Strong transcription blockage mediated by R-loop formation within a G-rich homopurine-homopyrimidine sequence localized in the vicinity of the promoter.

Guanine-rich (G-rich) homopurine-homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence. Remarkably, the blockage is not pronounced if transcription is performed in the presence of RNase H, which specifically digests the RNA strands within RNA-DNA hybrids. The blockage also becomes less pronounced upon reduced RNA polymerase concentration. Based upon these observations and those from control experiments, we conclude that the blockage is primarily due to the formation of stable RNA-DNA hybrids (R-loops), which inhibit successive rounds of transcription. Our results could be relevant to transcription dynamics in vivo (e.g. transcription 'bursting') and may also have practical implications for the design of expression vectors.

Transcription blockage by homopurine DNA sequences: role of sequence composition and single-strand breaks.

The ability of DNA to adopt non-canonical structures can affect transcription and has broad implications for genome functioning. We have recently reported that guanine-rich (G-rich) homopurine-homopyrimidine sequences cause significant blockage of transcription in vitro in a strictly orientation-dependent manner: when the G-rich strand serves as the non-template strand [Belotserkovskii et al. (2010) Mechanisms and implications of transcription blockage by guanine-rich DNA sequences., Proc. Natl Acad. Sci. USA, 107, 12816-12821]. We have now systematically studied the effect of the sequence composition and single-stranded breaks on this blockage. Although substitution of guanine by any other base reduced the blockage, cytosine and thymine reduced the blockage more significantly than adenine substitutions, affirming the importance of both G-richness and the homopurine-homopyrimidine character of the sequence for this effect. A single-strand break in the non-template strand adjacent to the G-rich stretch dramatically increased the blockage. Breaks in the non-template strand result in much weaker blockage signals extending downstream from the break even in the absence of the G-rich stretch. Our combined data support the notion that transcription blockage at homopurine-homopyrimidine sequences is caused by R-loop formation.

Comparing Breast Hemangiomas to Breast Angiosarcomas: An Imaging Based Review.

Vascular tumors of the breast are rare, and most can be classified as either hemangiomas or angiosarcomas. Breast hemangiomas are benign vascular tumors that are often found incidentally at pathologic examination. Breast hemangiomas may mimic low grade angiosarcomas, which are malignant vascular tumors. This article illustrates the imaging characteristics of breast hemangiomas and breast angiosarcomas across multiple imaging modalities.

Differential uptake of MRI contrast agents indicates charge-selective blood-brain interface in the crayfish.

This study provides a new perspective on the long-standing problem of the nature of the decapod crustacean blood-brain interface. Previous studies of crustacean blood-brain interface permeability have relied on invasive histological, immunohistochemical and electrophysiological techniques, indicating a leaky non-selective blood-brain barrier. The present investigation involves the use of magnetic resonance imaging (MRI), a method for non-invasive longitudinal tracking of tracers in real-time. Differential uptake rates of two molecularly distinct MRI contrast agents, namely manganese (Mn(II)) and Magnevist® (Gd-DTPA), were observed and quantified in the crayfish, Cherax destructor. Contrast agents were injected into the pericardium and uptake was observed with longitudinal MRI for approximately 14.5 h. Mn(II) was taken up quickly into neural tissue (within 6.5 min), whereas Gd-DTPA was not taken up into neural tissue and was instead restricted to the intracerebral vasculature or excreted into nearby sinuses. Our results provide evidence for a charge-selective intracerebral blood-brain interface in the crustacean nervous system, a structural characteristic once considered too complex for a lower-order arthropod.

Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System.

Homeostasis of vascular tone is intricately and delicately maintained systemically and locally, by autonomic nerves and hormones in the blood and by intimal vasoactive substances, respectively. The balance can be acutely or chronically interrupted secondary to many alterations, especially under pathological conditions. Excessive matricellular glycoprotein thrombospondin 1 (TSP1) levels in circulation have been found to play an important role in ischemia-reperfusion injuries of different organs, by acutely suppressing vasorelaxation and chronically remodeling vascular bed. Our laboratory has been interested in identifying new drug moieties, which can selectively and effectively counteract TSP1-induced vascular dysfunction, in order to address associated clinical complications. Preliminary studies using computational docking and molecular models revealed potential drug candidates for further evaluation via vascular functional bioassay to prove the antagonism using an ex vivo vascular model. Herein, we described an efficient screening method for the identification of active drug candidates, by adapting a multiwire myograph system to perform a protocol with different treatments, in the presence of pathological levels of TSP1. We discussed the promising pharmacological evaluation results and suggested suitable modification for versatile applications. We also described the necessity of pre-determination of optimal resting tension to obtain the maximal response, if the experimental test model is different from those with determined optimal resting tension.

