Two target gene activation pathways for orphan ERR nuclear receptors.

Estrogen-related receptors (ERRα/ß/γ) are orphan nuclear receptors that function in energy-demanding physiological processes, as well as in development and stem cell maintenance, but mechanisms underlying target gene activation by ERRs are largely unknown. Here, reconstituted biochemical assays that manifest ERR-dependent transcription have revealed two complementary mechanisms. On DNA templates, ERRs activate transcription with just the normal complement of general initiation factors through an interaction of the ERR DNA-binding domain with the p52 subunit of initiation factor TFIIH. On chromatin templates, activation by ERRs is dependent on AF2 domain interactions with the cell-specific coactivator PGC-1α, which in turn recruits the ubiquitous p300 and MED1/Mediator coactivators. This role of PGC-1α may also be fulfilled by other AF2-interacting coactivators like NCOA3, which is shown to recruit Mediator selectively to ERRß and ERRγ. Importantly, combined genetic and RNA-seq analyses establish that both the TFIIH and the AF2 interaction-dependent pathways are essential for ERRß/γ-selective gene expression and pluripotency maintenance in embryonic stem cells in which NCOA3 is a critical coactivator.

Mediator subunit MED1 is required for E2A-PBX1-mediated oncogenic transcription and leukemic cell growth.

The chimeric transcription factor E2A-PBX1, containing the N-terminal activation domains of E2A fused to the C-terminal DNA-binding domain of PBX1, results in 5% of pediatric acute lymphoblastic leukemias (ALL). We recently have reported a mechanism for RUNX1-dependent recruitment of E2A-PBX1 to chromatin in pre-B leukemic cells; but the subsequent E2A-PBX1 functions through various coactivators and the general transcriptional machinery remain unclear. The Mediator complex plays a critical role in cell-specific gene activation by serving as a key coactivator for gene-specific transcription factors that facilitates their function through the RNA polymerase II transcriptional machinery, but whether Mediator contributes to aberrant expression of E2A-PBX1 target genes remains largely unexplored. Here we show that Mediator interacts directly with E2A-PBX1 through an interaction of the MED1 subunit with an E2A activation domain. Results of MED1 depletion by CRISPR/Cas9 further indicate that MED1 is specifically required for E2A-PBX1-dependent gene activation and leukemic cell growth. Integrated transcriptome and cistrome analyses identify pre-B cell receptor and cell cycle regulatory genes as direct cotargets of MED1 and E2A-PBX1. Notably, complementary biochemical analyses also demonstrate that recruitment of E2A-PBX1 to a target DNA template involves a direct interaction with DNA-bound RUNX1 that can be further stabilized by EBF1. These findings suggest that E2A-PBX1 interactions with RUNX1 and MED1/Mediator are of functional importance for both gene-specific transcriptional activation and maintenance of E2A-PBX1-driven leukemia. The MED1 dependency for E2A-PBX1-mediated gene activation and leukemogenesis may provide a potential therapeutic opportunity by targeting MED1 in E2A-PBX1+ pre-B leukemia.

Efficacy of a small molecule inhibitor of the transcriptional cofactor PC4 in prevention and treatment of non-small cell lung cancer.

The human positive coactivator 4 (PC4) was originally identified as a multi-functional cofactor capable of mediating transcription activation by diverse gene- and tissue-specific activators. Recent studies suggest that PC4 might also function as a novel cancer biomarker and therapeutic target for different types of cancers. siRNA knockdown studies indicated that down-regulation of PC4 expression could inhibit tumorigeneicity of A549 non-small cell lung cancer tumor model in nude mice. Here we show that AG-1031, a small molecule identified by high throughput screening, can inhibit the double-stranded DNA binding activity of PC4, more effectively than its single-stranded DNA binding activity. AG-1031 also specifically inhibited PC4-dependent transcriptional activation in vitro using purified transcription factors. AG-1031 inhibited proliferation of several cultured cell lines derived from non-small cell lung cancers (NSCLC) and growth of tumors that formed from A549 cell xenografts in immuno-compromised mice. Moreover, pre-injection of AG-1031 in these mice not only reduced tumor size, but also prevented tumor formation in 20% of the animals. AG-1031 treated A549 cells and tumors from AG-1031 treated animals showed a significant decrease in the levels of both PC4 and VEGFC, a key mediator of angiogenesis in cancer. On the other hand, all tested mice remained constant weight during animal trials. These results demonstrated that AG-1031 could be a potential therapy for PC4-positive NSCLC.

