Alu elements occurs during human stem cell aging and hinders efficient DNA repair causing entry into senescence. We will discuss how this fine balance between transcription and genomic instability may be regulated by the dramatic changes to chromatin structure that accompany cellular senescence.It is well accepted that non-coding RNAs play a critical role in regulating gene expression. Recent paradigm-setting studies are now revealing that non-coding RNAs, other than microRNAs, also play intriguing roles in the maintenance of chromatin structure, in the DNA damage response, and in adult human stem cell aging. In this review, we will discuss the complex inter-dependent relationships among non-coding RNA transcription, maintenance of genomic stability, chromatin structure, and adult stem cell senescence. DNA damage-induced non-coding RNAs transcribed in the vicinity of the DNA break regulate recruitment of the DNA damage machinery and DNA repair efficiency. We will discuss the correlation between non-coding RNAs and DNA damage repair efficiency and the potential role of changing chromatin structures around double-strand break sites. On the other hand, induction of non-coding RNA transcription from the repetitive  Various signaling pathway and regulatory factors of senescence have been reported over the last four decades  to trigger apoptosis if there is too much DNA damage to be repaired. At an early stage of the DDR phosphatidylinositol 3-kinase-like protein kinase (PIKKs) family  and MRN (Mre11–Rad50–Nbs1) complexes are recruited to the DNA breaks. The PIKKs phosphorylate downstream checkpoint proteins, such as Chk1 and Chk2, and the histone variant H2A.X that is present in the chromatin around the breaks. Phosphorylated Chk1 and Chk2 phosphorylate various effector proteins to arrest the cell cycle to allow time for DNA repair. Phosphorylated H2A.X  assists to recruit other downstream signaling molecules that facilitate the DDR. The SWI/SNF (switch/sucrose non-fermentable) complex (ATP-dependent chromatin remodeling factor) triggers chromatin relaxation after UV treatment. This chromatin relaxation is involved in late stages of DNA repair because it does not affect the recruitment of early DDR response factors but later DDR response factors, such as xeroderma pigmentosum complementation group G (XPG) and proliferating cell nuclear antigen (PCNA) to UV damage sites (DNA damage is induced by various genotoxic stresses from inside the cell or from the environment. Arguably the most dangerous type of DNA lesion is a double-strand break (DSB) because a single DSB can lead to the loss of a chromosome arm during mitosis if not repaired, while its inaccurate repair can lead to chromosomal translocations or mutations. DSBs are repaired by either homologous recombination (HR) which copies the identical undamaged DNA information usually from the sister chromatid, or non-homologous end joining (NHEJ) in which the two DNA ends are ligated back together . Moreover, other non-coding RNAs are more directly involved in the DDR. Over 5% of the mammalian genome consists of short interspersed elements (SINEs), typified by the human Alu repeat. These are RNA polymerase III transcribed sequences whose expression is usually silenced. However, upon prolonged exposure to DNA damaging agents that predominantly cause DSBs, transcription from the human Alu elements and murine SINEs is strongly induced  regulate genes that are involved in the DDR (reviewed in  induced . The reag agents . Periceng agents . Similarg agents . TranscrIt has been reported that new types of non-coding RNAs (rather than miRNAs or Alu elements) were induced by DNA damage, that are actually beneficial for DNA repair, in the filamentous fungus Neurospora crassa . HoweverOne may ask: Why are small DNA damage-induced RNAs generated from the rDNA locus? The answer to this question may lie in the requirement for the single-strand DNA binding protein replication protein A (RPA) and the Neurospora counterpart of the Werner/Bloom syndrome helicase, QDE-1, for making small DNA damage-induced RNAs . A likelArabidopsis. This work showed that small RNAs, referred to as diRNA , are induced in the vicinity of DSB sites, and revealed that these diRNAs promote repair of the DNA lesion .Mammalian cells also show a similar local induction of small RNAs upon DNA damage. in vivo NHEJ and HR reporter cell lines. Both RBMX and NONO are recruited to DNA lesions following laser-microirradiation in a poly-ADP-ribose polymerase (PARP)-dependent manner, where PARP mediates one of the earliest chromatin modifications in response to DNA damage . Using a reporter that allows for visualization of the nascent transcript containing the MS2 RNA stem loop by binding to YFP-MS2 protein, silencing of transcription in cis to the DSB site has been observed .Small non-coding RNAs are known to be involved in the formation of repressive heterochromatin structures over repetitive DNA elements, such as centromeric repeats and retrotransposons . AlthougIn previous sections, we discussed the possibility of chromatin decompaction and/or chromatin compaction by diRNA during the DDR. But what is the real function of diRNAs for regulation of chromatin structure around DSB sites? The chromatin structure around DSB site should be opened for protein recruitment  and compacted for transcription repression . It is not clear which aspects of chromatin structure is regulated by diRNAs yet. There are several possibilities: (i) diRNAs could promote either open and closed chromatin structure around DSB sites. (ii) diRNAs could help make a boundary between closed and open chromatin structures around DSB sites. (iii) Compaction and/or decompaction of chromatin structures around DSBs by diRNAs may occur at different times during repair, or (iv) diRNAs may not directly regulate chromatin structure around DSBs but rather function through regulating the recruitment of DDR factors.asf1 deletion mutants do not have a significant DNA repair deficiency (slightly delayed) but they do have reduced levels of histone H3 over the repaired DNA and defective recovery from checkpoint arrest after DNA repair .Not all regions of the genome show relaxation of chromatin structure during aging, because senescent cells show heterochromatin foci called senescence-associated heterochromatin foci SAHF; . The forAlu and SINE retrotransposons of human adult stem cells also occurs during aging . However, in response to a potential decay of the chromatin structure during aging, excess retrotransposon transcription prevents recruitment of cohesion and condensin, causing persistent DNA damage checkpoint activation and senescence.Increased transcription from the normally silenced ng aging . Whether regions . Alu/SINrase III . FurtherBP1 foci . The ageBP1 foci . Given tBP1 foci , 2007, foperties . This leper se results in a more open chromatin structure, which would indirectly help recruit the DNA repair machinery such as 53BP1. Conversely, it is also a possibility that diRNA transcription around a DSB may be the consequence, not the cause, of local chromatin relaxation that occurs around the DSB. To determine if this is the case, it would be interesting to determine if the diRNAs are only produced over the domain that has lost histones or has H2AZ incorporation.The recent papers discussed here have hinted at a complex interplay between the chromatin structure, transcription, DDR, and cellular senescence. However, many questions remain to be answered. These include: What is the function of non-coding RNAs in the DDR and cellular senescence? Are they involved in regulation of chromatin structures and/or recruitment of proteins to chromatin during the DDR and cellular senescence? What is the mechanism to induce non-coding RNAs during DDR and cellular senescence? Also, it will be interesting to determine whether the diRNAs that are transcribed from the vicinity of DSBs remain in the vicinity of the DSB. If so, this would be consistent with the diRNAs helping to physically establish a repressive chromatin structure? By contrast, if the diRNAs do not remain in the vicinity of the DSB, this would be suggestive that the act of transcription A better understanding of how diRNAs are induced in response to DNA damage may also be relevant for understanding how retrotransposon RNA transcription is induced during adult human stem cell aging. Is it a response to DNA breaks or a consequence of altered chromatin structure allowing access to the general transcription machinery? Do retrotransposon transcripts play a structural role in the maintenance of chromatin structure over these regions of the genome? Investigation of these and many other pertinent questions will doubtless provide mechanistic insight and more important discoveries.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Transcranial direct current stimulation (tDCS) is a technique that can systematically modify behaviour by inducing changes in the underlying brain function. In order to better understand the neuromodulatory effect of tDCS, the present study examined the impact of tDCS on performance in a working memory (WM) task and its underlying neural activity. In two experimental sessions, participants performed a letter two-back WM task after sham and either anodal or cathodal tDCS over the left dorsolateral prefrontal cortex (DLPFC).Results showed that tDCS modulated WM performance by altering the underlying oscillatory brain activity in a polarity-specific way. We observed an increase in WM performance and amplified oscillatory power in the theta and alpha bands after anodal tDCS whereas cathodal tDCS interfered with WM performance and decreased oscillatory power in the theta and alpha bands under posterior electrode sides.The present study demonstrates that tDCS can alter WM performance by modulating the underlying neural oscillations. This result can be considered an important step towards a better understanding of the mechanisms involved in tDCS-induced modulations of WM performance, which is of particular importance, given the proposal to use electrical brain stimulation for the therapeutic treatment of memory deficits in clinical settings. N-methyl-D-aspartate (NMDA) receptor efficacy [Transcranial direct current stimulation (tDCS) is a technique that stimulates the cerebral cortex with a weak constant electric current in a non-invasive and painless manner . The curefficacy ,9-14.The neuromodulatory changes induced by tDCS have been associated with modifications of a variety of behavioural brain functions. In animal studies, anodal tDCS of the cortical surface has been linked with facilitation of an unconditioned response ,16 and iIn general, WM refers to a set of basic mental operations that define the ability to hold an item of information transiently in mind, in order to recall, manipulate and associate this information to incoming new information . WM is cThe present study examined the impact of tDCS on WM performance and the underlying neural activity. In particular, we explored the effect of tDCS applied over the left DLPFC on oscillatory brain activity during a letter n-back WM task. Based on previous findings we hypothesized tDCS-dependent alteration of WM performance ,29. FurtΔA, delta cathodal ΔC) and for the control group (delta ΔCG). The behavioural improvement from sham to