilic organisms. In S. solfataricus, mRNA half-lives are longer than those seen in bacteria, which may reflect the increased stability and protection provided by RNA-binding proteins (Bini et al. SsoSSB plays a role in RNA remodelling in conjunction with RNA helicases, for example in ribosome biogenesis, as SSB binding could protect unfolded rRNA and act as an RNA chaperone. We have shown previously that SsoSSB forms a tight physical interaction with RNA polymerase via the C-terminal tail, and can stimulate transcription in vitro, consistent with a role as an mRNA chaperone (Richard et al. There is a good reason to suppose that the OB fold evolved originally as an RNA-binding module, as RNA is thought to have predated DNA early in evolution Orgel , and sev


NL4-3 5′UTR substitutions were designed to individually stabilize the dimer-prone or monomer-prone conformations, validated in-solution, and introduced to molecular clones. The effect of 5′UTR conformation on ribosome loading to HIV unspliced RNA and rate of Gag polypeptide synthesis was quantified in cellulo. Monomer- and dimer-prone 5′UTRs displayed equivalent, basal rate of translation. Gain-of-function substitution U103, in conjunction with previously defined nt-pairings that reorient AUG to flexible nt-pairing, significantly activated the translation rate, indicating the basal translation rate is under positive selection. The observed translation up-mutation focuses attention to nt-pairings at the junction of R and U5, a poorly characterized structure upstream of the characterized HIV riboswitch and demonstrates the basal translation rate of authentic HIV RNA is regulated independently of monomer:dimer equilibrium of the 5′UTR.The paradigm protein synthesis rate is regulated by structural complexity of the 5′untranslated region (UTR) derives from bacterial and other riboswitches. In-solution, HIV-1 5′UTR forms two interchangeable long-range nucleotide (nt) -pairings, one sequesters the gag start codon promoting dimerization while the other sequesters the dimer initiation signal preventing dimerization. While the effect of these nt-pairings on dimerization and packaging has been documented their effect on authentic HIV translation in cellulo has remained elusive until now. HIV This dual utility of identical primary sequence is attributable to switching between higher-order conformations of the 5′UTR by a process that is poorly reconciled.RNA viruses require structural information conserved in noncoding sequences to carry-out critical events in their biogenesis. The 5′ untranslated region (UTR) of retroviruses is a bevy of cis-acting information that is communicated through various higher order structures whose primary sequences overlap. Genetic, biophysical and virological studies have dissected which sequences are essential for early and late viral activities. For instance, HIV-1 5′UTR has activity as mRNA template translated to viral structural proteins, and as dimerized genomic ribonucleoprotein (RNP) packaged into the virion17. Long-distance pairings between the unique 5′ (U5) nucleotides (nts) of HIV primary RNA and downstream nts around the gag start codon (AUG), function in common purpose to orient a palindromic dimer initiation sequence (DIS) for intermolecular dimerization. The higher order, dimeric conformation induced by U5:AUG nt - pairings facilitates packaging of gRNP to nascent virions18.A major accomplishment over the past twenty-years is the genetic and structural basis retroviral noncoding sequences dimerize and foster packaged diploid genomic RNA10. Documented by nuclear magnetic resonance (NMR), the U5:DIS pairings induce rearrangement of nts around AUG into a relatively flexible stem loop that is characteristic of a monomer conformation in-solution10. The NMR indicates U5:DIS pairings may also reorient higher order structure upstream of U5, although those details remain to be characterized10.Ironically, an alternative conformation of HIV 5′UTR is induced by reorientation of U5 nts to pair with DIS nts; this conformation abrogates intermolecular dimerization and downregulates selective packaging of virion precursor RNA10. Designated the HIV riboswitch or RNA switch model, this hypothesis invoked principles established for bacterial riboswitches, in which translation is attenuated by specific conformations in noncoding RNA induced by ligand binding20. The corollary components of an HIV RNA switch are 5′UTR binding to Gag structural protein via the nucleocapsid domain, NC. NC exhibits preferential affinity to the 5′UTR experiencing U5:AUG pairings21. Hence, the HIV RNA switch model construed U5:DIS pairings manifest the mRNA template for Gag synthesis, while U5:AUG pairings attenuate translation and activate dimerization and packaging of diploid gRNP.Given that the U5:DIS pairings: i) reorient AUG nts into flexible conformation; ii) downregulate 5′UTR dimerization; and iii) reduce gRNP packaged to virions, the monomer-prone 5′UTR in authentic HIV RNA was postulated to be the RNA template for translation to Gag and Gag-Pol23. Using actinomycin D to downregulate gene transcription revealed MLV transcripts were exclusively downregulated as template for translation, but not a substrate for intermolecular dimerization and gRNP biogenesis23. These mutually exclusive fates of MLV nascent transcripts were attributed to alternative conformations of the 5′UTR induced by nuclear RNA binding proteins and potentially Gag24. Since HIV Rev/RRE activity is downregulated by actinomycin D, alternative approaches, including use of translation inhibitors, were employed to evaluate the fate of HIV RNA. Studies documented HIV RNAs packaged to virions did not require prior usage as mRNA template, positing nascent HIV experiences mutually exclusive fates similar to MLV transcripts29.Resonating with the HIV RNA switch model are observations of murine leukemia virus (MLV), in which genome-length RNA segregated into two physically distinct pools32. The authors concluded nascent HIV RNA exhibiting monomer-prone 5′UTR conformation templates Gag synthesis, and binding by the nascent Gag polypeptide induces its intramolecular conversion and utility as substrate to be packaged gRNP32. This intramolecular conversion occurs in cis- and switches U5:DIS pairings to release DIS and sequester the gag start codon by U5:AUG pairings. This scenario, also designated the cis-packaging model33, resonates with results of experiments on avian sarcoma virus elucidating a single functional pool of the retroviral transcript used for translation and packaging35. Whether or not a conformational change exists on the same HIV RNA molecule or a distinct molecule remains controversial, and has been challenging to address in cell-based assays.Alternative approaches to address the HIV RNA switch hypothesis demonstrated sequence changes in HIV gag open reading frame that eliminated synthesis of Gag diminished the packaging efficiency of genomic RNAin vitro translation activity4, hence supporting the prediction that U5:AUG conformation attenuates translation. Also, introduction of HIV 5′UTR sequences to synthetic reporter RNAs was sufficient to diminish reporter protein output36. By comparison, thermal denaturation or gross deletions that perturbed the U5:AUG conformer upregulated 35S-cysteine incorporation to in vitro translated Gag polypeptide37.The central tenet of the HIV RNA switch model, translation attenuation by dimer-prone 5′UTR, has been abundantly addressed in context of synthetic transcripts and reporter RNAs. Substitution mutations were designed to favor the thermodynamic stability of either monomer- or dimer-prone RNA and evidence for translation attenuation was ascertained. Synthetic transcripts that were pre-dimerized demonstrated a dearth of 3. Results in a follow up-study identified issues in the post-transcriptional expression of select 5′UTR-luc reporter RNAs, which called into question the conclusions made previously38.Cell-based studies of the Luciferase reporter RNAs documented equivocal translation activity between native HIV LAI 5′UTR, which is prone to dimerize in-solution and variant 5′UTRs favoring monomer