Lower ambulatory blood pressure in chronic fatigue syndrome.

OBJECTIVE: To examine blood pressure circadian rhythm in subjects with chronic fatigue syndrome (CFS) and appropriate normal and fatigued controls to correlate parameters of blood pressure regulation with perception of fatigue in an observational cohort study. The cause of CFS remains unknown and there are no effective treatments. METHODS: To address whether inactivity was a confounder, we performed a 24-hour ambulatory blood pressure monitoring in the following three subject groups: 1) CFS patients (Fukuda Diagnostic criteria) (n = 38); 2) normal controls (n = 120); and 3) a fatigue comparison group (n = 47) with the autoimmune liver disease primary biliary cirrhosis (PBC). All patients completed a measure of fatigue severity (Fatigue Impact Scale). In view of the different demographics between the patient groups, patients were age- and sex-matched on a case-by-case basis to normal controls and blood pressure parameters were compared. RESULTS: Compared with the control population, the CFS group had significantly lower systolic blood pressure (p < .0001) and mean arterial blood pressure (p = .0002) and exaggerated diurnal variation (p = .009). There was a significant inverse relationship between increasing fatigue and diurnal variation of blood pressure in both the CFS and PBC groups (p < .05). CONCLUSION: Lower blood pressure and abnormal diurnal blood pressure regulation occur in patients with CFS. We would suggest the need for a randomized, placebo-controlled trial of agents to increase blood pressure such as midodrine in CFS patients with an autonomic phenotype.

Lutetium 177 PSMA radionuclide therapy for men with prostate cancer: a review of the current literature and discussion of practical aspects of therapy.

Prostate-specific membrane antigen (PSMA) is a receptor on the surface of prostate cancer cells that is revolutionising the way we image and treat men with prostate cancer. New small molecule peptides with high-binding affinity for the PSMA receptor have allowed high quality, highly specific PET imaging, in addition to the development of targeted radionuclide therapy for men with prostate cancer. This targeted therapy for prostate cancer has, to date, predominately used Lutetium 177 (Lu) labelled PSMA peptides. Early clinical studies evaluating the safety and efficacy of Lu PSMA therapy have demonstrated promising results with a significant proportion of men with metastatic prostate cancer, who have already failed other therapies, responding clinically to Lu PSMA. This review discusses the practical issues of administering Lu PSMA, and gives an overview of the findings from currently published trials in regards to treatment response rates, expected toxicities and safety.

Frusemide aids diagnostic interpretation of 68 Ga-PSMA positron emission tomography/CT in men with prostate cancer.

INTRODUCTION: 68 Ga-PSMA positron emission tomography-computed tomography (PET/CT) is useful for both staging and assessment of biochemical relapse in men with prostate cancer. Renal excretion of 68 Ga-PSMA can lead to difficulties in scan interpretation, particularly in the pelvis. We evaluated if intravenous Frusemide at the time of 68 Ga-PSMA injection reduces excreted activity artefact and improves diagnostic certainty. METHODS: Sixty-two men with prostate cancer undergoing clinically indicated 68 Ga-PSMA PET/CT were prospectively included, 30 men receiving Frusemide at the time of radiotracer injection. Clinical information and reasons for the scan were documented. Intensity of excreted activity was assessed semi quantitatively for each patient (SUV max). PET/CT images were interpreted by two experienced readers for image quality, and presence/absence of PSMA-positive disease. RESULTS: Twenty-nine percent (18/62) were staging scans, and 71% (44/62) re-staging. PSMA-positive findings were identified in 95% (59/62). Staging scans had PSMA-positive findings within the prostate in 100% (18/18) and re-staging scans, 50% (22/44) in the prostate fossa, 64% (28/44) in lymph nodes and 21% (9/44) in viscera and bone. Administration of Frusemide had a significant impact on intensity of excreted activity in the ureters and bladder. Impaired image quality was noted in 33% (10/30) of men not given Frusemide, compared to only 3% (1/32) in men given Frusemide (P = 0.002). Reporter confidence on the presence/absence of PSMA avid disease in the prostate fossa improved from 63% (19/30) without Frusemide, to 91% (29/32) with Frusemide (P < 0.015). CONCLUSION: Intravenous Frusemide given with 68 Ga-PSMA reduces excretion artefact, and improves diagnostic certainty. Frusemide should be considered for all 68 Ga-PSMA PET/CT imaging protocols.

Factors influencing dental students' specialty choice: a survey of ten graduating classes at one institution.