active tDCS was stronger after anodal than after cathodal stimulation . Analysis of reaction times (RT) showed that tDCS-treated participants responded faster after active tDCS compared to sham for Hits, but not for False Alarms , neither for Hits  nor for False Alarms .Pre-to-post measurements revealed that all participants improved their performance during the experiment. Table ΔA-d' >ΔCG-d', t30 = 0.99, P = 0.33), and a weaker d' increase after cathodal stimulation  compared to the control group. Analysis of RT showed that pre to post decreases did not differ between the control group and the tDCS-treated group for the anodal or cathodal session, either for Hits  or for False Alarms .In order to separate tDCS-induced alterations of WM performance from repetition-related learning effects, we compared the behavioural pre-to-post measurements between the tDCS-treated group and the control group  and for the anodal compared to the shamA condition , and P3 latencies were significantly shorter for the cathodal compared to the shamC condition .Figure t15 ≥ 2.13, P < 0.05). Anodal tDCS as compared to cathodal tDCS increased the total power in the theta, alpha, and lower beta band (7-24 Hz) at the latency range of -72 - 380 ms . Finally, a significant cluster  was found for the overall differences between sham and stimulation conditions  vs. ). This cluster had a latency range of -34 - 710 ms and revealed a significant increase in the theta, alpha, and lower beta band (6 - 19 Hz) for the anodal  compared to the cathodal session .Figure We assessed the impact of transcranial direct current stimulation (tDCS) on working memory (WM) performance and the underlying neural activity. In two experimental sessions participants performed a letter two-back WM task after sham and either anodal or cathodal tDCS. The results showed that WM performance was generally enhanced from the sham to the active stimulation condition, indicating a repetition-related learning effect on WM task performance. Importantly, this regular improvement in WM performance was influenced by active tDCS in a polarity-specific way. The improvement in WM performance was significantly stronger after the application of anodal than after cathodal tDCS over the left dorsolateral prefrontal cortex (DLPFC), while the WM improvement was intermediate when participants received no electrical stimulation. Thus, anodal tDCS improved the regular repetition-related increase in WM performance, whereas cathodal tDCS interfered with this effect. These tDCS-induced effects were reflected in the neural oscillatory activity, showing polarity-specific alterations as a function of tDCS. Anodal tDCS increased whereas cathodal tDCS decreased the event-related oscillatory power in the theta and alpha range. Our results suggest that tDCS altered WM performance by modulating the underlying oscillatory brain activity in the theta and alpha frequency bands. These results we consider an important step towards a better understanding of the mechanisms involved in the tDCS-induced modulations of WM performance, which is particularly relevant as electrical brain stimulation has been proposed as a useful therapeutic modality for the treatment of memory deficits in a clinical context ,52. MoreThe effect of tDCS on WM performance is consistent with recent findings on the modulatory effects of tDCS on WM functions ,28,29,52In this study we assessed oscillatory brain activity during the WM task in order to investigate the underlying neural mechanisms mediating the tDCS-induced behavioural effects. To date, reports of electrophysiological correlates of tDCS effects are sparse. Using visual event-related potentials (ERPs), a previous study has demonstrated that the amplitude of the N70 ERP component is increased by anodal tDCS, while it is decreased by cathodal tDCS . The oppChanges in oscillatory brain activity play an important role in the formation of perception and memory and thus are essential for higher cognitive functions ,67. AccoIn sum, previous studies have shown that alterations of rhythmic activity, specifically in the theta and alpha range, are associated with proper WM performance. These oscillations increase with behavioural enhancement. In the present study we show that tDCS over the left DLPFC induces altered WM performance by modulating its underlying theta and alpha activity. This result further highlights the importance of the left DLPFC and the specific role of theta and alpha activity during WM performance. Even though this interpretation implies that the modulatory effects of tDCS on WM are specifically related to the responsiveness of the left DLPFC, it may be assumed that altered local cortical excitability in one part of the responsible network influences the whole neural network associated with WM functions beyond the site of stimulation. Indeed, widespread tDCS-induced changes in cortical activity have been demonstrated by a previous neuroimaging study . Thus, iThe present investigation studied the impact of anodal, cathodal or sham tDCS over the left DLPFC on the oscillatory brain activity associated with higher-order cognitive processing. Our results show that tDCS can change the organized cortical activity associated with WM in concert with systematic alterations of WM performance. To our knowledge, this is the first study investigating the effects of tDCS on oscillatory brain activity in the context of a WM task. The results of the study will provide a better understanding of the neuromodulatory effects of tDCS and demonstrate its potential both at fostering knowledge on the functional significance of brain oscillations and for therapeutic application.Sixteen university students  participated in the tDCS study. All participants were consistent right-handers  and had The participants were seated comfortably in a recliner in front of a personal computer screen in an electromagnetically shielded room. TDCS was delivered by a battery-driven constant current stimulator  using a pair of rubber electrodes in a 5 × 7 cm saline-soaked synthetic sponge. For stimulation of the left DLPFC the active electrode  was placed over F3 according to the International 10-20 system for electroencephalography (EEG) electrode placement . The usePresentation software . Participants were stimulated with a sequence of white letters  which were presented on a black background in the centre of the screen. Each letter was presented for 500 ms with an interstimulus interval of 2 s. Each letter was followed by a white fixation cross that remained until the next letter was presented. A target letter was any letter repeated after one intervening letter. Participants were asked to respond to each letter as quickly and accurately as possible and to indicate whether the currently presented letter matched (left button) or did not match (right button) the letter which was presented two trials before  task with concurrent EEG recording. Stimulus presentation was controlled by the Brain Vision Analyzer 2.0 , EEGLAB 6.01 [FieldTrip http://fieldtrip.fcdonders.nl/. EEG data were off-line treated with a 24 dB zero-phase Butterworth filter from 0.1 to 30 Hz and were segmented into epochs from -312 to 712 ms relative to stimulus onset. After baseline correction (-312 to 0 ms), epochs were automatically screened for unique and non-stereotyped artifacts using a probability function built into EEGLAB [During the WM task, EEG was recorded continuously with thirty-two electrodes  located according to the International 10-20 system . The eleLAB 6.01  and Fielo EEGLAB . With tho EEGLAB ,93. AfteHits and False Alarms (FA) were analyzed. To evaluate the impact of tDCS on the repetition-related increase in WM performance, we analyzed the pre-to-post measurements by computing the difference between the behavioural values assessed after active stimulation  and the preceding sham stimulation (shamA/shamC), separately for the anodal  and cathodal session . We compared these pre-to-post measurements, ΔA and ΔC, for each behavioural parameter using paired t-tests. Prior to this calculation, we evaluated the equality of the two sham conditions using paired t-test. This statistical analysis revealed no significant difference between the two sham conditions for d' . Similarly, RTs for Hits  and False Alarms  were not significantly different between the two sham conditions. To determine tDCS-induced modulations on the repetition-related increase in WM performance, we compared the pre-post differences (ΔA and ΔC) of the tDCS-treated group with pre-post measurements of a separate control group (ΔCG) that also performed the WM task twice but without receiving a tDCS treatment during the break in between . Again, prior to this calculation, we evaluated the equality of the two experimental sham conditions of the tDCS-treated group and the pre-measurement  in the control group by means of independent-samples t-tests. This statistical analysis revealed no significant differences between the sham conditions of the tDCS-treated group and the pre-measurement of the control group  were measured relative to the pre-stimulus baseline (-312 - 0 ms) for each condition , sham preceding cathodal (shamC)). Peak detection was performed for the occipito-parietal ROI  and for the Pz electrode (P3 component). This procedure entailed the detection of the most positive  or negative (N1 component) peaks within specific latency bands . The time windows for peak analysis were defined on the basis of the global field power.Brain Vision Analyzer 2.0 software. We used a continuous wavelet transform (WT) with complex Morlet wavelets  to examine the frequency composition of single-trial epochs. The magnitudes of the WTs of single-trial epochs were then averaged to compute the total power of activity, which contains signal components that are phase-locked and non-phase-locked to the stimulus event. For each scale of the WT a baseline correction was applied by subtracting the mean amplitude within the -200 to -100 ms time window from each data point after stimulus onset. Similar to the procedures used in the ERP analysis, the data were subsequently pooled into an occipito-parietal ROI by averaging the ERSPs across different electrode sites .For each subject and each channel, event-related spectral perturbation (ERSP) was calculated using a wavelet-based analysis implemented in FieldTrip. This procedure defined clusters on the basis of the actual distribution of the data and tested the statistical significance of these clusters using a Monte-Carlo randomization method with correction for multiple comparisons [TDCS effects on oscillatory brain activity were analyzed by computing ERSP differences between the separate conditions. For statistical comparisons, we used a nonparametric cluster-based randomization approach built into parisons . The cluTZ: conceived of the study, designed the experimental paradigm, performed the statistical analysis and drafted the manuscriptPS: conceived of the study, performed the data acquisition and the statistical analysis and drafted the manuscriptJDT: performed statistical analysis and contributed to the manuscriptLJ: contributed to the hypothesis, and to the preparation of the manuscriptCSH: contributed to the design, discussion, and to the preparation of the manuscriptAll authors read and approved the final manuscript.