conformation42. Taken together and despite extensive reporter RNA analyses in vitro and in cell-based assays, a significant knowledge gap exists of whether or not dimer-prone (U5:AUG) 5′UTR attenuates HIV translation or if monomer-prone (U5:DIS) conformation is necessary to authentic viral protein synthesis. This conundrum engenders impetus to revisit the HIV RNA switch model and will require sensitive measurements of translation rate on authentic HIV genome length RNAs.A potential caveat to the HIV RNA switch model comes from studies of the HIV 5′UTR in context of bicistronic reporter RNAs. Herein, HIV 5′UTR sequences were placed in the intergenic position of vector RNAs and engendered equivocal reporter gene activity, indicative of detectable internal ribosome entry and cap-independent translationNL4-3 molecular clones. The molecular clones were introduced to cells that were subjected to kinetic measurement of Gag translation and quantitation of steady state and polysomal RNA. The molecular determinants of Gag translation rate in authentic HIV RNA were characterized for the first time.Herein, HIV 5′UTR substitutions favoring U5:AUG pairings or U5:DIS pairings, which are diagnostic of the dimer-prone or the monomeric 5′UTR, respectively, were introduced to HIV10. As summarized in Fig. In vitro results posit U103 and G104 are at the bottom of the loosely paired PolyA stem, while U105 participates in the U5:DIS or U5:AUG interactions43. The NMR of dimer-prone 5′UTR detects U5 nts in long-range pairings with nts around AUG, instead of DIS , nt substitutions were designed proximal and distal to U5 that increased stability of U5:DIS nt - pairings. As previously probed by NMR, SubA stabilizes the U5:DIS nt - pairings, Fig.  and SubBNL4-3 5′UTR exhibited two molecular conformations designated monomer and dimer. Stabilizing U5:DIS pairing with or without the U103 substitution  changed its mobility to the lower molecular weight monomer conformation  with random primers and the quantitative PCR (qPCR) with HIV gene-specific primers. The accumulation of unspliced, singly spliced, and multiply spliced HIV RNAs was similar between parental HIVNL4-3 and derivatives. As compiled in Table Next, the substitutions were introduced to HIV35S-methionine and cysteine (35S-cys/met) to support new protein synthesis from activated mRNA templates. Newly synthesized polypeptides were isolated by immunoprecipitation (IP) with specific antisera, subjected to SDS-PAGE and phosphorimaged to quantify 35S-incorporation to specific polypeptide. The 35S-cys/met incorporation into Gag measured the translation activity of HIV 5′UTRs.To compare translation activity of the 5′UTRs in context of authentic viral RNA, a sensitive, cell-based kinetic assay was developed, as summarized in Fig. Control experiments established new polypeptide synthesis was reliably detectable within 15 min incubation and de novo synthesis of Gag and loading control beta-Catenin increased during successive 15-min intervals Fig. . Differe35S-incorporation to Gag, indicating these 5′UTR conformations increased the rate of translation  and monomer-prone 5′UTRs that were identical except for destabilized U5:AUG pairings and nonsequestered AUG (SubA and SubB). The results demonstrate U5:AUG pairings were not sufficient to attenuate Gag translation, as postulated in the HIV RNA switch modelion Figs , positin254 and transcripts in each fraction were collected using Trizol. Guided by the profile of ribosomal RNA (UV254), the fractions were collected into designated groups: the low density non-ribosomal RNPs, and 40 S, 60 S, 80 S RNPs, light polysomes (two and three ribosomes) and heavy polysomes (four or more ribosomes)  Fig. . The amoNL4-3 and synthetic transcripts were subjected to the gel mobility shift assay. Compared to WT, U103C exhibited preference for monomer conformation, indicating this single nt upstream of U5 was sufficient to destabilize dimer conformation and presumably the U5:AUG pairings , has proven unwieldy in otherwise highly informative biophysical assays, including FRET-based analysis by Lever and colleagues15.Unexpectedly, U103 substitution is sufficient to shift monomer:dimer equilibrium toward monomer conformation in-solution. This perturbation of nt-pairings at the base of the PolyA stem (nt 59–103) can be explained by shifted register of localized nt-nt pairings. The U103 substitution modestly increased HIV translation activity alone, but significantly enhanced translation when in combination with SubA or SubB substitutions that release U5:AUG nt-pairings, implicating a synergistic effect on 5′UTR structure. Recently a similar structural perturbation at the base of PolyA stem was documented and proposed to be a consequence of heterogeneity in the HIV transcription start site49. Studies comparing HIV-1 and HIV-2 translation activity revealed HIV-2 TAR structure is more inhibitory than HIV-150. Given unprecedented translational activity uncovered at the junction of HIV-1 R and U5, structural parameters of this segment of the 5′UTR, which has evaded characterization in other solved HIV RNA structures, will be critical to identify.In refocusing attention to the role of RU5, this segment of HIV, HTLV-1 and several other retroviruses has been shown to activate polysome loading and increase Gag protein synthesis in conjunction with DHX9/RNA helicase A, and potentially other cellular RNA binding proteins51; what is the structural consequence to adenine methylation within the 5′UTR54 or the trimethylation of the 5-terminal 7-methylguanosine cap55. Finally, mechanistic studies are warranted to characterize the unprecedented role of DHX9/RHA in translational control of the 5′-proximal residues of HIV RNA situated between TAR and the PolyA stem47.In closing, our study documented complexity of HIV translational control is intertwined with 5′UTR conformations broader than monomer:dimer equilibrium. We are now asking the questions: how does 5′UTR structure influence interaction with the nuclear cap binding complex and its retention on HIV RNA during downregulation of eIF4ENL4-3 5′UTR residues (+1 to +356) in pUC19 and sequences were introduced by site-directed mutagenesis (Agilent). In vitro transcription reactions contained T7 RNA polymerase, PCR amplified template DNA (1.5 µg) in 30.8 mM Tris-HCl, 6 mM MgCl2•6H2O, 1 mM spermidine, 5 mM DTT, 1.1 µM Triton X-100, and NTPs for 120 min at 37 °C56. Reactions were loaded to 6% denaturing urea gels (SequaGel) and the transcripts were eluted by Elutrap Electroelution (Whatman). The RNA dimerization assay used aliquots of 0.65 µM RNA in 10 mM Tris-HCl (pH 7.5) in boiling water for 3 min and then transferred to ice. Samples were adjusted to 0.5 µM in 1x physiological ionic strength buffer  and incubated at 37 °C for 24 h. Electrophoresis at 4 °C used 2.5% native agarose gels prepared in TB buffer  and RNA was visualized by ethidium bromide staining.Template DNAs contained a T7 promoter adjacent to HIV5 per well). After overnight culture, cells were incubated with 500 ng pNL4-3 or derivatives and 1.5 µl XtremeGene (Invitrogen) in OptiMEM (100 µl). The 5′UTR sequences of SubA, SubB, SubC, SubA U103G, and SubB U103C were generated by site-directed mutagenesis (Stratagene) and verified by plasmid restriction and sequencing. After 24 h incubation, cells were lysed in 250 µl ice-cold RIPA buffer .HEK293 cells were maintained in EMEM (ATCC) plus 10% FBS (Gibco) and 1 × Anti-Anti (Gibco) and seeded in 12-well plates for transfections (2 × 10Cell lysate was mixed with Trizol (Invitrogen) and RNA isolated per manufacturer instructions and treated with (2 units) TurboDNAse (Ambion) for 60 min at 37 °C, followed by 5 µl DNAse inactivation reagent (Ambion). Samples were applied to RNeasy Clean-up columns (Qiagen) and RNA was eluted in DEPC-treated sterile water. Five-hundred ng of RNA per sample was incubated with 2 µl random hexamers (Invitrogen) and 4 units Omniscript RT (Qiagen) in 20 µl reactions for 60 min at 37 °C. Typically, 2 µl of each sample was subjected to real-time quantitative PCR with BioRad SYBR Green and HIV NL4-3 gene-specific primers: gag/unspliced KB1614 