ZBTB1 Regulates Asparagine Synthesis and Leukemia Cell Response to L-Asparaginase.

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response (ISR) that enables cell survival under nutrient stress. The mechanisms by which ATF4 couples metabolic stresses to specific transcriptional outputs remain unknown. Using functional genomics, we identified transcription factors that regulate the responses to distinct amino acid deprivation conditions. While ATF4 is universally required under amino acid starvation, our screens yielded a transcription factor, Zinc Finger and BTB domain-containing protein 1 (ZBTB1), as uniquely essential under asparagine deprivation. ZBTB1 knockout cells are unable to synthesize asparagine due to reduced expression of asparagine synthetase (ASNS), the enzyme responsible for asparagine synthesis. Mechanistically, ZBTB1 binds to the ASNS promoter and promotes ASNS transcription. Finally, loss of ZBTB1 sensitizes therapy-resistant T cell leukemia cells to L-asparaginase, a chemotherapeutic that depletes serum asparagine. Our work reveals a critical regulator of the nutrient stress response that may be of therapeutic value.

Diagnostic Accuracy Of Plain X-Ray Lateral Neck In The Diagnosis Of Cervical Esophageal Foreign Bodies Keeping Oesophagoscopy As Gold Standard.

BACKGROUND: Detection of foreign body oesophagus has always been a challenge for the otolaryngologists. Among different investigations available X -ray is valuable for detection of foreign bodies as it is readily available, inexpensive and easy to operate. However, this still remains to be decided that how accurate it is? The objective of the study is to determine the diagnostic accuracy of plain X ray lateral neck in the diagnosis of foreign bodies in cervical oesophagus keeping esophagoscopy as the gold standard. METHODS: This descriptive study was conducted at department of ENT, Ayub Medical Institute (AMI) Abbottabad, from Mar to Sep 2016. A total of 290 patients were included in this study and all the patients had X-ray lateral view of neck, followed by oesophagoscopy (gold standard). Diagnostic accuracy of plain X-ray lateral view of neck was detected by determining sensitivity, specificity and accuracy. RESULTS: The sensitivity, specificity and accuracy of plain X-ray lateral view of neck was 91.7%, 80%, and 89.7%, respectively. CONCLUSIONS: X-Ray lateral view of neck is a reliable investigation and should be advised among all the patients with history of foreign body ingestion.

Impact of Toupet Versus Nissen Fundoplication on Dysphagia in Patients With Gastroesophageal Reflux Disease and Associated Preoperative Esophageal Dysmotility: A Systematic Review and Meta-Analysis.

