Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis

Here we present an X-ray crystallography structure of the clinically relevant tigecycline antibiotic bound to the 70S ribosome. Our structural and biochemical analysis indicate that the enhanced potency of tigecycline results from a stacking interaction with nucleobase C1054 within the decoding site of the ribosome. Single-molecule fluorescence resonance energy transfer studies reveal that, during decoding, tigecycline inhibits the initial codon recognition step of tRNA accommodation and prevents rescue by the tetracycline-resistance protein TetM.Tetracyclines are broad-spectrum antibiotic agents that bind to elongating ribosomes and inhibit delivery of the ternary complex elongation factor thermo unstable (EF-Tu)⋅GTP⋅aminoacyl (aa)-tRNA to the ribosomal A site (1). Crystal structures of tetracyclines bound to the 30S subunit identified one common primary binding site that overlaps with the anticodon stem–loop of an A-site–bound tRNA (2–4). The widespread use of tetracyclines in the past has led to an increase in acquired tetracycline-resistance determinants among clinically relevant pathogenic bacteria, limiting the utility of many members of this class. Of the variety of tetracycline-specific resistance mechanisms, efflux and ribosome protection are the most common (5). Ribosome protection is mediated by ribosome protection proteins, with the best characterized being TetO and TetM (6). Ribosome protection proteins bind to tetracycline-stalled translating ribosomes and chase the drug from the ribosome, thus allowing translation to continue. The third generation of tetracycline derivatives, such as tigecycline, display enhanced antimicrobial activity compared with tetracycline, as well as overcoming efflux and ribosome protection mechanisms (7, 8).     

Cell-Surface labeling and internalization by a fluorescent inhibitor of prostate-specific membrane antigen

BACKGROUND: [corrected] Prostate-specific membrane antigen (PSMA) remains an attractive target for imaging and therapeutic applications for prostate cancer. Recent efforts have been made to conjugate inhibitors of PSMA with imaging agents. Compared to antibodies, small-molecule inhibitors of PSMA possess apparent advantages for in vivo applications. To date, there are no reports on the cellular fate of such constructs once bound the extracellular domain of PSMA. The present study was focused on precisely defining the binding specificity, time-dependent internalization, cellular localization, and retention of inhibitor conjugates targeted to PSMA on LNCaP cells. A novel fluorescent inhibitor was prepared as a model to examine these processes. METHODS: Fluorescence microscopy of LNCaP and PC-3 cell lines was used to monitor the specificity, time-dependent internalization, cellular localization, and retention of a fluorescent PSMA inhibitor. RESULTS: Fluorescent inhibitor 2 was found to be a potent inhibitor (IC50 = 0.35 nM) of purified PSMA. Its high affinity for PSMA on living cells was confirmed by antibody blocking and competitive binding experiments. Specificity for LNCaP cells was demonstrated as no labeling by 2 was observed for negative control PC-3 cells. Internalization of 2 by viable LNCaP cells was detected after 30 min incubation at 37 degrees C, followed by accumulation in the perinuclear endosomes. It was noted that internalized fluorescent inhibitor can be retained within endosomes for up to 150 min without loss of signal. CONCLUSIONS: Our results suggest that potent, small-molecule inhibitors of PSMA can be utilized as carriers for targeted delivery for prostate cancer for future imaging and therapeutic applications.     

Solitary rectal ulcer syndrome: clinical findings, surgical treatment, and outcomes