GTAAGAAAAAGGCACAGCAAGCAGC and KB1615 CATTTGCCCCTGGAGGTTCTG; singly spliced KB2299 GGCGGCGACTGGAAGAAGC and KB2300 CTATGATTACTATGGACCACAC; and doubly spliced KB 2301 GACTCATCAAGTTTCTCTATCAAA and KB2302 AGTCTCTCAAGCGGTGGT.35S-labeled cysteine/methionine (Perkin Elmer). At 15 min intervals, the medium was decanted and cells were harvested in ice-cold 250 μl RIPA buffer by gentle pipetting, and soluble proteins were collected by the centrifugation step. The entire sample was mixed with 20 µl pretreated Dynabeads and incubated at 4 °C for 3 h on a rotating platform. The pretreated Dynabeads were conjugated to an antiserum as described57. We used 2 µl HIV Gag 24–4 (NIH AIDS Reagent Program)59 or 2 µl beta-Catenin (Sigma) antiserum for each immunoprecipitation. Using a magnet to collect immunoprecipitates on the Dynabeads, supernatant was decanted and beads were washed in 1 ml NENT-150  and 1 ml wash buffer , and resuspended in 30 μl of 2 × Laemmli sample buffer. Samples were placed in boiling water bath for 5 minutes and centrifuged for 1 min at 10,000 rpm. The supernatant was decanted and 15 µl aliquots were loaded to 4–20% gradient gels for SDS-PAGE. The gel was applied to Whatman paper, treated under vacuum for 2 h at 80 °C and exposed to a phosphor screen. Radiolabeled proteins were detected on a Typhoon imager and data were processed by ImageQuant software.Transfected cells were cultured in 1 ml cysteine/methionine-free EMEM (Gibco) for 30 min. This medium was replaced with 300 µl EMEM/10% dialyzed fetal bovine serum/1 × L-glutamine (Gibco) supplemented with 100 μCi/ml 2, 10 mM NaCl, 2 mM DTT, Protease inhibitor, 100 µg/ml cycloheximide, 5 µl/ml RNase Out) and placed on ice for 5 min, diluted in 500 µl lysis buffer  and treated by ten strokes of a Dounce homogenizer. Following centrifugation to retain cell debris, typically ~1200 µl samples were collected. Eight-percent was extracted with Trizol to ascertain equivalent input RNA. After reserving an aliquot of the lysate, 900 µl was loaded to 13 ml continuous sucrose gradients (10–50%) and centrifuged in an SW41 rotor for 2 h at 25,000 rpm. The Brandel system that collects from the top of the gradient generated twenty-four 0.5 ml fractions. Every other fraction was stored or treated with warm ethanol and 3 M NaOAc overnight to collect nucleic acid. After centrifugation, 100 µl DEPC-treated water was used to resuspend each pellet and the samples were extracted by Trizol and chloroform and precipitated in isopropanol. This pellet was washed with 75% ethanol and resuspended in 11 µl of DEPC-treated water and 10 µl was treated with Omniscript RT. Approximately 20% of the cDNA reaction was used for qPCR with HIV-specific primers, as above. The value of 1/CT was assessed against plasmid standard curves to determine relative copy number per sample. Gradients were evaluated to ensure similar total abundance of gag RNA and the distribution of the transcripts was evaluated on the basis of percentage.Cells were transfected for 24 h with indicated molecular clone in two 100 mm plates and then treated with 100 µg/ml cycloheximide (CHX) for 5 min. Cells were harvested in 1 ml PBS/CHX by pipeting, pelleted, and the PBS/CHX was removed. Cells were resuspended in 450 µl ice cold low salt buffer  units with time, sample and their interaction as fixed effects and the replicate and the molecular clone within replicate as random effects. For each time point separately and when averaged over the time points, pairwise comparisons between each molecular clone and WT were computed, and p-values corrected for multiple comparisons using Dunnett’s method. Differences ± standard errors were computed on the log scale and back-transformed to ratios. As a control, a similar model was fit using both 35S-Gag and 35S-beta-Catenin with additional terms for the protein and interactions with protein and time and sample.To assess differences in To assess differences in proportion of gag transcript between the molecular clones for each RNP, ANOVA models were fit on the log of the proportion, and pairwise comparisons between each molecular clone and WT were computed, with p-values again corrected for multiple comparisons using Dunnett’s method. Data are available upon request.Supplementary information


Tissue distribution following intravenous administration revealed that LHNPs  achieved increased delivery to the metastatic brain at 5.43 and 4.36 times the levels of Tykerb  and lapatinib solution , respectively. Compared to the marketed Tykerb group, LHNPs had markedly better inhibition effects on brain micrometastasis and significantly extended the median survival time of 4T1 brain metastatic mice in consequence. The improved anti-tumor efficacy of LHNPs could be partly ascribed to down-regulating metastasis-related proteins. Therefore, these results clearly indicated that LHNPs could become a promising candidate for clinical applications against brain metastasis of TNBC.Brain metastasis from triple-negative breast cancer (TNBC) has continued to lack effective clinical treatments until present. However, the feature of epidermal growth factor receptor (EGFR) frequently overexpressed in TNBC offers the opportunity to employ lapatinib, a dual-tyrosine kinase inhibitor of human epidermal growth factor receptor-2 (HER2) and EGFR, in the treatment of brain metastasis of TNBC. Unfortunately, the low oral bioavailability of lapatinib and drug efflux by blood-brain barrier have resulted in low drug delivery efficiency into the brain and limited therapeutic effects for patients with brain metastasis in clinical trials. To overcome such disadvantages, we developed lapatinib-loaded human serum albumin (HSA) nanoparticles, named LHNPs, by modified nanoparticle albumin-bound (Nab) technology. LHNPs had a core-shell structure and the new HSA/phosphatidylcholine sheath made LHNPs stable in bloodstream. Compared to free lapatinib, LHNPs could inhibit the adhesion, migration and invasion ability of high brain-metastatic 4T1 cells more effectively  Breast cancer is the most common cancer in women, with more than 1.7 million new cases every year reported by the International Agency for Research on Cancer (IARC) . Brain mAmong all breast cancer subtypes, triple-negative breast cancer (TNBC) has the highest incidence of brain metastasis up to 46%) and the poorest prognosis 6% and th, both TNIn the past few years, many studies have proved that the epidermal growth factor receptor (EGFR) is frequently overexpressed in TNBC –9, in asLapatinib, a selective small-molecule dual-tyrosine kinase inhibitor of HER2 and EGFR, was approved in combination with capecitabine for use in HER2-positive patients with advanced metastatic breast cancer . Its selin vivo, and it can be preferentially taken up by tumors [Currently, nanoparticle-based drug delivery has been shown to avoid P-gp-mediated drug efflux, thus elevating drug bioavailability by systemic administration , 16. Mory tumors , 18. In y tumors , 20, thuy tumors . In addiin vivo biodistribution were conducted. Finally, the antitumor efficacy of LHNPs in 4T1 brain metastatic mouse models was investigated, and its mechanisms of action were elucidated by biochemical index assays.Regarding this strategy, we constructed lapatinib–loaded HSA nanoparticles (LHNPs) for treating TNBC brain metastasis. Phosphatidylcholine (PC), a safe material approved by FDA, was introduced into the nano-system to solubilize lapatinib and achieve the appropriate particle size. The possible interaction among the components of the nanoparticles was clarified using differential scanning calorimetry (DSC) analysis and the stability of LHNPs was evaluated in simulated human plasma. Since metastasis is an extremely complicated process involving local invasion, intravasation and extravasation of vascular system, growth at secondary site and angiogenesis , 23, adhNab technology, a mature technology for nanoparticles industrialization, was modified to extend its range of application and prepare lapatinib-loaded HSA nanoparticles in this study. PC was introduced into the formulation due to the low solubility of lapatinib in chloroform (1.18 mg/mL). The nanoparticles