BACKGROUND: Controversy exists regarding the best surgical approach for the management of gastroesophageal reflux disease (GORD) and associated preoperative esophageal dysmotility. Our aim was to conduct a systematic review and meta-analysis to compare the outcomes of Toupet fundoplication (TF) and Nissen fundoplication (NF) in patients with GORD and coexistent preoperative esophageal dysmotility. METHODS: We conducted a systematic search of electronic information sources, including MEDLINE, EMBASE, CINAHL, CENTRAL, ClinicalTrials.gov , and bibliographic reference lists. We applied a combination of free text search and controlled vocabulary search adapted to thesaurus headings, search operators, and limits in each of the above-mentioned databases. Postoperative dysphagia and improvement in dysphagia were primary outcome parameters. RESULTS: We identified 3 randomized controlled trials and 1 observational study reporting a total of 220 patients, of whom 126 underwent TF and the remaining 94 patients had NF. Despite the existence of significantly higher preoperative dysphagia in the TF group (29.3% vs 4.2%, P = .05), TF was associated with significantly lower postoperative dysphagia (odds ratio [OR] = 0.31, P = .002) with low between-study heterogeneity ( I2 = 11%, P = .34), and significantly higher improved dysphagia (OR = 10.32, P < .0001) with moderate between-study heterogeneity ( I2 = 31%, P = .23) compared with NF. CONCLUSION: TF may be associated with significantly lower postoperative dysphagia than NF in patients with GORD and associated preoperative esophageal dysmotility. However, no definite conclusions can be drawn as the best available evidence comes mainly from a limited number of heterogeneous randomized controlled trials. Future studies are encouraged to include patients with similar preoperative dysphagia status and report the outcomes with respect to recurrence of acid reflux symptoms.

Irrigation Versus Suction Alone in Laparoscopic Appendectomy: Is Dilution the Solution to Pollution? A Systematic Review and Meta-Analysis.

OBJECTIVES: To investigate outcomes of peritoneal irrigation versus suction without irrigation in patients undergoing emergency laparoscopic appendectomy. METHODS: We performed a systematic review and conducted a search of electronic information sources to identify all randomized controlled trials (RCTs) and observational studies investigating outcomes of irrigation versus suction alone in patients undergoing emergency laparoscopic appendectomy. We used the Cochrane risk of bias tool and the Newcastle-Ottawa scale to assess the risk of bias of RCTs and observational studies, respectively. Random-effects models were applied to calculate pooled outcome data. RESULTS: We identified 3 RCTs and 2 retrospective observational studies, enrolling 2511 patients. Our results suggested that there was no difference between peritoneal irrigation and suction alone in terms of intraabdominal abscess rate (odds ratio = 2.39, 95% confidence interval [CI] = 0.49-11.74, P = .28), wound infection (risk difference = 0.00, 95% CI = -0.04 to 0.05, P = .85), and length of stay (mean difference = -1.02, 95% CI = -3.10 to 1.07, P = .34); however, peritoneal irrigation was associated with longer operative time (mean difference = 7.12, 95% CI = 4.33 to 9.92, P < .00001). Our results remained consistent when randomized trials, adult patients, and pediatric patients were analyzed separately. CONCLUSIONS: The best available evidence suggests that the peritoneal irrigation with normal saline during laparoscopic appendectomy does not provide additional benefits compared with suction alone in terms of intraabdominal abscess, wound infection, and length of stay but it may prolong the operative time. The quality of the best available evidence is moderate; therefore, high-quality RCTs, which are adequately powered, are required to provide more robust basis for definite conclusions.

Stapled anastomosis versus hand-sewn anastomosis of gastro/duodenojejunostomy in pancreaticoduodenectomy: A systematic review and meta-analysis.

