To Assess Sleep Quality among Pakistani Junior Physicians (House Officers): A Cross‑sectional Study

Background: Sleep deprivation among junior physicians (house officers) is of growing concern. In developed countries, duty hours are now mandated, but in developing countries, junior physicians are highly susceptible to develop sleep impairment due to long working hours, on‑call duties and shift work schedule. Aim: We undertook the study to assess sleep quality among Pakistani junior physicians. Subjects and Methods: A cross‑sectional study was conducted at private and public hospitals in Karachi, Pakistan, from June 2012 to January 2013. The study population comprised of junior doctors (house physicians and house surgeons). A consecutive sample of 350 physicians was drawn from the above‑mentioned study setting. The subject underwent two validated self‑administered questionnaires, that is, Pittsburgh Sleep Quality Index (PSQI) and Epworth Sleepiness Scale (ESS). Results: A total of 334 physicians completely filled out the questionnaire with a response rate of 95.4% (334/350). Of 334 physicians, 36.8% (123/334) were classified as “poor sleepers” (global PSQI score > 5). Poor sleep quality was associated with female gender (P = 0.01), excessive daytime sleepiness (P < 0.01), lower total sleep time (P < 0.001), increased sleep onset latency (P < 0.001), and increased frequency of sleep disturbances (P < 0.001). Abnormal ESS scores (ESS > 10) were more prevalent among poor sleepers (P < 0.01) signifying increased level of daytime hypersomnolence.Conclusion: Sleep quality among Pakistani junior physicians is significantly poor. Efforts must be directed towards proper sleep hygiene education. Regulations regarding duty hour limitations need to be considered.Keywords: Epworth sleepiness scale, Excessive daytime sleepiness, House officers, Junior physicians, Pittsburgh sleep quality index, Poor sleepers, Sleep disturbances, Sleep qualit

Transcription Start Site selection within a single cluster and G quadruplex structures: a novel mechanism regulating gene expression

The 5' untranslated region (UTR) in a messenger RNA (mRNA) can greatly influence translation. Depending on where the transcription starts, the 5’ UTR will contain (or not) regulatory elements that can modulate mRNA translation. Although studies have demonstrated the effect of differential transcription start sites (TSSs) usage on translation efficiency, these are mainly restricted to TSSs in different clusters many nucleotides apart. However, it is currently unknown if there is any relevance to the TSS variation within a single cluster. Here, we present our findings on single cluster TSS-mediated regulation of protein expression, using mainly AGAP2 as an example. Using 5' RLM-RACE, we have identified different TSSs usage for AGAP2 mRNA in chronic myeloid leukaemia (CML) and prostate cancer (PC) cell lines, giving rise to populations of transcripts with variable lengths of 5' UTR. The population of longer 5' UTR were relatively higher in CML cell lines (P < 0.05), and those extra nucleotides contained the consensus sequence for a G-quadruplex (G4). The G4 formation was verified by CD spectroscopy. Additionally, we developed an immunoprecipitation method termed 'GRIP' [G4 RNA Immunoprecipitation] and demonstrated the existence of these RNA secondary structures in the living cells. To study the impact of the longer 5' UTR and the G4 on translation efficiency, we cloned three 5' UTR isoforms (shorter, longer and mutated-longer version) into a bicistronic plasmid and reported a significant decrease in luciferase activity by the G4 in the longer 5' UTR (P < 0.001). This result coincides with the discrepancy noted in AGAP2 mRNA and protein levels in these cell lines. Furthermore, polysome fractionation studies also confirmed that mRNA with longer 5' UTR associated less prominently with polyribosomes (P < 0.001). Our bioinformatics pipeline has identified 4,920 transcripts in the FANTOM database that contained putative G4 sequences between the major and upstream TSSs within the same TSS cluster. By integrating the NCI-60 microarray and SWATH-MS database, we curated a list of genes that displayed discrepancies in RNA and protein expression with a significantly higher level of G4 forming TSS; and validated our findings in another gene target (HK1). This highlights that the TSS-G4 mediated mechanism is not only limited to AGAP2 expression regulation but is also implicated in controlling the expression of other genes