presented spherical and uniform characteristics under transmission electronic microscopy (TEM), with a core-shell structure Figure . The meaThe possible interaction among lapatinib, PC and HSA can be detected by DSC experiments.As shown in Figure It was reported that Abraxane was unstable after intravenous injection, and disassociate into individual albumin molecules in several seconds and then circulated in bloodstream as paclitaxel-albumin complexes without desired advantages of nanoparticles . Howeverin vitro adhesion, migration and invasive assays were performed.While the exact mechanism of brain metastasis remains unclear, accumulated studies have proved that the brain metastatic process is a complex process involving multiple steps . First, We examined the influence of LS and LHNPs on the adhesion of 4T1 cells to the plates coated with Matrigel, which is basement member matrix. To avoid interference effects, the concentrations of LS and LHNPs were set at the level of 0.1 μg/mL, which exerted no effects on cell viability as demonstrated in MTT assays . As showIn addition to adhesion ability, the motility of tumor cells was also considered an important measurement for evaluating the metastatic potential of cells. Wound healing assay was used to investigate the lateral migration of cells, while transwell assay focused on the vertical mobility. As shown in Figure in vivo.All of these results demonstrated that LHNPs could more effectively inhibit the lateral and vertical mobility of 4T1 cells compared with free drug, which could form the basis for inhibition of the spontaneous metastasis of breast cancer Invasive assay requires tumor cells to migrate through transwell filters precoated with Matrigel, a model of an extracellular matrix barrier. After enzymatically degrading that barrier, the tumor cells traversed the membrane and proliferated in a new location . As showin vivo model of brain metastasis [in vivo model of brain metastasis.Breast cancer cells have been reported to follow a hematogenous route for metastasis to the brain because there is barely a lymphatic system in the brain , 33. Thetastasis , 35. The0–48 h 125013.3 ± 4475.3 mg/L × h vs 113718.2 ± 2111.1 mg/L × h) [To clarify the tumor-targeting efficacy of LHNPs, the distribution of drugs in normal tissues and in the brain lesions was detected after three formulations of lapatinib treatment. At the same time, to determine the integrity of the BBB in the brain metastasis animal models, the distribution of drugs in normal mice was used as a control. The dose determination of Tykerb and LHNPs was based on the oral dose of human and our pharmacokinetic study, both has similar AUC in plasma (AUCg/L × h) , suggestIn normal mice, the lapatinib concentrations in the brain in the LS group were quite low and could be negligible after 2 h post-injection Figure , primaryAfter being administered to the model mice with brain metastasis, LS displayed slightly increased accumulation in the brains, suggesting that the BBB became leaky, along with the progress of brain metastasis. Several researchers have demonstrated that the BBB is intact in and around brain metastases that are smaller than 0.25 mm in diameter, but larger metastasis can affect the integrity of the BBB . This phbrain to AUCblood for LHNPs(0.144) was higher than that for LS(0.050) and Tykerb(0.019) after the effects of plasma concentration was excluded, indicating the great superiority of LHNPs at brain tumor targets in vivo, resulting from the advantages of EPR effect and HSA nanoparticles, as stated in the Introduction part.The lapatinib accumulations of LHNPs and LS in the brains with metastasis were 5.43 and 1.25 times the level of Tykerb, respectively. In addition, the ratio of AUCAnti-tumor and anti-metastasis efficacy of LHNPs was evaluated by survival experiment performed on brain metastatic nude mice. As shown in Figure p < 0.05), Tykerb , LS , and NPL   was significantly longer than those of mice treated with physiological saline  Figure . ImportaAt the end of the study, the metastatic brains were excised and imaged. Figure Brain metastasis of breast cancer is a complex process involving multiple molecular mechanisms. MMP-2, a classical member of the MMP family, is vital in the progression of intravasation by degrading the extracellular matrix and basement membrane . uPA, a As shown in Figure Lapatinib ditosylate and Gefitinib were purchased from Melonepharma . Tykerb was purchased from GlaxoSmithKline. Phosphatidylcholine (PC) was purchased from Shanghai Advanced Vehicle Technology Pharmaceutical Ltd. . 3--2,5-diphenyltetra-zoplium bromide (MTT), coumarin-6 and Hoechst 33342 dye were purchased from Beyotime . D-luciferin  was obtained from Gold Biotechnology . The 4T1 cell line was obtained from the Chinese Academy of Sciences Cell Bank. The 4T1-luc cell line was kindly donated by the Shanghai Institute of Materia Medica . RMPI medium, fetal bovine serum (FBS) and trypsin-EDTA solutions were obtained from Gibco . All of the other chemicals were analytical or reagent grade.Female nude Balb/c mice (18–20 g) and ICR mice (20–25 g) were obtained from Shanghai Sino-British Sippr/BK Lab Animal Ltd.  and were maintained at a constant temperature (25 ± 1°C). The animal studies were conducted in accordance with the protocols approved by institutional animal care and use committee (IACUC), school of pharmacy, Fudan University.Lapatinib-loaded human serum albumin nanoparticles (LHNPs) were prepared using modified Nab technology. Lapatinib and phosphatidylcholine (PC), at a weight ratio of 1:4, were dissolved in chloroform and then were dripped into 0.6% w/v HSA aqueous solution (pH 4~6.5) under high speed stirring to form an oil-in-water (o/w) emulsion. The primary emulsion was further passed through a Micro fluidizer  6–14 times at 100–170 MPa. After evaporation by rotary vacuum, the nanoparticle suspension was filtered through filter membranes  and freeze-dried .The particle sizes and zeta potential of the nanoparticles were determined using the Malvern Zetasizer , and the morphological examination was performed using transmission electron microscopy . The encapsulation efficiency of the LHNPs was measured using High Performance Liquid Chromatography (HPLC) method.The possible interaction among the components of LHNPs was confirmed by DSC experiments. Approximately 5 mg of the five samples  was put in crimped aluminum pans and heated from 20 to 300°C at a rate of 10 K/min under constant purging of nitrogen at 10 mL/min using DSC 204/1/G, Phoenix .n = 4) by the Malvern Zetasizer.A variety of ingredients in human bloodstream might induce the dissociation of HSA nanoparticles, thus leading to the reduction of particle size of nanoparticles . AccordiIn order to better evaluate the efficacy of LHNPs in brain metastasis, 4T1, a highly metastatic murine breast cancer cell line, characterized as ER-/PR-/HER-2 negative and EGFR-expressing, was used to mimic human brain metastasis in this study , 44.The effects of LHNPs on the adhesive ability of 4T1 cells were assayed as reported previously , 46 withThe effects of LHNPs on cell lateral migration ability were assessed by wound-healing assay . 