South Africa endorses the global policy shift from primarily client-initiated voluntary counselling and testing (VCT) to routine/provider-initiated testing and counselling (PITC). The reason for this policy shift has been to facilitate uptake of HIV testing amongst at-risk populations in high-prevalence settings. Despite ostensible implementation of routine/PITC, uptake amongst tuberculosis (TB) patients in this country remains a challenge. This study presents the reasons that non-tested TB patients offered for their refusal of HIV testing and reflects on all TB patients' suggestions as to how this situation may be alleviated.In February-March 2008, a cross-sectional survey was conducted amongst 600 TB patients across 61 primary health care facilities in four sub-districts in the Free State. Patient selection was done proportionally to the numbers registered at each facility in 2007. Data were subjected to bivariate tests and content analysis of open-ended questions.undecided' (37.0%). Other self-reported reasons for non-uptake included: fear ; perception of being at low risk of HIV infection (13.4%); desire first to deal with TB 'on its own' (12.5%); and because HIV testing had not been offered to them (12.0%). Many patients expressed the need for support and motivation not only from health care workers (33.3%), but also from their significant others (56.6%). Patients further expressed a need for (increased) dissemination of TB-HIV information by health care workers (46.1%).Almost one-third (32.5%) of the respondents reported that they had not undertaken HIV testing, with the most often offered explanation being that they were 'Patients did not undergo HIV testing for various patient-/individual-related reasons. Non-uptake of HIV testing was also due to health system limitations such as the non-offer of HIV testing. Other measures may be necessary to supplement routine/provider-initiation of HIV testing. From the TB patient's perspective, there is a need for (improved) dissemination of information on the TB-HIV link. Patients also require (repeated) motivation and support to undergo HIV testing, the onus for which rests not only on the public health authority and health care workers, but also on other people in the patients' social support networks. Tuberculosis Strategic Plan, 2007-2011 /61 or more days [follow-up or continued treatment phase]); whether a condom was used during most recent sex (yes/no); worry about acquiring HIV (worried/not worried); worry about already being infected with HIV (worried/not worried); worry about infecting others if already infected with HIV (worried/not worried); whether patients received information on the link between TB and HIV from the PHC facility (yes/no); and whether HIV testing was recommended at the PHC facility (yes/no). Differences between tested and non-tested patients were examined using chi-square tests.All closed-ended questions were classified as binary variables: other'. Next, the categories were grouped under the three main themes that emerged from previous studies conducted on uptake of HIV testing amongst TB patients elsewhere  ready'. All explanations eliciting some form of expressed fear or apprehension were grouped under the category 'fear' (19.0%). These fear-related reasons included fear of HIV testing as such, fear of stigmatisation attached to HIV, and/or fear of the consequences of testing HIV-positive. A further category included patients who reportedly were still grappling with being ill with TB and did not wish to subject themselves to the potential burden of also having to deal with HIV (12.5%), e.g. 'I'm still on TB treatment' or 'I'm still very sick'.Patient-/individual-level reasons comprised the leading self-reported explanations for patients' non-uptake of HIV testing. Reasons were categorised as 'I know I am negative because my girlfriend tested and was negative'. Others stated that they did not need to test because they were cautious or because they trusted their sexual partners, e.g. 'I am always careful', 'I do not do [have sex with] HIV girls', and 'I trust my [partner]'. Since these patients did not perceive themselves as being at risk regarding HIV infection, their responses were grouped under the category 'no/low risk perception' (13.4%).Patients also advanced reasons for non-uptake of HIV testing that reflected the importance of relational factors. Some patients used their sexual partners' HIV-test results as proxy for knowing their own HIV status, e.g. 'The nurse did not tell me' and 'No one invited me'. Others cited service-related explanations (6.0%) like: 'The lay counsellor is always late', and 'The clinic is always full'.The most frequent health service-related barrier identified was that some patients had reportedly not (yet) been advised to undergo HIV testing (12.0%), e.g. 'Both tested and non-tested patients perceived relational and health systems factors as potential facilitators of HIV-test uptake.They should stress the importance of HIV testing', '...encourage very ill patients to consider HIV testing', and '...create support groups'. The second most frequent category of suggestions implored other people to be more involved in creating awareness around TB and HIV (14.3%), e.g. 'They should read pamphlets to patients' and '...involve influential people in TB-HIV education'. Some suggestions entreated other people to refrain from stigmatising/discriminating against TB patients (8.6%), e.g. 'They should not judge patients' and '...should not gossip about patients'. Suggestions categorised as 'other' included statements such as 'They should help patients without transport to [reach] clinics' and '...accompany patients to clinics'.Figure They should give health talks', '...inform us about the TB-HIV relationship' and '...show us pictures of people who are sick [with AIDS]'.Figure They should emphasise the importance of testing when patients come for treatment' and '...form support groups'. Less frequent propositions (8.9%) included: 'They should test all patients', '...should not give [TB] treatment without an HIV-test result', '...should provide mobile testing', and '...provide incentives to test'.As was the case with their suggestions regarding the role of family, friends and communities in facilitating uptake of HIV testing, patients also requested service providers to motivate and to be more supportive of TB patients (33.3%), e.g. 'undecided' about undergoing HIV testing. However, drawing from previous findings amongst pregnant women in the USA [indecisiveness' could be that they were not adequately informed about the TB-HIV link. In line with research in Cambodia [Almost one-third (32.5%) of TB patients participating in our study had, by their own report, not undergone HIV testing. These patients' reasons for non-uptake of HIV testing mostly amounted to patient-/individual-level factors. It had not been foreseen that many of the untested patients would be ' the USA  and a qu the USA  on the sCambodia , most ofundecided' actually had no intention of undergoing HIV testing - perhaps for fear of being portrayed as 'promiscuous'. Again, the possibility exists that some of these patients might in fact actually already have undergone HIV testing but did not wish to reveal this, or indeed wished to keep their HIV status secret. Our findings showed a substantial proportion of the patient sample to be worried about either contracting or already having contracted and unknowingly been spreading HIV. This seemed to be a reasonable concern since more than half of all patients by their own report had not used condom protection during their most recent sexual encounter. Hence non-tested patients' supposed 'indecision' could have been a way to avoid further interrogation from the interviewers.Alternatively, given that testing for HIV positions people as being 'at risk' , it is pAs with studies amongst TB populations elsewhere -15, someThough research has indicated that TB patients are generally enthusiastic about collaborative TB and HIV activities , some paSimilar to findings of a Cambodian study , more thAs far as improving uptake of HIV testing is concerned, most patients asserted that it could be increased by motivation and support of TB patients by both health care workers and significant others in their communities. Also, providing (more) TB-HIV information to patients was perceived to be a salient role for health care providers. Our findings align with evidence from research in Kenya, which established that combined facility and community efforts are feasible and do indeed contribute to the scaling up of HIV testing .Convenience sampling of patients limits the extent to which the findings may be generalised to populations of TB patients beyond the four sub-districts surveyed in the present study. However, as previously mentioned, comparison revealed that the patient sample did not differ significantly from the larger population of TB patients in respect of key biographical variables including sex and age.Like all research based on self-reports, the current findings are subject to bias. The possibility of interviewer bias such as unintentional errors resulting from interviewers omitting questions or misunderstanding respondents is acknowledged. To reduce such bias we exercised in-field quality control including the immediate editing of questionnaires for completeness and accuracy. Where necessary, patients were traced to provide or clarify missing information. Our study does however offer insight into TB patients' perspectives regarding non-uptake of HIV testing in a specific context.An important question left unanswered by the current research is that one of the reasons for non-uptake of HIV testing during a current episode of TB might be that patients had been tested for HIV at an earlier stage but due to fear of stigmatisation or other reasons, wished to conceal that and keep their HIV status secret. Our research also did not fully investigate the roles