The aim of this study was to survey ten graduating classes at Harvard School of Dental Medicine regarding students' specialty choice and factors influencing that choice. Students were surveyed once in 2008 (for the Classes of 2007-11) and again in 2013 (for the Classes of 2012-16). A prior article reported results regarding students' interest in and experiences with prosthodontics; this article presents results regarding their interest in all dental specialties and factors influencing those interests. Of a total 176 students in the Classes of 2012-16, 143 responded to the survey, for a response rate of 81%, compared to a 95% response rate (167 of total 176 students) for the Classes of 2007-11. The results showed that orthodontics was the most popular specialty choice, followed by oral and maxillofacial surgery. From the 2008 to the 2013 survey groups, there was an increase in the percentages of students planning to pursue oral and maxillofacial surgery, pediatric dentistry, and postdoctoral general dentistry. The educational debt these students expected to accrue by graduation also increased. The largest percentage of students chose "enjoyment of providing the specialty service" as the factor most influencing their specialty choice. "Prior dental school experience" and "faculty influence" were greater influences for students pursuing specialties than those pursuing postdoctoral general dentistry. Increased interest in particular disciplines may be driven by high debt burdens students face upon graduation. Factors related to mentoring especially influenced students pursuing specialties, demonstrating the importance of student experiences outside direct patient care for exposure to the work of specialists beyond the scope of predoctoral training. This finding suggests that dental schools should increase mentoring efforts to help students make career decisions based not on financial burden but rather on personal interest in the specialty, which is likely to have a more satisfying result for them in the long run.

Dental students' perceptions of and experiences with prosthodontics: ten graduating classes at one institution.

It is important for members of the dental specialties to understand what motivates students to enter the specialty in order to ensure its continuing development and ability to meet patient needs. The aim of this study was to compare ten graduating classes at Harvard School of Dental Medicine (HSDM) regarding students' experiences with and perceptions of prosthodontics and factors influencing those interested in pursuing prosthodontics as a specialty. In 2013, HSDM students in the classes of 2012-16 were surveyed, achieving a response rate of 81%. Survey questions sought information regarding specialty choice, factors influencing the choice, student experiences with prosthodontics, and student perceptions of the dental disciplines. Responses were compared to those from a prior study of the HSDM classes of 2007-11. The responses showed a decrease in negative student experiences with prosthodontics. The students regarded prosthodontics highly for its impact on patient quality of life; however, students interested in pursuing prosthodontics as a specialty decreased. All students said provider enjoyment was most important in choice of specialty. Cost of program, patient type, and program location were factors that especially influenced students interested in prosthodontics. The improved student experiences with and perspectives on prosthodontics may be a result of a curriculum change that led to more prosthodontics procedures and case completions by students. The fall in students interested in prosthodontics may have resulted from prosthodontic faculty transitions that occurred when the survey was conducted, as well as large debt burdens in spite of the fact that prosthodontists' earnings are among the highest in dentistry. Faculty must educate and mentor students about the realities of the profession, provide positive learning experiences in the field, and encourage students who enjoy prosthodontics to pursue specialty training.

The antimicrobial activity of the appetite peptide hormone ghrelin.

The present study examined the antimicrobial activity of the peptide ghrelin. Both major forms of ghrelin, acylated ghrelin (AG) and desacylated ghrelin (DAG), demonstrated the same degree of bactericidal activity against Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), while bactericidal effects against Gram-positive Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis) were minimal or absent, respectively. To elucidate the bactericidal mechanism of AG and DAG against bacteria, we monitored the effect of the cationic peptides on the zeta potential of E. coli. Our results show that AG and DAG similarly quenched the negative surface charge of E. coli, suggesting that ghrelin-mediated bactericidal effects are influenced by charge-dependent binding and not by acyl modification. Like most cationic antimicrobial peptides (CAMPs), we also found that the antibacterial activity of AG was attenuated in physiological NaCl concentration (150mM). Nonetheless, these findings indicate that both AG and DAG can act as CAMPs against Gram-negative bacteria.

Additive effects of orexin B and vasoactive intestinal polypeptide on LL-37-mediated antimicrobial activities.

The present study examined the bactericidal effects of orexin B (ORXB) and vasoactive intestinal peptide (VIP) alone or combined with cationic antimicrobial peptides, such as LL-37, on Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans and Staphylococcus aureus. The bactericidal effect of ORXB or VIP alone was detected in low NaCl concentration, but attenuated in physiological NaCl concentration (150 mM). However, such attenuated bactericidal activities of ORXB and VIP in 150 mM NaCl were regained by adding LL-37. Therefore, our results indicate that VIP and ORXB appear to mediate bactericidal effects in concert with LL-37 in the physiological context of mucosal tissue.