4T1 cel5 cells in 100 μL were plated in the top chamber of 24-well transwell . Before seeding, cells were plated in serum-free culture medium supplemented with 0.1% BSA and pre-incubated with LS or LHNPs (lapatinib concentration of 0.1 μg/mL) for 24 h, respectively. The lower chamber contained culture medium with 10% FBS, which served as a chemoattractant. After 24 h of incubation at 37°C, the migrated cells remaining on the lower surface of the insert membranes were fixed and stained with crystal violet solution for 30 min at room temperature. After drying in the air, the migrated cells were photographed in five random fields, and quantified by absorbance at 570 nm in a plate reader after washing with 33% acetic acid, respectively.Transwell migration assay was used to test the vertical movement of cells. A total of 5 × 106/mL) with LS or LHNPs (lapatinib concentration 0.1 μg/mL) were suspended in 100 μL of serum-free culture medium and were added to the tops of the Matrigel-coated transwell filters. Cells incubated without any treatment served as the controls. All of the cells were incubated at 37°C and were allowed to invade for 24 h. After incubation, cells invaded through the transwell filters were fixed, stained and counted as per the protocols for migration assay.For cell invasion, the upper surfaces of transwell filters were precoated with Matrigel (60 μL) as protocols for adhesion assay. Then, pretreated 4T1 cells  in a lentiviral construct. Luc-tagged cancer cells (4T1-luc) were injected into the right internal carotid artery of female BALB/c mice, as per a protocol described previously [7/mL) were suspended in PBS at a total volume of 100 μL and were slowly injected into the internal carotid artery from the common carotid artery using a small syringe with a 32 G needle . Subsequently, the ligature of the common carotid artery was pulled for hemostasis and was then removed before the wound was sealed.For eviously , 31 within vivo PET/CT was used for metastasis detection (Supplementary Information).The location and growth of brain metastasis were monitored by repeated noninvasive bioluminescence imaging, using an IVIS Spectrum Imaging System  after luciferin injection  on days 0, 2, 4, 6, 8, 10 and 12 after cell injection. To confirm the successful establishment of brain metastasis models, in vivo, 30 normal mice were also divided into two groups, receiving LS or LHNPs at same dose, and they served as control groups. Subsequently, the mice were sacrificed, and blood and tissues  were collected at 0.5, 1, 2, 4, and 8 h after drug administration.To clarify the tumor-targeting efficacy of LHNPs, the concentration of lapatinib in different tissues was measured after administration. Sixty brain metastasis mice were randomly divided into three groups, receiving Tykerb , LS  or LHNPs . To determine the integrity of the BBB in brain metastasis animal models and the penetration ability of LHNPs across the BBB 0-t) was calculated using the trapezoidal method.Lapatinib concentrations in plasma and tissue samples were analyzed by LC/MS/MS . Then thn = 16). The mice were injected with NPH , NPM , NPL , LS (10 mg/kg) or saline (control group) via the tail vein at 3-day intervals. The compared group was orally administered Tykerb (100 mg/kg) every day. Bioluminescence signals were recorded at days 5, 10, 20, and the inhibition efficacy of brain metastasis for each formulation was evaluated by measuring the survival time of the animals after the treatments.Nude brain metastasis mice were established as described above. After being examined under the IVIS Spectrum Imaging System , mice with similar bioluminescence signals were randomly divided into six groups  and were observed using an optical microscope.n = 3 for each group) were anesthetized and transcardially perfused with saline. The samples of brain metastasis were rapidly isolated and stored at −80°C. The expressions of urokinase-type plasminogen activator (uPA), osteopontin (OPN), matrix metalloproteinase-2 (MMP-2) and CXCR4 in the location with brain metastasis were determined using western blot analysis. The same location in the brains of normal mice was also analyzed as a negative group. The following antibodies were used: anti-urokinase-type plasminogen activator (uPA) , anti-OPN , anti-matrix metalloproteinase-2 , and anti-CXCR4 .Animals (post hoc analysis, was used for multi-group comparison. Student's t-test was used for two-group comparison. Statistical significance was set at p < 0.05.All of the data are expressed as the mean ± S.D. One-way ANOVA, followed by Dunnett's in vitro experimental results demonstrated that LHNPs could remain the integrated structure of nanoparticles in bloodstream for hours and effectively inhibit the adhesion, migration and invasion ability of 4T1 cells. Following intravenous administration in 4T1 brain metastatic mice, LHNPs could remarkably increase lapatinib accumulation in metastatic brains and down-regulate metastasis-related proteins, which could be beneficial for blocking the metastasis process and reducing the occurrence of underlying distant metastasis. Additionally, increased drug delivery to the brain maximized the cytotoxic potential of lapatinib to cancer cells and exhibited excellent therapeutic effects in vivo. All of these results suggested that LHNPs might be a promising candidate for the prevention and treatment of brain metastasis of TNBC.In the present study, we prepared lapatinib-loaded HSA nanoparticles which have much possibility for clinical applications due to the safe adjuvants and mature Nab-technology for industrialization. The


Xist mediated silencing has been a key goal in X inactivation research. Early studies implicated the Polycomb proteins, a family of factors linked to one of two major multiprotein complexes, PRC1 and PRC2 ; Silva 2003 Dev. Cell4, 481–495 (doi:10.1016/S1534-5807(03)00068-6); de Napoles 2004 Dev. Cell7, 663–676 (doi:10.1016/j.devcel.2004.10.005); Plath 2003 Science300, 131–135 (doi:10.1126/science.1084274)). PRC1 and PRC2 complexes catalyse specific histone post-translational modifications (PTMs), ubiquitylation of histone H2A at position lysine 119 (H2AK119u1) and methylation of histone H3 at position lysine 27 (H3K27me3), respectively, and accordingly, these modifications are highly enriched over the length of the inactive X chromosome (Xi). A key study proposed that PRC2 subunits bind directly to Xist RNA A-repeat element, a region located at the 5′ end of the transcript known to be required for Xist mediated silencing ). Subsequent recruitment of PRC1 was assumed to occur via recognition of PRC2 mediated H3K27me3 by the CBX subunit of PRC1, as has been shown to be the case at other Polycomb target loci ). More recently, several reports have questioned aspects of the prevailing view, both in relation to the mechanism for Polycomb recruitment by Xist RNA and the contribution of the Polycomb pathway to Xist mediated silencing. In this article I provide an overview of our recent progress towards resolving these discrepancies.Identifying the critical RNA binding proteins (RBPs) that elicit This article is part of the themed issue ‘X-chromosome inactivation: a tribute to Mary Lyon’. The requirement for ongoing Xist RNA expression has been interpreted to indicate that Xist RNA recruits Polycomb complexes either directly or through an intermediary RNA binding protein (RBP). In support of this view, conventional and super-resolution microscopy studies indicate that Xist RNA and Polycomb subunits localize closely with one another [Xist RNA binding [Xist RNA  directly, via the A-repeat, an element at the 5′ end of the transcript that is critical for Xist mediated chromosome silencing [in vitro interaction studies, established a model in which Polycomb recruitment to Xi is initiated by PRC2 binding to the Xist A-repeat. PRC1 recruitment was inferred to occur through the classical hierarchical pathway in which the CBX subunit present in canonical PRC1 complexes binds to PRC2 mediated H3K27me3  indicates that the spatial separation is in fact is greater than would be expected for directly interacting factors [A further consideration is that although microscopy studies point to there being a close association of  factors . This co factors . In thisXist interactome failed to identify core PRC2 subunits, although interestingly PRC1 proteins were detected [Xist RNA [A final argument is that proteomic analysis of the detected ,20. In tdetected . A domaidetected  but deleXist RNA .2.et al. [Xist dependent recruitment of PRC2 and of non-canonical PRC1. However, an alternative interpretation came to light with reports demonstrating that PRC2 can recognize PRC1 mediated H2AK119u1 [A further topic of debate has been the basis for recruitment of PRC1 to Xi. As indicated in §1, this was initially attributed to the classical hierarchical model for PRC1 recruitment . Howeveret al.  found thet al. ,25, acco2AK119u1 –28, refeXist RNA, previously implicated in PRC2 recruitment. Linked to these findings we have recently shown that Jarid2, the PRC2 cofactor implicated in interactions with