of gender and age. While it was observed that male and older TB patients were less likely to report having been tested for HIV, the reasons for such tendencies and how they can be addressed need to be understood in order to develop appropriate interventions for groups at high risk not to test for HIV.Our study found patient-/individual-related factors or personal reasons to figure prominently in TB patients' explanations for non-uptake of HIV testing. This finding contributes to efforts to understand the limited success, to date, of routine/PITC implementation in South Africa. The only health system-related factor that featured - albeit much less prominently - in the patients' accounts of their non-uptake of HIV testing, was the reported non-offer of HIV testing at PHC facilities.The study findings suggest that from the TB patients' perspective, initiatives to scale up HIV testing by TB patients in the Free State need to consider additional measures to augment routine/provider-initiation of HIV testing. The surveyed patients expressed a need for (more) motivation by and support from both health care workers and significant others, including family, friends and the community. Health care workers also need to (more) consistently and repeatedly inform and educate patients about the link between TB and HIV.AIDS: acquired immunodeficiency syndrome; HCT: HIV counselling and testing; PITC: provider-initiated testing and counselling; HIV: human immunodeficiency virus; TB: tuberculosis; VCT: voluntary counselling and testingThe authors declare that they have no competing interests.NGK undertook instrument development, fieldwork management, data analysis, initial drafting and revision of the manuscript. JCH was involved in the study design, instrument development, fieldwork management and revision of the manuscript. EW critiqued and contributed inputs towards improvement of the manuscript. HSvdB contributed towards instrument development and commented on the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/11/110/prepub


Many patients describe pain and other adverse feeling from their ICU stay and the impact of such feelings impacts long-term psychological morbidity. Presently no objective method for detecting pain or distress is available. Skin conductance variability has been investigated as a monitor of perioperative pain. The method has not been studied in adult ICU patients.Twenty-five (13 intubated and 12 non-intubated) patients were included in this observational study. Patients were monitored with the MED-STORM Stress Detector for 1 hour of intensive care treatment and care. Skin conductance variability (number of skin conductance fluctuations per second (NSCF)) was measured and patients were observed in parallel during rest and during procedures and staff-patient interactions. The sedation-agitation level was monitored with the Motor Activity Assessment Scale. Pain was monitored with the Numeric Rating Scale (0 to 10) in communicating patients and by observation of expressions of pain in patients unable to communicate verbally.In non-intubated patients, NSCF values were low when patients were unstimulated and comfortable and increased with increasing stimulation but also with increasing agitation without any apparent pain. The highest NSCF values were noted during combined pain and agitation. In intubated patients, a similar pattern was observed but with generally lower values, most likely due to sedation. Sensitivity and specificity of NSCF at a cut-off value > 0.13 for detecting expressed pain/discomfort were 74% and 55% for non-intubated patients and 61.5% and 68% for intubated patients.Skin conductance variability increases in critically ill patients with increasing stimulation but is also affected by the level of sedation/agitation, making the method unsuitable for detecting pain alone in critically ill patients, but possibly of value to more generally monitor emotional stress with different etiology. Further studies of the method in critically ill patients, over longer time and with validated pain instruments are warranted.HS is a co-owner of Med-Storm AS, the company responsible for the production and distribution of the Med-Storm Stress Detector. The other authors declare that they have no conflicts of interest.


Autism spectrum disorders (ASD) manifest with neurodevelopmental phenotypes including communicative, social and behavioral impairments that affect as many as 1 in 88 children. The majority of autism cases have no known genetic cause, suggesting complex genetics of the disorder, but a few genes of large effect have been identified.In order to identify novel ASD genetic correlates, we investigated non-protein coding RNAs (ncRNAs) which are abundantly transcribed from the human genome, enriched in the brain, and have been implicated in neurodevelopmental disorders. Using an algorithm that we developed, we examined a publicly available transcriptomics database, AceView, to identify the natural antisense transcripts (NATs) that overlap with known autism-related genes. We validated the presence and differential expression of NATs in different brain regions of ASD and control brains using qRT-PCR. Additionally, we investigated the subcellular localization of these transcripts in a neuronal cell line using RNA-sequencing (RNA-seq).SYNGAP1 locus (SYNGAP1-AS) was found to be differentially expressed in brain regions of patients with ASD compared to control individuals. RNA-seq analysis of subcellular compartments from SH-SY5Y human neuroblastoma cells demonstrated that antisense RNAs to ASD candidate genes are predominantly expressed in the nucleoplasmic or chromatin compartments, implying their involvement in nuclear-associated processes.We found noncoding antisense RNA transcripts at approximately 40% of loci previously implicated in ASD. We confirmed the expression of 10 antisense RNAs in different postmortem human brain tissues. The expression of five antisense transcripts was found to be region-specific, suggesting a role for these ncRNAs in the development and function of specific brain regions. Some antisense RNAs overlapping suspected ASD genes exhibited concordant expression relative to their sense protein-coding genes, while other sense-antisense pairs demonstrate a discordant relationship. Interestingly, the antisense RNA corresponding to the Our data suggests that NATs are abundantly expressed from ASD-related loci and provide evidence for their roles in target gene regulation, neurodevelopment and autism pathogenesis. This class of RNA should therefore be considered in functional studies aimed at understanding genetic risk factors for ASD. Autism spectrum disorders (ASD) are heterogeneous neurodevelopmental disorders, both in terms of clinical manifestations and genetic risk factors . Diseasecis-regulatory functions to increase (concordant) or decrease (discordant) expression levels of their corresponding sense mRNA [in trans by affecting genes from distant genomic loci.Large transcriptomic consortiums such as ENCODE  and FANTnse mRNA . The genHere, we developed an algorithm to mine existing public transcriptomic repositories for the presence of NATs that are produced from ASD candidate genes. We believe that ncRNA information processing systems involving such transcripts represent a critical but under-appreciated dimension of the cell machinery that must be considered in order to identify pathological events and facilitate novel therapeutic development strategies for ASD.The University of Miami Institutional Review Board has deemed this study exempt from the full review due to the use of de-identified human post-mortem brain samples, with no possibility to track back the identity of the donors. There is no animal study involved in this paper.Tissue samples were provided by the National Institute of Child Health and Development (NICHD) at the University of Maryland. A complete description of the samples is provided in Additional file For RNA extraction ~100 mg of brain tissue was lysed in trizol (Life Technologies), 200 μL of chloroform were added and the sample was incubated at room temperature for 10 minutes. The samples were then centrifuged for 20 minutes at 4°C. The supernatant (aqueous phase) was then transferred to a new tube containing 1.5 volumes of 100% ethanol. The ethanol/RNA mixture was then loaded onto a RNeasy column (Qiagen) and purified as per the manufacturer’s instructions, including on-column DNase treatment. Typical yields from both non-ASD and Autism subjects were about 10–12 μg of total RNA from 100 mg of tissue.Primers were designed using Primer 3 software with the sequences from AceView and synthesized by Integrated DNA Technologies  were used to measure gene expression of the endogenous control. Three technical replicates were performed for each reaction. No-template controls were included in each reaction and the melting curve was analyzed to assess the specificity of each primer . The cDNA was then diluted 1:5 and was used as a template for both SYBR Green  and TaqMan qPCR using the ABI 7900 (Life Technologies). TaqMan probes for human To perform strand-specific measurement of antisense transcript expression, we designed primers for a region of antisense transcript that overlaps with an intron or the promoter of the sense gene. Next, we used one-step RNA-to-Ct SYBR Green Kit . We performed reverse transcription (RT) step in a 384-well optical plate using reverse primers to specifically reverse-transcribe antisense RNA and to exclude the possibility of measuring the expression of the sense pre-mRNA. Samples were then incubated at 95°C for 5 minutes to inactivate the reverse transcriptase enzyme. Forward primers were then added to the reaction and quantitative PCR was performed on the same plate. We included no-RT control and no-template controls for each set of primers to control for non-specific binding.A p value of below 0.05 was considered as statistically significant. The Student’s