BACKGROUND: Controversy exists regarding the best anastomotic method for pancreaticoduodenectomy (PD). We aimed to evaluate the perioperative outcomes of PD with stapled anastomosis (SA) versus hand-sewn anastomosis (HA) of gastrojejunostomy or duodenojejunostomy. METHODS: We conducted a systematic search of electronic information sources, including MEDLINE; EMBASE; CINAHL; the Cochrane Central Register of Controlled Trials (CENTRAL); the World Health Organization International Clinical Trials Registry; ClinicalTrials.gov; ISRCTN Register, and bibliographic reference lists. We applied a combination of free text and controlled vocabulary search adapted to thesaurus headings, search operators and limits in each of the above databases. Delayed gastric emptying (DGE), postoperative pancreatic fistula (POPF), anastomotic bleeding, anastomotic leak, intra-abdominal abscess and mortality were defined as the outcome parameters. Combined overall effect sizes were calculated using fixed-effect or random-effects models. RESULTS: We identified 1 randomised controlled trial (RCT) and 5 observational studies reporting a total of 890 patients who underwent PD with SA (n = 300) or conventional HA (n = 590). Our analysis demonstrated that SA significantly reduced postoperative DGE (OR: 0.37, 95% CI 0.25-0.54, P < 0.00001) but significantly increased anastomotic bleeding (OR: 13.4, 95% CI 2.96-57.41, P = 0.0007) compared to HA. No significant difference was found in POPF (OR: 0.83, 95% CI 0.56-1.21, P = 0.33); anastomotic leak (OR: 0.50, 95% CI 0.09-3.79, P = 0.58); intra-abdominal abscess (OR: 1.39, 95% CI 0.71-2.70, P = 0.34); or mortality (RD: -0.01, 95% CI 0.03-0.02, P = 0.65) between two groups. CONCLUSIONS: Our analysis demonstrated that compared to conventional HA, SA may be associated with lower incidence of DGE after PD without increasing the risk of clinically significant POPF, anastomotic leak or mortality. However, it is associated with higher rate of anastomotic bleeding which mandates careful and precise haemostasis of the stapled line. Considering the current limited evidence, no definitive conclusion can be drawn. Future research is required.

Operative versus non-operative management of adhesive small bowel obstruction: A systematic review and meta-analysis.

OBJECTIVES: To investigate outcomes of operative and non-operative management of adhesive small bowel obstruction (SBO). METHODS: We performed a systematic review in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement standards. We conducted a search of electronic information sources to identify all randomised controlled trials (RCTs) and observational studies investigating outcomes of operative versus non-operative management of patients with adhesive SBO. We used the Cochrane risk of bias tool and the Newcastle-Ottawa scale to assess the risk of bias of RCTs and observational studies, respectively. Fixed-effect or random-effects models were applied to calculate pooled outcome data. RESULTS: We found one RCT, two prospective and three retrospective observational studies, enrolling a total of 876 patients. The analyses showed that operative management of adhesive SBO was associated with a lower risk of future recurrence [odds ratio (OR) 0.54, 95% confidence interval (CI) 0.38-0.76, P = 0.0005] but a higher risk of mortality [risk difference (RD) 0.03, 95% CI 0.01-0.06, P = 0.01] and complications (OR 5.39, 95% CI 2.97-9.78, P < 0.00001). There was no difference in need for surgical re-intervention rate (OR 0.72, 95% CI 0.35-1.47, P = 0.36) and length of stay [mean difference (MD) 5.07, 95% CI -2.36-12.49, P = 1.0] between operative and non-operative managements. The baseline suspicion of strangulation was a major confounding factor. When the baseline suspicion of strangulation was higher in the operative group, the risk of mortality (RD 0.04, 95% CI 0.02-0.07, P = 0.0006) and complications (OR 8.14, 95% CI 4.16-15.94, P = 0.00001) were higher in the operative group but the risk of recurrence was lower (OR 0.62, 95% CI 0.43-0.90, P = 0.01). When the baseline suspicion of strangulation was low in both groups, there was no difference in any of the outcomes except recurrence (OR 0.09, 95% CI 0.02-0.37, P = 0.0009) which was lower in the operative group. CONCLUSIONS: The difference in baseline suspicion of strangulation between operative and non-operative groups is a major confounding factor in current literature. The benefit of surgical treatment should be balanced with the risks associated with surgery, patient's co-morbidities, and presence or absence of strangulation. Based on the best available evidence it could be argued that surgical intervention could be preserved for cases with high suspicion or evidence of bowel strangulation. The controversy still remains for optimum length of conservative management and timing of surgery (early or late) for cases with low baseline suspicion of strangulation. Randomised controlled trials are required to compare outcomes of early operation (<24 h) versus late operation (>24 h) and early operation versus conservative management in patients with low suspicion of strangulation.

Effectiveness of conservative treatment in the management of secretory otitis media.