Native RNA G quadruplex Immunoprecipitation (rG4IP) from mammalian cells

G quadruplex structures play an important role in regulating DNA replication and transcription and mRNA translation. Although there are several techniques that can determine its formation in vitro, the study of RNA G quadruplexes in vivo is not simple. In the current protocol, we describe an optimized technique (RNA G quadruplex immunoprecipitation [rG4IP]) to selectively pull down native cytoplasmic RNAs containing G quadruplex structures in mammalian cells. We also use a bicistronic plasmid to confirm and pinpoint the structure location. For complete details on the use and execution of this protocol, please refer to Surani et al. (2022)

Implications of differential Transcription Start Site selection on CML and prostate cancer cell protein expression

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukaemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5’ UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumour cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role on protein expression and should not be ignored

SP1 and RARα regulate AGAP2 expression in cancer

AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is considered a proto-oncogene, but not much is known about AGAP2 gene expression regulation. To get some insight into this process, AGAP2 proximal promoter was cloned and characterised using reporter assays. We have identified SP1 as a transcription factor bound to AGAP2 promoter and required for AGAP2 expression in two different types of cancer cells (KU812, a chronic myeloid leukaemia cell line; and DU145, a prostate cancer cell line): silencing SP1 decreased AGAP2 protein levels. We have also found that all-trans retinoic acid (ATRA) treatment increased AGAP2 protein levels in both cell lines whilst curcumin treatment reduced ATRA-mediated AGAP2 increase. Furthermore, chromatin immunoprecipitation studies revealed the presence of RARα, RXRα and the lysine acetyl transferase PCAF in AGAP2 promoter. Our results provide a novel understanding of AGAP2 expression regulation that could be beneficial to those patients with cancers where AGAP2 is overexpressed

Defective germline reprogramming rewires the spermatogonial transcriptome.

Defective germline reprogramming in Piwil4 (Miwi2)- and Dnmt3l-deficient mice results in the failure to reestablish transposon silencing, meiotic arrest and progressive loss of spermatogonia. Here we sought to understand the molecular basis for this spermatogonial dysfunction. Through a combination of imaging, conditional genetics and transcriptome analysis, we demonstrate that germ cell elimination in the respective mutants arises as a result of defective de novo genome methylation during reprogramming rather than because of a function for the respective factors within spermatogonia. In both Miwi2-/- and Dnmt3l-/- spermatogonia, the intracisternal-A particle (IAP) family of endogenous retroviruses is derepressed, but, in contrast to meiotic cells, DNA damage is not observed. Instead, we find that unmethylated IAP promoters rewire the spermatogonial transcriptome by driving expression of neighboring genes. Finally, spermatogonial numbers, proliferation and differentiation are altered in Miwi2-/- and Dnmt3l-/- mice. In summary, defective reprogramming deregulates the spermatogonial transcriptome and may underlie spermatogonial dysfunction

Systems biology discoveries using non-human primate pluripotent stem and germ cells: novel gene and genomic imprinting interactions as well as unique expression patterns

The study of pluripotent stem cells has generated much interest in both biology and medicine. Understanding the fundamentals of biological decisions, including what permits a cell to maintain pluripotency, that is, its ability to self-renew and thereby remain immortal, or to differentiate into multiple types of cells, is of profound importance. For clinical applications, pluripotent cells, including both embryonic stem cells and adult stem cells, have been proposed for cell replacement therapy for a number of human diseases and disorders, including Alzheimer's, Parkinson's, spinal cord injury and diabetes. One challenge in their usage for such therapies is understanding the mechanisms that allow the maintenance of pluripotency and controlling the specific differentiation into required functional target cells. Because of regulatory restrictions and biological feasibilities, there are many crucial investigations that are just impossible to perform using pluripotent stem cells (PSCs) from humans (for example, direct comparisons among panels of inbred embryonic stem cells from prime embryos obtained from pedigreed and fertile donors; genomic analysis of parent versus progeny PSCs and their identical differentiated tissues; intraspecific chimera analyses for pluripotency testing; and so on). However, PSCs from nonhuman primates are being investigated to bridge these knowledge gaps between discoveries in mice and vital information necessary for appropriate clinical evaluations. In this review, we consider the mRNAs and novel genes with unique expression and imprinting patterns that were discovered using systems biology approaches with primate pluripotent stem and germ cells