t-test was used to compare the expression between the normal brain and ASD.For all qRT-PCR reactions, three technical replicates were performed. To compare the expression of antisense RNAs across the three brain regions, GraphPad prism software was used to perform ANOVA followed by Tukey post-hoc test. SH-SY5Y cells were fractionated using a modified NE-PER Kit (PIERCE) to isolate RNA from the cytosol, nucleoplasm and chromatin. Briefly, the cells were collected and washed twice with PBS. Cell membranes were lysed using a hypotonic buffer and cells were ultra-centrifuged to pellet nuclei, and the cytosol was recovered from the supernatant. Nuclei were further lysed and centrifuged in order to pellet the insoluble chromatin and recover the nuclear extract in the supernatant. The insoluble chromatin pellet was solubilized in PBS with mild sonication. RNA was extracted from each of the three compartments using a combination of two protocols: Trizol LS (Invitrogen) and RNeasy Mini Kit (QIAGEN). Each sample was dissolved in the appropriate amount of Trizol LS (1 mL for 300 μL sample) and incubated for 10 min at room temperature. Chloroform (200 μL for 1 mL Trizol) was added to the mix and the sample was centrifuged for 20 minutes at 4°C. The aqueous phase of the supernatant was transferred into a new tube and mixed with 1.5 volumes of absolute ethanol. The sample was then loaded onto the cartridge provided by the QIAGEN kit and on-column DNase treatment was performed as per the manufacturer’s protocol. RNA quality was verified using the Agilent Bioanalyzer RNA6000 nano kit.RNA samples were prepared for directional RNA sequencing using a modified version of the Illumina sample preparation protocol. Briefly, 1 μg of total RNA was processed using Ribo-ZeroTM rRNA Removal Kits to remove ribosomal RNAs. Ribosome-depleted RNA was treated with phosphatase before being treated with T4 polynucleotide kinase (PNK). PNK-treated RNA was then purified with the QIAGEN RNeasy column purification kit and 3’ and 5’ RNA adapters were ligated to both ends of the RNA in separate reactions. Next, the RNA was reverse transcribed and PCR amplified. PCR products were purified using AMPure beads. RNA sequencing libraries were validated using the Agilent Bioanalyzer High Sensitivity DNA kit and sequenced using the Illumina HiSeq2000 platform at the Genomics sequencing core at the University of Miami. Each sample was run in a single flowcell to increase depth of sequencing.ab initio transcriptome assembly. The assembled fragments were then annotated using the Cuffcompare module of Cufflinks and AceView database file as a reference. The fragments that originated from introns and incompletely spliced RNAs were filtered out, and Fragments Per Kilobase of transcript per Million reads Mapped (FPKM) values for fragments transcribed from each locus were added to obtain locus expression.The sequencing reads were pre-processed with a custom Python script to trim library adapters. This allowed the generation of 62,500,000 reads per sample on average, which provided an acceptable coverage and sequencing depth. The trimmed reads were then aligned to the human transcriptome assembly GRCh37 from ENSEMBL using TopHat version 2.0.4 . TopHat AceView is a transcriptome database created and supported by the National Center for Biotechnology Information (NCBI) that represents a curated non-redundant collection of RNA transcripts derived from public cDNA collections  . AceViewWe have developed a bioinformatics pipeline to mine the AceView and to perform high-throughput searches of noncoding RNAs in the antisense orientation to genes of interest Figure a. This pFOXG1 (FOXG1-AS) was found in the cortex but not in the cerebellum, implying a certain level of region-specificity in the expression of some ASD-NATs. FOXG1 encodes a transcription factor thought to play a role in the development of the cortex [Of the 71 identified ASD-NATs, 18 were selected for qRT-PCR validation studies using commercially available RNAs from total brain extract, frontal cortex, and cerebellum. The expression of 12 transcripts was confirmed in at least one brain region or in the total brain extract , superior temporal gyrus (STG), and cerebellum  and in the cerebella of 13 .SYNGAP1-AS to be significantly upregulated (p<0.05) in the PFC and STG of autistic patients  show an appreciable trend of differential expression in ASD  of SH-SY5Y neuroblastoma cells and performed RNA sequencing (RNA-seq). FPKM reflecting expression levels of individual loci were used to further compare the expression of antisense RNAs between different compartments  are involved in ASD [Despite overwhelming evidence for the genetic causes of ASD, an exact mode of inheritance has not been elucidated and the wide phenotypic variability of ASD likely reflects the disruption of multiple gene networks and complex regulatory circuits within the genome. Recent data indicate that multiple genomic loci and several rare and highly penetrant gene variants  is a NAT overlapping the DISC1 gene and has been implicated in schizophrenia, bipolar disorder [PTCHD1 (Patched Domain Containing 1) were reported in several families with ASD and intellectual disability. Interestingly, deletion of 5’-flanking region of the gene containing a non-coding RNA were detected in several males with ASD while not present in controls [FMR1) locus. Fragile X syndrome (FXS) is the leading genetic cause of autism and intellectual disability among boys [FMR1 locus, FMR4, that is a 2.4 kb long, primate-specific transcript residing upstream of FMR1 and which may have an anti-apoptotic function [Most eukaryotic genomes are transcribed as ncRNAs of various sizes ranging from 20 nucleotides to over 100 kb . The numsubtypes ,56,57. Tdisorder  and autidisorder . A more disorder . This ancontrols . Non-proong boys . Althougong boys . We prevcis and trans and can be affected by mutations. Knockdown or blockade of endogenous antisense transcripts can have multiple outcomes, with the corresponding sense transcript concentration showing either an increase (discordant regulation) or a decrease (concordant regulation). It has been proposed that discordant de-repression of sense transcript expression, resulting in upregulation of sense RNA expression, can be achieved by removal or steric blockade of many but not all antisense transcripts. Here, we noticed that two exonic antisense RNAs, SYNGAP1-AS and PQBP1-AS, have tissue expression patterns that are discordant to that of their protein-coding partners, whereas two other promoter-associated antisense RNAs, NIBPL-AS and FOXG1-AS, have concordant tissue expression patterns with their sense genes. These findings suggest a possible functional regulation exerted by these antisense RNAs on their sense counterparts, a phenomenon already described for a subset of sense-antisense pairs [In the current study, we explored and validated the expression of ncRNAs in several reported ASD-related genomic loci utilizing bioinformatics and molecular biology approaches. Our bioinformatics pipeline allowed us to identify 71 noncoding antisense RNAs that overlap 38 of 103 genes previously implicated in ASD. These findings indicate that a large proportion of genomic loci implicated in ASD have a complex structure with transcription arising from both the plus and minus strands of DNA. Antisense transcripts can exert regulatory roles on gene expression in se pairs . Discordse pairs . Thus, tSYNGAP1-AS expression in the PFC and STG of autistic patients compared to control individuals. We also observed a statistically significant negative correlation of SYNGAP1-AS and SYNGAP1 expression in the PFC of non-ASD individuals and a similar trend in the PFC of ASD patients. These data, together with the observed discordant regulation of SYNGAP1-AS and SYNGAP1 mRNA, suggest a possible scenario in which upregulation of antisense RNAs lead to the dysregulation of the protein-coding gene expression.We demonstrated that 12 of the 18 randomly selected antisense RNAs overlapping ASD-NATs are expressed in the human brain where they can have specific regional expression, suggesting a possible region-specific function of these RNAs. Differential expression analysis of NATs in the PFC, STG and cerebellum revealed a significant increase in SYNGAP1-AS, providing additional support to the hypothesis that this NAT might have a regulatory function on its sense mRNA partner by mediating the epigenetic modifications of the regulatory elements controlling SYNGAP1 expression.Many noncoding RNAs function at the chromatin level, acting as scaffolds for the recruitment of functionally related epigenetic enzymes to specific loci ,64-66. TThe data presented here provide strong evidence that the molecular network underlying ASD pathology is far more complex than anticipated and may involve dysregulation of ncRNAs. These regulatory elements, which are mostly ignored from current ASD genetics and functional studies, must be taken into account in order to obtain a more holistic view of the interplay of factors that lead to the disease state. We initiated a comprehensive genomic study of ASD that is not dependent solely on protein coding genes, and we demonstrate the expression of NATs in ASD-related genomic loci. Abundant transcriptions of regulatory ncRNAs in ASD-related genomic regions indicate that, in addition to conventional protein coding genes, disruption of RNA regulatory elements may contribute to the pathogenesis of ASD. Identification of disease specific RNAs , as wellADI-R: Autism diagnostic interview revised; ASD: Autism spectrum disorders; FPKM: Fragments per kilobase of transcript per million reads mapped; NATs: Natural antisense transcripts; ncRNAs: Non-protein-coding RNAs.The authors declare that they have no competing interests.DV developed bioinformatics pipeline, performed data mining, qRT-PCR, RNA-seq analysis, statistical analysis, prepared figures and drafted the manuscript. MM processed