BACKGROUND: Secretory otitis media is a very important and common ENT disease, especially in the children. The aetiology of Secretory otitis media is multifocal, and the treatment is initially medical or conservative and if it fails then surgery is indicated. The objective of the study was to determine the efficacy of medical treatment in the management of Secretory otitis media. METHODS: This cross sectional descriptive study was conducted at the outpatient department of ENT, Ayub Medical Institute (AMI) Abbottabad, from Mar to Sep 2013. A total of 40 patients were included in this study and standard medical treatment of secretory otitis media was given. The patients were followed up at 2nd and then 4th week and results were analysed. RESULTS: Both clinical and audiological diagnosis of secretory otitis media was made and patients were followed up at 2nd and 4th week to see the effect of treatment. Among 40 patients, 26 (65%) completely recovered from the disease while 14 patients (35%) did not improve. CONCLUSION: Conservative treatment is effective in the management of Secretory otitis media.

Anatomical relationship between recurrent laryngeal nerve and inferior thyroid artery in thyroidectomy patients.

BACKGROUND: Thyroid surgery is performed very frequently now a day. Previously it was used to be associated with high morbidity especially hoarseness. This complication is now almost negligible as most of the surgeons are well acquainted with the anatomical knowledge of the nerves in relation to the gland. The objective of this study was to find out variable anatomical relationships between Recurrent Laryngeal Nerve and Inferior Thyroid Artery in patients undergoing thyroid surgery. METHODS: This cross-sectional retrospective study was conducted in Government Lady Reading Hospital Peshawar and Abasyn Hospital (Private) Peshawar from May 2010 to June 2014. Patients undergoing surgery for benign goiters, T1, T2 well differentiated thyroid cancers without lymph node involvement was included. Data on various types of relationships between RLN and ITA were recorded. RESULTS: In total 271 patients operated and included in the study, 117 were male and 154 were female. Total of 398 RLNs were identified in 416 sides operated. In 55.27% cases the nerve was found to be anterior to inferior thyroid artery while it was posterior to the artery in 34.67% cases. In the remaining 10.05% cases the nerve was observed passing within the branches of inferior thyroid artery. CONCLUSIONS: The anatomical relationship between Recurrent Laryngeal Nerve RLN and Inferior Thyroid Artery ITA is highly variable. For all head and neck surgeons to perform safe surgery on thyroid, it is necessary to have sound anatomical knowledge of these variable relationships between recurrent laryngeal nerve and inferior thyroid artery.

Laser and incisional myringotomy in otitis media with effusion-a comparative study.

BACKGROUND: Otitis media with effusion (OME) or middle ear effusion (MEE) is a common cause of hearing difficulty in children. MEE must be detected early and managed properly to prevent conductive hearing loss in children. It was aimed to compare results of laser myringotomy and conventional myringotomy in terms of hearing improvement, recurrence of MEE and time to put ventilation tube. METHODS: This randomized control trial was conducted from February 2012 to April 2014. Children of 4 years age or older with MEE were included in the study. These children were investigated with Pure tone audiometry (PTA) and tympanometry to confirm conductive hearing loss. X-Ray nasopharynx lateral view was performed for adenoids. Sixty-six patients were randomly assigned in to 2 groups, (1) treated with laser myringotomy and (2) treated with classical myringotomy. The ears were evaluated for MEE, for presence of perforation and level of hearing. RESULTS: A total of 98 ears in 66 patients underwent intervention. Middle ear effusion cleared in 44 out of 48 ears with laser myringotomy (LM) as compared to34 out of 50 ears with incisional myringotomy. The perforation was still patent in 36 ears treated with LM while it was found closed in all 50 ears with conventional myringotomy after 2 weeks. The hearing level improved with LM by 10-15 dB after first 3 months. CONCLUSION: The aim of management in otitis media with effusion is ventilation of tympanic cavity. Laser myringotomy is a best alternative to conventional one. It also has comparable results with ventilation tubes (VT). The ears with refractory or recurrent MEE should have VT insertion.