the XN region [Xist RNA, based on the aforementioned results, is illustrated in In recent work we set out to determine if Polycomb recruitment to Xi is initiated by PRC1, PRC2 or both complexes together. Interestingly we found that PRC2 recruitment is entirely dependent on prior deposition of H2AK119u1 by non-canonical PRC1 complexes . MoreoveN region , directlN region . Gene knN region . A revis3.The first evidence linking the Polycomb system and X inactivation was the finding that embryos with a null mutation in the gene encoding the PRC2 subunit EED, display a female specific phenotype in which trophoblast giant cells (TGCs), an extraembryonic cell type found in the placenta and other extra-embryonic tissues, are largely absent . This reXist transgenes. Deletion of the A-repeat element strongly abrogates silencing [Xist RNA domains, including the XN region, was reported to have little or no effect on silencing [Xist transgenes to silence genes located in cis [Xist mediated silencing [A second body of evidence indicating that Polycomb plays only a limited role in Xi gene silencing in embryonic lineages arises from studies in mESCs using inducible ilencing , yet recilencing ,14,16. Milencing . Additiod in cis . Similarilencing .Xist mediated silencing in embryonic cells, there are some caveats that need to be considered. The assay to determine the silencing function of different Xist regions is relatively crude, relying on the occurrence (or not) of significant levels of cell death following induction of Xist expression from the single X chromosome in XY mESCs [Xist RNA is entirely abolished [Xist mediated silencing is affected by complete abrogation of both PRC1 and PRC2 recruitment. Are these caveats relevant? The answer it would appear is very much in the affirmative. In our recent work we found that in ncPRC1 knockout embryos in which recruitment of both PRC1 and PRC2 to Xi is abolished, there is female specific lethality at around E7.5–8.5. Male embryos were also affected but died at a later stage, around E10.5. Female lethality at E7.5–8.5 is unlikely to be attributable simply to effects on the TGC lineage, as the dependence on a functional placenta is apparent only from E9.5 onwards. Moreover, in equivalent mutant mESCs expressing an inducible Xist transgene, we observed significantly reduced silencing of most genes across the entire inactivated chromosome [While the aforementioned experiments indicate that Polycomb has at most a marginal effect on XY mESCs . The absXY mESCs ,25. AddiXY mESCs , the latbolished . Importaromosome .4.Xist RNA overturns previous models based on initiation by PRC2. The classical model gained traction largely because of the reported biochemical interaction of PRC2 with A-repeat sequences in vitro [Our recent findings demonstrating that non-canonical PRC1 initiates Polycomb recruitment by in vitro . A key iin vitro –40.Xist RNA. A key area for future studies is to identify the critical element(s) within the XN region, and to define the biochemical interactions that recruit non-canonical PRC1. Here it should be noted that the interaction may be direct, with a non-canonical PRC1 subunit(s) binding the XN region, or alternatively, via an adaptor protein that binds both the XN region and PRC1 complexes. The latter possibility is more probable as known RNA binding domains are not present in core non-canonical PRC1 subunits. Moreover, non-canonical PRC1 subunits were identified in the Xist interactome determined using formaldehyde but not UV cross-linking [Xist RNA is more likely indirect, i.e. via an adaptor protein.As noted in §2, we find that non-canonical PRC1 complexes initiate Polycomb recruitment through interaction with a sequence element in the XN region of -linking ,20, suggXist RNA. As discussed in §1, Polycomb recruitment lags significantly behind expression of Xist RNA in early mouse embryos, and the reason for this remains to be determined. Additionally, experiments using Xist transgenes revealed that inducing expression in differentiated ESCs does not lead to Polycomb recruitment. However, if the Xist transgene is expressed transiently prior to differentiation, Polycomb recruitment does occur following differentiation [It will be important in the future to determine the degree to which the revised recruitment model can account for all of the experimental observations relating to Polycomb recruitment by ntiation . Again, Xist mediated silencing, our recent findings provide substantive evidence of the importance of Polycomb, not only in a specialized extraembryonic lineage, TGCs, but in all lineages derived from the early embryo. The fact that this was not detected previously is likely attributable to the fact that we used a relatively sensitive chromosome wide transcriptome assay, and also, that in our experiments both PRC1 and PRC2 activity is entirely abrogated.In relation to the contribution of Polycomb to Xist mediated silencing activity. There is little or no detectable silencing in the absence of the A-repeat, yet Polycomb recruitment occurs similarly to wild-type Xist RNA. This observation indicates that Polycomb functions downstream of, or somehow supports primary silencing by A-repeat binding factors. We envisage two possible scenarios: Polycomb modifications may serve to stabilize gene silencing established by A-repeat binding factors. Thus, aberrant silencing in the absence of Polycomb would represent a defect in maintenance of gene silencing. Alternatively, by affecting the compaction state of Xi chromatin, Polycomb may function to facilitate the spread of gene silencing mediated by the A-repeats. This latter idea is predicated on evidence that Polycomb proteins, through self-association, play an important role in higher order folding of the chromatin fibre [Xist RNA accumulates [in cis spreading of primary silencing mediated by the A-repeat binding factors.An important question for the future is to understand how Polycomb facilitates in fibre –44. Whilin fibre –47. On Xumulates ,7,10. Th


Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.The online version of this article (doi:10.1007/s11033-016-4043-6) contains supplementary material, which is available to authorized users. A sinThe measurement and exact quantification of miRNAs from serum are hampered by low yields of RNA due to its low concentration (0.1–1 ng/ml) . HoweverIn this study, we evaluated different extraction procedures, with and without addition of a carrier, on serum “test samples” that were obtained from the Department of Defense Serum Repository (DoDSR). These specimens are linked with the Defense Medical Surveillance System (DMSS) data and are augmented by epidemiological studies for military and operational relevance, and upon additional approval, the samples can be applied for research purposes. The rich details related to military service that have been collected provide health professionals with unique opportunities to utilize these archived samples to find solutions for both demographical groups and individuals. It is important to optimize the different aspects of sample management, such as sample storage conditions and extraction procedures, and to demonstrate that the miRNA obtained is of sufficient quality for NGS.The serum samples were obtained from the DoDSR where they were stored at −30 °C. These samples were called “test samples” and were used to conduct a pilot study with the purpose of determining suitable methods for these compromised samples before setting up a larger study. In the current study, a subset of samples was thawed on ice, pooled, and divided into 350 μL aliquots, frozen and stored at −80 °C to be used in extractions as “pooled” samples Fig.  and we uDifferent miRNA extraction methods were evaluated with either unpooled or pooled serum samples used in triplicate per extraction method. The extraction methods TRIzol Reagent  and TRIzol LS Reagent (Invitrogen) were tested with or without a different carrier, such as glycogen (Invitrogen), bacteriophage MS2  and yeast tRNA (Invitrogen). Extractions were also performed using the miRNeasy Mini Kit . Other extraction methodologies that were tested only