clinical samples, performed RNA extraction, helped in coordination of the project and drafted the manuscript. MAF conceived of the study, helped in its design and coordination, drafted the manuscript and performed RNA-sequencing. All authors read and approved the final manuscript.Patients informations.Click here for filePrimer sequences for qRT-PCR studies.Click here for fileSchematic representation of Antisense and Sense RNA partners. Diagram showing the genomic location of Antisense (in blue) and Sense (in black) RNA partners. The primers used to measure antisense RNAs expression by qRT-PCR are shown as red arrows.Click here for fileFormer appendix1. Melting temperatures.Click here for file Antisense RNAs to ASD-related genes.Click here for filePGK1. Strand-specific qRT-PCR was used to measure expression of ZNF81-AS and NHS-AS.ASD-related NATs expression in different human brain regions. qRT-PCR analysis of ASD-related NATs in the prefrontal cortex (PFC), superior temporal gyrus (STG) and cerebellum of non-ASD human postmortem brain samples. Transcripts expression is normalized to Click here for fileFOXG1-AS, VPS13B-AS and NHS-AS in the non-ASD brain and in the brain of patients affected by ASD. qRT-PCR analysis of antisense RNAs expression in the non-ASD brain and brain affected by ASD pathology. (a) Expression of FOXG1-AS in the prefrontal cortex (PFC), (b) Expression of VPS13B-AS in the PFC and (c) Strand-specific qPCR analysis of NHS-AS in the superior temporal gyrus (STG). Antisense RNA expression is normalized to PGK1. P value – Student’s t-test.Expression of Click here for fileDataset S1. Gene expression in the cytoplasm, nucleoplasm and chromatin of SH-SY5Y cells based on RNA-seq.Click here for file


Evidence-based screening, brief intervention, and referral to treatment (SBIRT) programs focusing on drug and alcohol use have successfully been implemented in a variety of general medical settings. Such programs require adaptations to function effectively in a high-volume hospital emergency department (ED) setting. We describe implementation and training procedures used in the six-site National Institute on Drug Abuse-Clinical Trials Network (NIDA-CTN) Screening, Motivational Assessment, Referral and Treatment (SMART-ED) trial and present lessons learned from the implementation of the study. The discussion is organized around issues of site selection, staff selection, research assistant and interventionist training, site preparation, and data collection. Several implementation components were particularly important in the SMART-ED trial: 1) Site selection—department and ED staff buy-in was central to decisions on which sites were chosen to participate. 2) Staff selection—interventionists/RAs needed to possess both the empathy necessary to deliver a motivational-interviewing intervention and the research knowledge necessary for protocol adherence. 3) Research assistant and interventionist training and ongoing coaching—in-person and webinar trainings ensured that research staff understood and were able to follow protocol procedures and were certified to deliver the intervention and study procedures that required it. Ongoing coaching based on reviews of the intervention recordings and feedback on compliance with study procedures is successfully preventing drift. 4) Site preparation—prior to beginning the main trial, each site had real-world practice conducting study procedures through standardized patient visits. 5) Data collection—screening data is collected using direct entry into tablet computers to facilitate rapid screening and mobility in the ED setting. There are unique factors associated with effective implementation of SBIRT delivered in the ED. Some of the procedures used in this clinical trial may be useful in the successful implementation of clinical SBIRT programs in EDs.


Classification of proteins into families based on remote homology often helps prediction of their biological function. Here we describe prediction of protein cargo receptors involved in vesicle formation and protein trafficking. Hidden Markov model profile-to-profile searches in protein databases using endoplasmic reticulum lumen protein retaining receptors  as query reveal a large and diverse family of proteins with seven transmembrane helices and common topology and, most likely, similar function. Their coding genes exist in all eukaryota and in several prokaryota. Some are responsible for metabolic diseases , others are candidate genes for genetic disorders  or solute uptake and efflux (SWEETs) and many have not yet been assigned a function. Comparison with the properties of KDEL receptors suggests that the family members could be involved in protein trafficking and serve as cargo receptors. This prediction sheds new light on a range of biologically, medically and agronomically important proteins and could open the way to discovering the function of many genes not yet annotated. Experimental testing is suggested. One of the best described sorting machineries delivering specialised proteins to their appropriate subcellular locations is the early secretory pathway The sorting proteins with the highest known number of TM helices (7) named Erd2 or KDEL receptors are responsible for the retrograde transport from GA to ER. They package proteins with a KDEL amino acid sequence (or variations thereof) that have escaped from ER to GA and return them as a part of the COPI complex. The difference in pH between ER and GA lumen regulates the dissociation/association of the cargo from its receptor. Human cells contain three closely related Erd receptors (numbered 1 to 3). The predicted receptor topology has been confirmed experimentally The mechanism of cargo-receptor-COPI-vesicle formation is relatively well understood  [3]. It It can be expected that a great variety of cargo receptors may exist to accommodate the diversity of various cargos. In the present report, an extensive search for homologues of KDEL receptors uncovers a large and diverse family named PQ-loop proteins. Discussion of those family members that have been previously investigated experimentally leads to a suggestion that PQ-loop proteins might function as cargo receptors in vesicle transport.Drosophila melanogaster, Caenorhabiditis elegans, Arabidopsis thaliana, yeast and several prokaryota) as implemented in MPI toolkit server -5. In addition, the sequences from the individual proteomes were accepted only if the hit matched all 7 TM helices of the query profile (exceptions discussed below). The HHpred searches were repeated with all human and yeast accepted sequences, with the 3 lowest scoring accepted proteins from the other proteomes and with the InterPro profiles that covered more then just one group of orthologous proteins. The repeated searches provided essentially identical hits as the initial ones, the only difference being the E values.The amino acid sequences of the three aligned human KDEL receptors were used for profile-to-sequence . The human and yeast proteins are listed in Korarchaeum cryptofilum) and the bacterial protein B2JBF6 (Nostoc punctiforme).HHpred searches identified 12 human, 8 The searches thus revealed a large, well-defined, self-contained and diverse gene/protein family. Many of its members are described in the curated database Swiss-Prot  to proteins shorter then a typical PQ-loop protein whose sequences covered just half of the query and in which 3 TM helices could be recognised. The human proteins are listed as the last three entries in HHpred searches also provided many significant hits over just half of the query (3 TM helices). Most were alternative alignments of the proteins already identified or even of the query itself. It appears that PQ-loop proteins display a very strong profile homology between their N- and C-terminal parts delineated by the 3 N- and the 3 C-terminal helices. This is an extension of the previous observation that the PQ-loop sequences contain two repeats; the duplication in fact encompasses the whole helical body including the interleaving loops. The finding strengthens the suggestion ERD2, CTNS, MPDU1 and RAG1AP1/SWEET1 have well-established orthologues in many eukaryota including plants. The phylogenic tree of proteins from human and model organisms proteins  and not in the cell membrane. Preliminary unpublished results indicate that TMEM44 is located in ER and probably also in cytoplasmic vesicles . The location of the proteins is consistent with their putative sorting function. Grouping them together based on their common structural features and evolutionary relationship may help to cast light on their function and help design new experiments.PQ-loop genes have never been considered, to the best of our knowledge, as being functionally related. Their common 7TM structure and topology and homology to the well-known protein trafficking receptors strongly suggest that they might all be cargo receptors. The subcellular location of several PQ-loop proteins is known from high throughput studies and/or focussed experiments and compiled in the Swiss-Prot database. They are found predominantly in membranes of organelles such as ER and GA , mitochondria (yeast RTC2), lysosomes (cystinosins), vacuoles (yeast ERS1 and YO092), chloroplasts (+ driven sympoter coupling its function with the vacuolar (H+)-ATPase Ers1 can be rescued with human CTNS. Ers1 localises in the vacuole  and in the punctate pattern (endosome), the compartment transporting molecules from outside the cell membrane to the vacuole. A high copy suppressor of deleted Ers1, protein Meh1 , that interacts with a GTPase Gtr1 was identified C.elegansErs1 was originally cloned as Erd1 suppressor Cystinosin is a key protein required for clearing cystine from lysosomes. Its point mutational defects cause cystinosis, a recessive autosomal disorder of cystine clearance leading to renal failure +/Pi contransporters in the apical membrane. It is tempting to suggest that Fanconi syndrome in