Transcriptional regulation by Pol II(G) involving mediator and competitive interactions of Gdown1 and TFIIF with Pol II.

Pol II(G) is a distinct form of RNA polymerase II that contains the tightly associated Gdown1 polypeptide (encoded by POLR2M). Unlike Pol II, Pol II(G) is highly dependent upon Mediator for robust activator-dependent transcription in a biochemically defined in vitro system. Here, in vitro studies show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II, thereby inhibiting an essential function of TFIIF in preinitiation complex assembly, but also that Mediator can actually facilitate Pol II(G) binding to the promoter prior to subsequent Mediator functions. Complementary ChIP and RNAi analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where it appears to restrict transcription. These and other results suggest that Pol II(G) may act to modulate some genes while simultaneously, as a poised (noninitiated) polymerase, setting the stage for Mediator-dependent enhancement of their activity.

Complex regulation of the transactivation function of hypoxia-inducible factor-1 alpha by direct interaction with two distinct domains of the CREB-binding protein/p300.

Activation of transcription in response to low oxygen tension is mediated by the hypoxia-inducible factor-1 (HIF-1). HIF-1 is a heterodimer of two proteins: aryl hydrocarbon receptor nuclear translocator and the oxygen-regulated HIF-1 alpha. The C-terminal activation domain of HIF-1 alpha has been shown to interact with cysteine/histidine-rich region 1 (CH1) of the coactivator CBP/p300 in a hypoxia-dependent manner. However, HIF forms lacking C-terminal activation domain (naturally occurring or genetically engineered) are still able to activate transcription of target genes in hypoxia. Here, we demonstrate that the N-terminal activation domain (N-TAD) of HIF-1 alpha interacts with endogenous CBP and that this interaction facilitates its transactivation function. Our results show that interaction of HIF-1 alpha N-TAD with CBP/p300 is mediated by the CH3 region of CBP known to interact with, among other factors, p53. Using fluorescence resonance energy transfer experiments, we demonstrate that N-TAD interacts with CH3 in vivo. Coimmunoprecipitation assays using endogenous proteins showed that immunoprecipitation of CBP in hypoxia results in the recovery of a larger fraction of HIF-1 alpha than of p53. Chromatin immunoprecipitation demonstrated that at 1% O(2) CBP is recruited to a HIF-1 alpha but not to a p53 target gene. Upon activation of both pathways, lower levels of chromatin-associated CBP were detected at either target gene promoter. These results identify CBP as the coactivator directly interacting with HIF-1 alpha N-TAD and mediating the transactivation function of this domain. Thus, we suggest that in hypoxia HIF-1 alpha is a major CBP-interacting transcription factor that may compete with other CBP-dependent factors, including p53, for limiting amounts of this coactivator, underscoring the complexity in the regulation of gene expression by HIF-1 alpha.

Correlation of operative findings with pre-operative clinical signs and analysis of x-rays in patients with airway foreign bodies.

BACKGROUND: Foreign body aspiration is one of the commonly encountered emergencies in ENT and still it remains a significant cause of mortality and morbidity. However with the development of modern endoscopic techniques and controlled anaesthesia, most foreign bodies can be removed safely with a bronchoscope. METHODS: This study was carried out at Department of ENT, Head and Neck Surgery, Ayub Medical College, Abbottabad from 1st Jan 2003 to 30th June 2005. Total 81 patients were registered in the study. Two patients, in whom the foreign body could not be removed, were excluded from the study. RESULTS: The most consistent finding was decreased air entry on chest auscultation on the side of impacted foreign body which was present in 72 (91.1%) patients. The second most consistent finding was audible wheeze on the side of impacted foreign body found in 42 (53.2%) patients. The most common finding on Chest X-ray was emphysema found in 19 (61.3%) patient, followed by atelactasis in 9 (28%) patients while 3 (9.7%) patients had normal Chest x-ray. CONCLUSION: The pre-operative clinical signs in patient with aspirated foreign body give an idea about the site of foreign body in an airway. Although chest x-ray gives an idea about the pathological changes in respiratory tract it has little impact in the management of a patient with aspirated foreign body.