on the unpooled samples were the mirVANA miRNA Isolation Kit , the ExoQuick Exosome precipitation  extraction in conjunction with the TRIzol procedure, and the ExoQuick Exosome precipitation in combination with mirVANA miRNA Isolation Kit. The detailed study plan is shown in Fig. g for 15 min at 4 °C. The aqueous phase containing RNA was transferred to a new tube and isopropanol  was added. The samples were incubated at room temperature for 10 min followed by centrifugation at 12,000xg for 15 min at 4 °C. The pellet was washed with 75 % Ethanol (Sigma-Aldrich), air-dried and resuspended in 20 μL nuclease-free water (Ambion). For addition of carriers, 2 μL of glycogen (5 μg/μL), 1 μL of bacteriophage MS2 RNA (500 ng/μL) or 1 μL of yeast tRNA (500 ng/μL) were added during the isopropanol precipitation of the extraction procedure. TRIzol LS was used instead of TRIzol in the procedure using the glycogen carrier.TRIzol extractions were conducted according to the manufacturer’s protocol (Invitrogen). Briefly, 700 μL of TRIzol was added to 350 μL of serum followed by incubation at room temperature for 5 min. Chloroform  was added, and the samples were vortexed and incubated for 5 min at room temperature followed by centrifugation at 12,000×The samples were processed using the mirVANA miRNA Isolation Kit following the manufacturer’s protocol.g for 30 min at room temperature and the supernatant was aspirated, followed by centrifugation of the pellet at 1500xg for 5 min at room temperature to remove all traces of residual Exoquick solution. The pellet was dissolved in 350 μL of TRIzol reagent and the TRIzol extraction procedure was followed.The samples were processed using the ExoQuick Exosome precipitation method by adding 90 μL of ExoQuick solution  and incubating overnight at 4 °C. The samples were centrifuged at 1500xThe miRNeasy Mini Kit was used to extract miRNA and total RNA following the manufacturer’s protocols.The quality of the RNA was evaluated by the Bioanalyzer 2100 instrument  using the small RNA chip (Agilent) according to the manufacturer’s protocol.Small RNA libraries were prepared using the TruSeq Small RNA Sample Preparation kit  following the manufacturer’s instructions. A fraction of five microliter of total RNA was used for library preparation as quantification is below the detection limit. Five microliters of total RNA was ligated with 3′ and 5′ adaptors followed by reverse transcription with SuperScript II Reverse Transcriptase (Invitrogen) and unique indexes were introduced during PCR amplification for 15 cycles. A multiplexed pool consisting of equimolar amounts of small RNA-derived libraries was prepared. The 140–150 nt region containing the microRNA fraction of the library was excised from a 6 % polyacrylamide gel after electrophoresis, ethanol-precipitated and quantified. The samples were subsequently sequenced on the Illumina HiScan SQ instrument using 50 + 7 (index) sequencing cycles.Our data analysis pipeline Fig.  performsThe standard open source tool CASAVA 1.8.2  was used to preprocess raw base calls and de-multiplex samples. Reads were processed further for quality assessment, quality filtering, and adaptor trimming, and quality filtered again using a Phred quality score threshold of 20. The ligated adaptor sequences were trimmed, leaving a minimum overlap of five bases while allowing an error rate of 0.1 using CutAdapt 1.2.1. (Martin 2011). Additional filtering was also applied to retain sequences with a minimum length of 15 base pairs and to discard sequences of fewer than 15 base pairs in length. The average read length distribution across protocols per purification method was further statistically assessed using Pearson Chi Square test using read length as bins for each protocol.http://broadinstitute.github.io/picard), SAMtools 0.1.19 [Using the short read aligner Bowtie 1.0.0 , filteres 0.1.19 , BEDtools 0.1.19  and shels 0.1.19 . The miRThe purpose of our study was to develop a method of RNA extraction from archived serum samples stored at −30 °C to obtain sufficient yields and acceptable quantities of miRNA for NGS. miRNA has been shown to be present in twelve body fluids tested , and higThe method used to purify RNA impacts both yield and the spectrum of RNA isolated , 39. miRSerum has low amounts of total RNA, and small RNA only constitutes 0.4–0.5 % of this total . To maxiThe use of glycogen in RNA isolation has been reported previously  to prepaMany studies have compared different methods for miRNA extraction . TRIzol-The samples obtained from the DoDSR cannot be controlled for the initial sample processing and storage conditions; therefore, the purpose of this study was to test for maximum RNA yield using the different isolation methods available.There is not a method currently available that can evaluate the exact amount or quality of small RNA. Extracellular miRNAs are less than 1 % of the total RNA recovered , and sinThe Agilent 2100 Bioanalyzer system quantifies RNA using an algorithm based on ribosomal RNA detection and assigns an RNA Integrity Number (RIN) to demonstrate RNA quality. However, this algorithm is not applicable to quantification of miRNA extracted from serum samples. Recently, it was reported that the percentage of RNA fragments >200 nt in a sample  is a more reliable predictor of RNA quality when analyzing highly-degraded RNA sample quality as compared to the traditional integrity score or value .There are conflicting opinions on whether biological carrier RNAs might be responsible for non-specific hybridization or amplification in downstream analytics . AdditioIt is known that RNA yield is affected by the specific purification methods used , 57. TheStudies have attempted to detect plasma/serum miRNA using microarrays and qRT-PCR . RecentlOur data analysis pipeline performs preprocessing of reads, aligns sequence reads against reference genome assembly, and quantifies miRNAs, followed by a quality control step. The alignment step generates alignment files and HTML files with mapping statistics for the percentage of reads mapped out of total number of reads against the reference genome/miRBase. Pooling has been used to examine the general characteristics of the reads  and for The miRNA profile was compared between different extraction methods with and without, as well as before and after, the addition of carrier  end of each read before alignment. The good quality processed reads were aligned to mature miRBase and only a proportion of sequences mapped to the database, as shown in stacked bar plot Fig. . The TRIMore than 95 % of the reads from most of the unpooled sample types were annotated with coding sequences. The TRIzol with carrier yeast tRNA procedure had 81 % of the reads in coding regions whereas TRIzol with MS2 had 92 % aligned to coding regions (data not shown). From the pooled samples, 90 % of the reads were in the coding regions for all the procedures tested. We did not observe an increase in reads mapping to the tRNA as a result of the extraction procedure including yeast tRNA as a carrier. The coding RNA species were the largest proportion when mapped to the genome, and furthermore, other RNA species involved in many biological processes were identified among the isolated RNAs. The proportions of the RNA species other than coding RNA are shown in Fig. We analyzed the associations among different replicates extracted by different miRNA isolation methods, and all of the samples were positively correlated to each other for pooled as well as for unpooled samples. The correlation of pooled samples ranged from 76 to 94 %, whereas for unpooled samples, it ranged from 88 to 99 %. We found that TRIzol with yeast tRNA gave a very poor correlation for miRNA counts with the rest of the procedures, for pooled as well as unpooled samples (data not shown). It has been reported that the addition of carrier tRNA during RNA or DNA extraction can lead to false positives during hybridization-based assays due to carry over of contaminating nucleic acids . We can log2 counts per million (logCPM) values Supplementary material 2 (XLSX 81 kb)Below is the link to the electronic supplementary material.