cystinosis could be due to the defective sorting of various solute contransporters to their correct place of action because of the defective sorting receptor Cystinosin.Cystinosis patients develop Fanconi syndrome caused by defective reabsorption of various solutes by the renal proximal tubule, e.g. phosphate, bicarbonate, amino acids and glucose C-mannosylation of tryptophanyl residues, and protein O-mannosylation MPDU1 and RFT1 are two additional genes behind CDG whose function is not clear. The growing oligosaccharide chain is attached to the ER membrane by a lipid carrier, dolichol. The synthesis is carried out first outside and subsequently inside ER. Dolichol needs therefore to be flipped to expose the oligosaccharide chain to the lumen. RFT1 is proposed to be the missing flippase that is needed to transfer the growing chain from outside to inside ER The function of MPDU1 is unknown. It is needed in all known classes of monosaccharide-P-dolichol-dependent glycosyltransferase reactions in mammals, i.e. mannosylation and glucosylation of lipid-linked oligosaccharides, the mannosylation of glycosylphosphatidylinositols, the RAG1AP1  when overexpressed in human or yeast cells support cell membrane uptake and ER efflux of sugars Determining the organelle location of PQ-loop proteins would be the first step towards identifying their function. They could be identified in two or more different compartments. As sorting depends crucially on ARF-like, GTPase activating protein-like and guanine exchange factor-like proteins , useful PQ-loop proteins, as members of one family, are likely to exercise similar function. The protein cargo function is firmly established for KDELR and RAG1AP1/SWEET1. The review of the literature indicates that the experimental data obtained for Cystinosin, MPDU1 and plant SWEETs are consistent with the sorting function of these proteins although they are currently annotated as metabolite (or solute) carriers. Experiments to test their participation in vesicle trafficking could be designed. If positive, identification of their cargo could throw new light on two debilitating congenital diseases. The phenotype of several genes in Sequences were extracted from UniProt database and are labelled with their UniProt or Swiss-Prot IDs. Only those absent from UniProt are from NR protein NCBI database and are labelled with their NCBI IDs. Gene names are printed in italics and protein names in plain text. Homology searches were performed by PSI-BLAST


Oocyte-derived maternal RNAs drive early embryogenesis when the newly formed embryo is transcriptionally inactive. Recent studies in zebrafish have identified the role of microRNAs during the maternal-to-embryonic transition (MET). MicroRNAs are short RNAs that bind to the 3' UTR of target mRNAs to repress their translation and accelerate their decay. Newborn ovary homeobox gene (NOBOX) is a transcription factor that is preferentially expressed in oocytes and essential for folliculogenesis in mice. NOBOX knockout mice are infertile and lack of NOBOX disrupts expression of many germ-cell specific genes and microRNAs. We recently reported the cloning and expression of bovine NOBOX during early embryonic development and our gene knockdown studies indicate that NOBOX is a maternal effect gene essential for early embryonic development. As NOBOX is a maternal transcript critical for development and NOBOX is depleted during early embryogenesis, we hypothesized that NOBOX is targeted by microRNAs for silencing and/or degradation.Using an algorithm "MicroInspector", a potential microRNA recognition element (MRE) for miR-196a was identified in the 3' UTR of the bovine NOBOX mRNA. Expression analysis of miR-196a in bovine oocytes and during early embryonic development indicated that it is expressed both in oocytes and embryos and tends to increase at the four-cell and eight-cell stages. Ectopic expression of NOBOX and miR-196a in HeLa cells inhibited the expression of NOBOX protein compared to the control cells without miR-196a. Similarly, the activity of a luciferase construct containing the entire 3' UTR of bovine NOBOX was suppressed, and the regulation was abolished by mutations in the miR-196a binding site indicating that the predicted MRE is critical for the direct and specific binding of miR-196a to the NOBOX mRNA. Furthermore, ectopic expression of miR-196a mimic in bovine early embryos significantly reduced the NOBOX expression at the both mRNA and protein levels.Collectively, our results demonstrate that miR-196a is a bona fide negative regulator of NOBOX during bovine early embryogenesis. The earliest stages of embryonic development in vertebrates primarily rely on the maternal RNA and proteins synthesized during oogenesis ,2. The pMultiple negative regulatory mechanisms are critical for post-transcriptional regulation of maternal transcripts, such as transcript deadenylation and interaction with RNA-binding proteins in a nonspecific or sequence-specific fashion . Recent In recent years, several studies have revealed the significance of miRNAs in reproduction and embryonic development. For example, targeted disruption of Dicer, a key enzyme involved in miRNA processing and the synthesis of small interfering RNAs from long double-stranded RNA ,16 in miin silico subtraction of expressed sequence tags (ESTs) derived from newborn ovaries in mice [Newborn ovary homeobox gene (NOBOX) is a transcription factor, identified by  in mice . NOBOX m in mice . Nobox k in mice ,26. Furt in mice ,28. We r in mice . Bovine MicroRNAs regulate mRNA translation rate by perfect or imperfect base pairing with the 3' UTR regions of their targets . It has To determine the tissue specific expression pattern of miR-196a, quantitative real-time PCR was performed. As shown in Figure in vitro, HeLa cell transfection studies were conducted. A significant inhibition of NOBOX expression was observed in HeLa cells ectopically expressing both NOBOX and miR-196a  in the NOBOX 3' UTR using Microinspector equences .Bovine tissue sample collection, total RNA isolation and miRNA expression analysis in multiple tissues, oocytes and early embryos were performed as described previously .The full-length bovine NOBOX mRNA sequence was amplified from bovine adult ovary cDNA samples by PCR using gene-specific primers containing restriction sites BamHI/XhoI  containing 10% FBS and 1% penicillin/streptomycin . For transient transfection, FuGENE6  was used according to manufacturer's instructions. Following transfection, cells were incubated for 48 h before harvest for western blotting and luciferase assay. Luciferase assay was performed using the Dual-Glo luciferase assay system  as described by the manufacturer. Firefly luciferase activity was normalized to renilla luciferase activity to adjust for variations in transfection efficiency among experiments. All transfection experiments were performed in quadruplicate (n = 4) with data averaged from four independent experiments.Western blot was performed as previously described  with minProcedures for in vitro maturation of oocytes (obtained from abattoir-derived ovaries) and in vitro fertilization to generate zygotes for microinjection and for subsequent embryo culture were conducted basically as described ,55. PresOne-way ANOVA using the general linear models (GLM) procedure of SAS were used to determine the significance of differences in mRNA abundance and between the treated samples and the controls where values resulted from the luciferase reporter assay, quantitative real-time PCR and western blots. Different letters indicate significant differences (P < 0.05).SKT designed and performed most of the experiments including expression analysis of miRNA, preparation of constructs, cell transfection and analysis of gene expression. SKT also drafted the manuscript. KBL and GW performed the microinjection experiments. GWS and JY designed the study and supervised the experimental work. All authors read and approved the final manuscript.Table 1. List of primers used in this study.Click here for file


To define a population-level cohort of individuals infected with the human immunodeficiency virus (HIV) in the province of British Columbia from available registries and administrative datasets using a validated case-finding algorithm.A priori hypotheses were generated and tested to verify excluded cases.Individuals were identified for possible cohort inclusion from the BC Centre for Excellence in HIV/AIDS (CfE) drug treatment program  and laboratory testing datasets  and CD4 diagnostic test results), the BC Centre for Disease Control (CDC) provincial HIV surveillance database (positive HIV tests), as well as databases held by the BC Ministry of Health (MoH); the Discharge Abstract Database , the Medical Services Plan (physician billing) and PharmaNet databases . A validated case-finding algorithm was applied to distinguish true HIV cases from those likely to have been misclassified. The sensitivity of the algorithms was assessed as the proportion of confirmed cases  positively identified by each algorithm. A total of 25,673 individuals were identified as having at least one HIV-related health record. Among 9,454 unconfirmed cases, the selected case-finding algorithm identified 849 individuals believed to be HIV-positive. The sensitivity of this algorithm among confirmed cases was 88%. Those excluded from the cohort were more likely to be female , had a lower mortality rate (2.18 per 100 person years (100PY) vs. 3.14/100PY; p<0.01), and had lower median rates of health service utilization .The application of validated case-finding algorithms and subsequent hypothesis testing provided a strong framework for defining a population-level cohort of HIV infected people in BC using administrative databases. The Public Health Agency of Canada (PHAC) estimated there were 11,700 individuals living with HIV/AIDS in British Columbia in 2011 As a result of continued incomplete access to care and the