Identification of DNA-dependent protein kinase as a cofactor for the forkhead transcription factor FoxA2.

Forkhead factors are important regulators of animal development and homeostasis. They are among the earliest to bind quiescent genes, which they activate in conjunction with other transcription factors. Many liver-specific genes are under the control of FoxA2, a liver-enriched forkhead protein. Here we confirmed by chromatin immunoprecipitation that FoxA2 is one of the factors bound to the promoter-proximal enhancer of the gene encoding apolipoprotein AI (a component of high density lipoprotein) and that it functions in synergy with the nuclear receptor hepatocyte nuclear factor-4alpha. Furthermore, toward identifying additional cofactors that could potentially regulate FoxA2 activity, we identified DNA-dependent protein kinase (DNA-PK) as a FoxA2-associated factor upon affinity purification of epitope-tagged FoxA2. We show that FoxA2, found to be a phosphoprotein in vivo, is also an efficient substrate for DNA-PK, which targets serine 283. This residue is contained within a conserved serine-glutamine phosphorylation signal for DNA-PK, located within the C-terminal third of the polypeptide, just distal to its winged-helix DNA binding domain. We establish that this residue is critical for FoxA2 function because FoxA2 bearing a mutation at this site is severely compromised in its ability to activate a reporter gene under the control of its cognate DNA-binding site (apoAI site B). Complementary experiments rule out that this mutation compromises the ability of FoxA2 to either translocate to the nucleus or to bind site B. We therefore conclude that DNA-PK-dependent phosphorylation of FoxA2 plays a critical role in its transcriptional activation function per se.

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator.

The multiprotein Mediator coactivator complex is universally required for transcription of metazoan genes. It has been proposed to function by interfacing between transcriptional activators and the RNA polymerase II machinery. However, in vitro transcription systems reconstituted from homogeneous preparations of RNA polymerase II, the general transcription initiation factors, and the cofactor PC4 display relatively robust activator (HNF-4)-dependent activity, which, nonetheless, can be further stimulated by Mediator. By contrast, an unfractionated nuclear extract-based system in which Mediator has been immunodepleted displays a near-absolute dependence on ectopic Mediator. Here, we identified and purified an activity, MSA-2, that confers extract-like Mediator responsiveness to our reconstituted system. Mass spectrometric analyses identified its two constituent polypeptides as hSpt5 and hSpt4, which also comprise the elongation factor DSIF. Mechanistically, MSA-2/DSIF acts by restricting overall transcription in the pure system, thereby imposing a strong Mediator dependence. Our data thus point to potential mechanisms for Mediator function beyond its presently believed role in promoting the initial formation of the RNA polymerase II-containing preinitiation complex.

Evaluation of efficacy of management protocol in allergic rhinitis.