Using our previously developed and tested Adolescent Sexual and Reproductive Health (SRH) Stigma Scale, we investigated factors associated with perceived SRH stigma among adolescent girls in Ghana.We drew upon data from our survey study of 1,063 females 15-24yrs recruited from community- and clinic-based sites in two Ghanaian cities. Our Adolescent SRH Stigma Scale comprised 20 items and 3 sub-scales  to measure stigma occurring with sexual activity, contraceptive use, pregnancy, abortion and family planning service use. We assessed relationships between a comprehensive set of demographic, health and social factors and SRH Stigma with multi-level multivariable linear regression models.In unadjusted bivariate analyses, compared to their counterparts, SRH stigma scores were higher among girls who were younger, Accra residents, Muslim, still in/dropped out of secondary school, unemployed, reporting excellent/very good health, not in a relationship, not sexually experienced, never received family planning services, never used contraception, but had been pregnant . In multivariable models, higher SRH stigma scores were associated with history of pregnancy  and excellent/very good self-rated health , while lower stigma scores were associated with older age , higher educational attainment , and sexual intercourse experience .Findings provide insight into factors contributing to SRH stigma among this young Ghanaian female sample. Further research disentangling the complex interrelationships between SRH stigma, health, and social context is needed to guide multi-level interventions to address SRH stigma and its causes and consequences for adolescents worldwide. Stigma “deeply discredits and transforms people from whole individuals to tainted, discounted ones” ,2. It isStigma occurring in the context of adolescent sexual behavior, pregnancy, early childbearing, abortion, and sexually transmitted infections (STIs) may lead to adverse health and social consequences, including shame, social marginalization, violence and mental health morbidity –21. Our While this work was useful in providing initial estimates of SRH stigma and documenting relationships between girls’ higher SRH stigma scores and nonuse of modern contraception –24, findWe investigated a diverse set of demographic, social and health factors associated with environmental SRH stigma perceived by adolescent girls from two urban communities in western Sub-Saharan Africa.The study was approved by the Institutional Ethics Review Boards of the Ghana Health Services, University of Ghana, Kwame Nkrumah University of Science and Technology, and University of Michigan. For this analysis, we drew upon data from a comprehensive survey study of adolescent and young adult females ages 15–24 from community- and clinic-based sites in Accra and Kumasi, Ghana. Our study design and sample have been described elsewhere [Trained Ghanaian research assistants (RAs) approached women at recruitment sites, provided study information, invitations to participate, and screened young women for eligibility. Eligible potential participants were: 1) females, 2) between the ages of 15 and 25, 3) residents of Accra or Kumasi or surrounding areas, and 4) able to speak English or one of two local languages (Ga or Twi). Following eligibility screening and enrollment, RAs obtained informed written consent; we obtained parental consent waivers from all Ghanaian IRBs given the sensitive nature of our survey and to ensure confidentiality. Enrolled participants then completed the confidential survey interview administered by RAs via tablets using Qualtrics Mobile, a secured, web-based data collection and management system. Our survey was informed by our preceding qualitative study, our prior reproductive health services and stigma-related research, an extensive review of the relevant literature on stigma and the social context of adolescent SRH, and drawing upon well-established existing survey items .–11,22–24The comprehensive survey included our new Adolescent SRH Stigma Scale, which we psychometrically tested and validated specifically for this project. The finaWe examined a series of items on physical and mental health and social wellbeing as potential predictors of SRH stigma. For health histories, women were asked whether they “suffer from any chronic medical condition or ongoing health problem, for example high blood pressure or asthma,” which we examined as a binary variable . We assessed current general and mental health status with a series of standardized items. We first used the 5-point Likert item for self-rated overall health  and present results as excellent/very good vs. <very good health. We assessed mental health symptoms with single items adapted from validated depression, anxiety and stress scales. Via the Patient Health Questionnaire (PHQ), women were asked how often, on a 5-pt scale , in the past month they had been bothered by the following depression and anxiety symptoms, respectively: 1) feeling sad, depressed, or hopeless, and 2) feeling excessive worry, nervous, anxious, or on edge. With theFor social wellbeing, we administered the abbreviated Everyday Discrimination Scale. Via fiveWe examined the following sociodemographic variables as “predictors” of SRH stigma: age, ethnic group , religious affiliation (Muslim vs. Christian/Other), religious importance , frequency of religious service attendance (≥weekly vs. <weekly), educational attainment (completed secondary school or more vs. <secondary school), relationships status , employment status (employed in last seven days vs. not employed) and health insurance status (insured vs. uninsured). We examined the following sexual and reproductive history characteristics: sexual intercourse with male partner , number of sex partners , sexual assault , physical assault by an intimate partner , receipt of family planning services , contraceptive use , and histories of pregnancy, live birth, miscarriage/stillbirth, and abortion .Our analytic sample included 1,063 of the 1,080 participants (98.5%) who completed 60% or more of survey items, including all SRH stigma items. We used descriptive (means with standard deviations (SD), frequencies with proportions (%)) and bivariate statistics  to describe and compare SRH stigma scores across sociodemographic, health, and reproductive history groups. We used multivariable linear regression with a recruitment site cluster effect to simultaneously examine all available sociodemographic, health, and reproductive history factors potentially associated with stigma. Variables were considered for inclusion in regression models if their p-values in bivariate analyses were <0.25. We employed a step-wise approach, modeling sociodemographics first, then adding health and social wellbeing factors, followed by reproductive history factors. For factors that were highly collinear , we retained those with the strongest effects in final models. We present regression results as adjusted beta coefficients (β) and 95% CIs. We used STATA 13.0  for all analyses.Characteristics of the sample are presented in Responses to the SRH Stigma Scale, sub-scales, and individual items are presented in In the unadjusted analysis , factorsIn multivariable linear regression analyses , factorsUsing a new validated Adolescent SRH Stigma Scale we developed and tested among adolescents in two urban, western Sub-Saharan African communities, we found high levels of environmental SRH stigma, especially reported by the youngest girls. Our nuanced measurement approach highlighted varying degrees of negative social and community norms, attitudes and beliefs occurring across different dimensions of SRH. While high levels of abortion stigma were not surprising, similar levels of abortion stigma reported for sexual intercourse and pregnancy–two distinct reproductive events that have been less studied in stigma research—were perhaps less expected. Building upon prior stigma work in the U.S. and Africa largely focused on HIV and abortion stigma, our findings add estimates of environmental SRH stigmas across a broader spectrum of reproductive life experiences. They also provide insight into SRH stigma specifically perceived by adolescents—a vulnerable but understudied group in African family planning and stigma research, despite the severe impact of stigma and its health and social consequences for this group –32.lower levels of stigma. It is not clear from our data whether girls had disclosed prior sexual experiences, such that sex itself may not have been a stigmatizing event but rather the perceived social manifestations and consequences of it were. Alternatively, it may be that sex without the consequence of pregnancy could foster stigma resilience, or even a sense of pride or accomplishment. This requires further study. Of note, our survey items were designed to measure SRH stigma within women’s social environments and not (necessarily) experienced stigma; although in this analysis we explicitly assessed girls’ histories of a range of SRH events. Our ongoing work in Africa and the U.S. is further investigating stigma directly attributable to sex, pregnancy, contraceptive use, abortion and family planning service use.We explored a diverse set of demographic, health, and social context factors associated with adolescent SRH stigma. Prior pregnancy was perhaps the clearest, strongest predictor among our young Ghanaian sample. This finding is consistent with those of studies (ours and others’) in which previously pregnant or parenting adolescents, both in U.S. and African cohorts, have reported discrimination, marginalization, and violence –17,33,34Additionally, given the cross-sectional nature of our data, we were unable to establish temporality or examine time-varying levels of SRH stigma that may change over time as a result of new exposures. It is likely that stigma and SRH perceptions and experiences are bi-directionally associated–that one’s perceptions of SRH shaped by the SRH experiences of one’s own, one’s friends, family, or community members, which in turn may or may not be accompanied by stigma –24. ConvSRH stigma specifically, or even to stigma in this geographic/cultural context. In our longitudinal analysis of a cohort of adolescent girls in the United States [Regarding social and health predictors, lack of education, unemployment, and religious affiliation (Muslim) were associated with adolescent SRH stigma, findings which align with results of other studies documenting high levels of reproductive and other types of stigmas among vulnerable or conservative groups –17,33,34In addition to the above-described limitations, our results were likely impacted by social desirability bias given the sensitive nature of our study and our young sample. Additionally, we focused on two major cities in Ghana and on participants recruited from schools and health facilities, such that our findings are not generalizable to all girls in Sub-Saharan Africa or other sociocultural or geographic contexts in which SRH stigma may be perceived or experienced differently. Although, the SRH stigma found here is perhaps notable given the relatively developed reproductive healthcare infrastructure in Ghana compared to other countries. We did not explore the perceptions or experiences of SRH stigma and its predictors among adolescent boys or of transgendered youth, which are highly relevant sub-populations for which SRH stigma has been understudied and deserve future research inquiry. Finally, our stigma scale did not capture the full spectrum of SRH stigmas, for example HIV, STIs or sexual minority stigmas, or an exhaustive list of potential predictors of stigma.Nonetheless, our study suggests that these adolescent girls endorse stigmatizing attitudes around issues of SRH and/or have observed or felt SRH stigma in their environments. Some life experiences or lack thereof appear to render some girls more vulnerable to stigmatizing beliefs , and others more resilient to stigma . While our findings provide insight into a diverse set of factors associated with SRH stigma in Ghana, they also highlight clear remaining gaps in adolescent health research and important directions for future study. Research is needed to inform more holistic, evidence-based practices, programs and policies that can foster stigma resilience at the individual level and counter adolescent SRH stigma at the environmental level in order to improve SRH outcomes for young women and communities across the globe.