newly-discovered preventive benefits of Highly Active Antiretroviral Treatment (HAART) Health administrative data are defined as information collected for the purpose of health care management, often by government and health care providers The cohort of interest for analysis included all HIV positive persons aged 18 months of age or older who tested HIV positive or otherwise had some HIV-related record in at least one of the databases described in Communicable Disease Regulation, where identifiers were insufficient for linkage. Individuals were identified in MoH datasets using ICD-9/10 diagnostic codes associated with HIV/AIDS .Individuals meeting the provincial HIV case definition, and testing HIV-positive for the first time in British Columbia, were included from the CDC database. This entails detection of HIV antibody by screening test  followed by positive confirmatory test , or Detection of HIV nucleic acid (RNA or DNA) or detection of p24 antigen with confirmation by neutralization assay, or isolation of HIV in culture. Tests were excluded when an individual chose non-nominal reporting as prescribed in the provincial Database linkage was executed by data stewards in each collaborating agency and coordinated by the Vancouver Coastal Health Authority. Clients were matched to the client registry by provincial health number (PHN). PHNs are mandatory for all BC residents Prior to application of the case-finding algorithm, we excluded individuals with one or more pVL tests with undetectable pVL and no other HIV-related records . These cases typically represent instances where a pVL test was ordered for HIV diagnostic purposes among selected individuals whose antibody based test was HIV-negative. Those meeting the above criteria and also receiving antiretroviral medications through the BC PharmaNet database  were considered to have received pre- or post-exposure prophylaxis and were also excluded. In addition, we excluded individuals receiving antiretrovirals (ARVs) for treatment of Hepatitis B, distinguished by the prescription of Lamivudine (3TC) or Tenofovir (TDF) or Truvada (TDF/FTC) alone as their only ARV ever prescribed (from the PharmaNet database), associated with no records of HAART treatment from the CFE. We also excluded infants receiving antiretrovirals up to 18 months of age, with no HIV-related records thereafter. Antiretroviral prophylaxis was prescribed up to a period of 18 months to prevent vertical HIV transmission A previously-validated set of case-finding algorithms Four case finding algorithms were considered: algorithm 1: indicating HIV-positivity with 3 HIV-related physician claims; algorithm 2: indicating HIV-positivity with 3 HIV-related physician claims OR 1 HIV-related hospital admission; algorithm 3: indicating HIV-positivity with 2 HIV-related physician claims or 1 HIV-related hospital admission; algorithm 4: indicating HIV-positivity with 1 HIV-related physician claim or 1 HIV-related hospitalization.We defined confirmed HIV-positive cases as having records of either an HIV-positive test (CDC database) or records of pVL/CD4 tests or HIV-related medications in the CfE database; unconfirmed cases therefore only had HIV-related records in the DAD and MSP administrative datasets.While we could not ascertain specificity , sensitivity  of each case-finding algorithm was assessed by applying the same case-finding algorithm to a subset of the cohort of individuals we classified as ‘gold standard’ HIV-positive cases, who had linked records from each of the data sources . These gold standard cases were used to assess the sensitivity of the algorithms.a priori hypotheses regarding the characteristics of cohorts considered for exclusion.The case finding algorithms could not provide an objective criterion for exclusion of unconfirmed cases. We therefore supported this analysis by testing a series of a priori hypotheses to assess the face validity of the algorithms. We hypothesized that proportion of females would be greater within a cohort of HIV-negative than HIV-positive individuals, as a result of high HIV prevalence among men who have sex with men in BC and elsewhere. Second, we hypothesized that the all-cause mortality rate would be lower within a cohort of HIV-negative compared to that of HIV-positive individuals. The mortality rate was defined as the number of deaths per 100 person years (100PY) of follow-up . Finally, we tested three hypotheses regarding rates of health service utilization. Specifically, we hypothesized that the rate of outpatient care utilization (MSP claims), the rate of inpatient care, and the rate of pharmaceutical dispensations, all median individual rates per 100PY of follow-up (in this case estimated as the time between 1st HIV-related diagnosis to death or censorship) would be lower in a cohort of HIV-negative compared to HIV-positive individuals. Statistical inference was conducted at an alpha level of 0.05, using non-parametric Kruskal-Wallis tests. All analyses were conducted using SAS version 9.2.We specified five The cohort selection process is illustrated in Results of the case-finding algorithms were presented in We tested five hypotheses to guide the choice between algorithms 2 or 3 . The cohWhile cases determined to be included in algorithm 2 (N = 849) were statistically significantly different from those excluded on each criterion on most dimensions (results not presented), these cases were more likely to be female  and received less outpatient care , but had higher levels of drug dispensation when compared to the gold standard cohort  and rates of inpatient care that were not statistically significantly different from the gold standard cohort . Further, the mortality rate of these individuals was higher than that of the gold standard cohort , though the difference was not statistically significant . As a reWe defined a cohort of individuals with HIV/AIDS in British Columbia from a systematic linkage of multiple population-level, province-wide health administrative datasets. Importantly, this cohort includes 3,576 (29%) individuals who had never accessed HAART, thus representing a critical target population requiring further study to inform efforts regarding engagement into HIV care. This new knowledge provides a rare opportunity to observe health care utilization patterns of HIV-positive individuals not engaged in regular HIV care, and provide a more complete basis for public health surveillance and monitoring.Of note, the cohort represents a prevalence of 12,349 for the study period (1996–2010); at the end of March 2010, 9,597 individuals with HIV/AIDS remained in the study cohort, after excluding decedents using linked vital statistics data. In contrast, PHAC point prevalence estimated 11,040 individuals living with HIV/AIDS in BC in 2008, and 11,700 in 2011 While the case-finding algorithms provided a structured means to select individuals for cohort inclusion, selecting the most relevant algorithm remains a subjective task. In the absence of a nested sub-group of cases and non-cases identified via chart review, the definition and testing of a priori hypotheses provide a means to confirm the results of the case-finding algorithms. In this application, the conclusiveness of these tests underlines their practical utility in applied settings. Further, individuals identified only through the administrative data may include some individuals diagnosed with HIV in BC but not linkable. This is a strength of this cohort in relation to others which have linked surveillance to treatment data only, and overcomes some of the known limitations due to incomplete identifiers in HIV surveillance data.Over 90% of unconfirmed cases identified only in the MoH administrative databases were excluded using the selected case-finding algorithm, indicating a high frequency of coding errors related to HIV, particularly in the Medical Services Plan dataset. Further, we found no distinct trend in erroneous HIV coding, indicating a persistent, rather than deteriorating level of misclassification. Diagnostic coding errors are commonly reported in administrative physician billing records databases The hypothesis tests provided strong evidence confirming the use of the chosen case-finding algorithms There were several limitations in the analyses presented. The inability to confirm, with certainty, the diagnosis of all selected cases was inherent and is indeed the premise of the manuscript. Nonetheless, we've outlined a pragmatic approach for cohort selection using health administrative datasets. Misclassification of unconfirmed HIV cases remains possible, and HIV-positive individuals with no HIV-related contact with the BC health system could not be captured. Our inability to link non-nominal HIV tests likely resulted in a smaller sample than otherwise possible, and may have resulted in misclassification of cases not linked to HIV care as some unlinked cases may have been captured among the confirmed and unconfirmed cases. A separate analysis by CDC database managers found unlinked  cases  were of younger age, more likely to be men who have sex with men, more likely to have resided in the Vancouver Coastal Health Authority region, and were diagnosed early in the study period (results not presented). Also, in-and-out migration was not observed, and therefore was not accounted for in our analysis. A recent BC study indicated a high level of within-province migration - nearly 50% of all individuals in treatment had migrated between local health areas during a median 3.9 years of follow-up In conclusion, as electronic medical records become more commonplace, the availability of large administrative and clinical databases for programmatic monitoring and evaluation, as well as for research purposes is likely to expand. In this analysis, we have demonstrated the ability to identify HIV-infected subjects in the HAART era using an existing algorithm, and validated this algorithm with a series of a priori hypothesis tests.