BACKGROUND: Allergic rhinitis represents a global health issue affecting between 10% to 25% of the world population, with increasing prevalence, resulting in a significant impact on quality of life, multiple comorbidities and the considerable socio-economic burden. Majority of the patients are younger than 30 years and it may be inherited. Symptoms typically occur as a result of exposure to dust, dander or certain seasonal pollen. Lack of standardized management protocol is one of the major factors responsible for poor control of this condition. This study was designed to evaluate the patients suffering with allergic rhinitis and manage them with a protocol based on the pathogenesis of the condition. METHOD: This prospective study was conducted in the Department of Ear, Nose & Throat and Head & Neck Surgery, Ayub Teaching Hospital, Abbottabad, over a period of two years (2005 - 2006), to assess the efficacy of a standard protocol of treatment developed and followed in the department. 1167 patients, clinically diagnosed as suffering with allergic rhinitis, were evaluated and managed. Age ranged from 08 months to 64 years. All the patients were prescribed medical treatment, divided into initial phase of 10 days to two weeks duration followed by a maintenance phase, and a regular follow-up schedule was maintained upto two years. RESULTS: 634 (54.32%) patients were male and 533 (45.67%) were female. Age ranged from 08 months to 64 years, 85% being between 05 and 45 years. 91% of patients had symptom duration of 06 months to 03 years. 96% of the patients were getting treated on as required basis, whereas 98% had no educational sessions with the treating doctor, especially regarding preventive measures. Typical presentation in most of the patients was nasal obstruction, runny & itchy nose, post nasal drip and bouts of sneezing. 90.57% patients reported improvement in symptoms. 53.21% patients had a relapse of symptoms at some stage during the study period. 37.53% patients had surgery done for associated pathologies, mostly a DNS. Compliance regarding medication was more than 90% in the initial phase of treatment that dropped to 50% in the maintenance phase. 93% of the patients tolerated the treatment well. CONCLUSION: Allergic rhinitis is a growing problem worldwide. Optimal treatment protocol is still lacking especially in the developing countries. Patient's education on avoidance of allergens must be stressed. Associated problems that may need surgical treatment. Regular follow-up must be ensured to monitor the progress of treatment as well as to identify patients who might be candidates for immunotherapy. Newer modalities of treatment need to be further explored. A team approach is mandatory in the presence of symptoms related to lower respiratory tract.

A Mediator-responsive form of metazoan RNA polymerase II.

RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of the machinery responsible for transcription of mRNA. Although associated general transcription factors impart promoter specificity, responsiveness to gene- and tissue-selective activators additionally depends on the multiprotein Mediator coactivator complex. We have isolated from tissue extracts a distinct and abundant mammalian Pol II subpopulation that contains an additional tightly associated polypeptide, Gdown1. Our results establish that Gdown1-containing Pol II, designated Pol II(G), is selectively dependent on and responsive to Mediator. Thus, in an in vitro assay with general transcription factors, Pol II lacking Gdown1 displays unfettered levels of activator-dependent transcription in the presence or absence of Mediator. In contrast, Pol II(G) is dramatically less efficient in responding to activators in the absence of Mediator yet is highly and efficiently responsive to activators in the presence of Mediator. Our results reveal a transcriptional control mechanism in which Mediator-dependent regulation is enforced by means of Gdown1, which likely restricts Pol II function only to be reversed by Mediator.

Mammalian mediator subunit mMED8 is an Elongin BC-interacting protein that can assemble with Cul2 and Rbx1 to reconstitute a ubiquitin ligase.

The heterodimeric Elongin BC complex has been shown to interact in vitro and in cells with a conserved BC-box motif found in an increasing number of proteins including RNA polymerase II elongation factor Elongin A, suppressor of cytokine signaling (SOCS)-box proteins, and the von Hippel-Lindau tumor suppressor protein. Recently, the Elongin BC complex was found to function as an adaptor that links these BC-box proteins to a module composed of Cullin family members Cul2 or Cul5 and RING-H2 finger protein Rbx1 to reconstitute a family of E3 ubiquitin ligases that activate ubiquitylation by the E2 ubiquitin-conjugating enzyme Ubc5. As part of our effort to understand the functions of Elongin BC-based ubiquitin ligases, we exploited a modified yeast two-hybrid screen to identify a mammalian BC-box protein similar in sequence to Saccharomyces cerevisiae Mediator subunit Med8p. In this report we demonstrate (i) that mammalian MED8 is a subunit of the mammalian Mediator complex and (ii) that MED8 can assemble with Elongins B and C, Cul2, and Rbx1 to reconstitute a ubiquitin ligase. Taken together, our findings are consistent with the model that MED8 could function to recruit ubiquitin ligase activity directly to the RNA polymerase II transcriptional machinery.