Background Solitary rectal ulcer syndrome (SRUS) is a rare disorder often misdiagnosed as a malignant ulcer. Histopathological features of SRUS are characteristic and pathognomonic; nevertheless, the endoscopic and clinical presentations may be confusing. The aim of the present study was to assess the clinical findings, surgical treatment, and outcomes in patients who suffer from SRUS. Materials and methods A retrospective chart review was undertaken, from January 1989 to May 2005 for all patients who were diagnosed with SRUS. Data recorded included: patient’s age, gender, clinical presentation, past surgical history, diagnostic and preoperative workup, operative procedure, complications, and outcomes. Results During the study period, 23 patients were diagnosed with SRUS. Seven patients received only medical treatment, and in three patients, the ulcer healed after medical treatment. Sixteen patients underwent surgical treatment. In four patients, the symptoms persisted after surgery. Two patients presented with postoperative rectal bleeding requiring surgical intervention. Three patients developed late postoperative sexual dysfunction. One patient continued suffering from rectal pain after a colostomy was constructed. Median follow-up was 14 (range 2–84) months. Conclusion The results of this study show clearly that every patient with SRUS must be assessed individually. Initial treatment should include conservative measures. In patients with refractory symptoms, surgical treatment should be considered. Results of anterior resection and protocolectomy are satisfactory for solitary rectal ulcer.     

Treatment strategies in obstructed defecation and fecal incontinence

Obstructed defecation (OD) and fecal incontinence (FI) are challenging clinical problems, which are commonly encountered in the practice of colorectal surgeons and gastroenterologists. These disorders socially and psychologically distress patients and greatly impair their quality of life. The underlying anatomical and pathophysiological changes are complex, often incompletely understood and cannot always be determined. As a consequence, many medical, surgical, and behavioral approaches have been described, with no panacea. Over the past decade, advances in an understanding of these disorders together with rational and similar methods of evaluation in anorectal physiology laboratories (ARP), radiology studies, and new surgical techniques have led to promising results. In this brief review, we discuss treatment strategies and recent updates on clinical and therapeutic aspects of obstructed defecation and fecal incontinence.     

The blunted loop diuretic response in acute heart failure is driven by reduced tubular responsiveness rather than insufficient tubular delivery. The role of furosemide urine excretion on diuretic and natriuretic response in acute heart failure

Aims

Diuretic response in heart failure is blunted when compared to healthy individuals, but the pathophysiology underlying this phenomenon is unclear. We aimed to investigate whether the diuretic resistance mechanism is related to insufficient furosemide tubular delivery or low tubular responsiveness.

Methods and results

We conducted a prospective, observational study of 50 patients with acute heart failure patients divided into two groups based on previous furosemide use (furosemide naïve: n = 28 [56%] and chronic furosemide users: n = 22 [44%]). Each patient received a protocol-derived, standardized furosemide dose based on body weight. We measured diuretic response and urine furosemide concentrations. The furosemide naïve group had significantly higher urine volumes and natriuresis when compared to chronic users at all timepoints (all p < 0.05). Urine furosemide delivery was similar in furosemide naïve versus chronic users after accounting for differences in estimated glomerular filtration rate (28.02 [21.03–35.89] vs. 29.70 [18.19–34.71] mg, p = 0.87). However, the tubular response to delivered diuretic was dramatically higher in naïve versus chronic users, that is the urine volume per 1 μg/ml of urine furosemide at 2 h was 148.6 ± 136.1 versus 50.6 ± 56.1 ml (p = 0.005).

Conclusions

Patients naïve to furosemide have significantly better diuresis and natriuresis when compared to chronic furosemide users. The blunted diuretic response in patients with chronic loop diuretic exposure is driven by decreased tubular responsiveness rather than insufficient furosemide tubular delivery.     

A comparison between size of the occluder device and two-dimensional transoesophageal echocardiographic sizing of the ostium secundum atrial septal defect

Objectives

Transcatheter closure of a secundum atrial septal defect (ASD II) has become an effective alternative for surgical treatment. In this study we evaluated the correlation between the two-dimensional transoesophageal echocardiographic (2D TEE) sizing of ASDs and the actual diameter of occluders in patients undergoing device closure.

Methods

The records of 54 patients who underwent transcatheter ASD closure were reviewed. ASD characteristics and maximum defect diameter were evaluated using preprocedure 2D TEE images. Appropriate device size was determined by the balloon sizing method, which measures the balloon occlusive diameter (BOD) via TEE and fluoroscopy. ASD closure was performed under continuous TEE monitoring using the Amplatzer occluder in all patients.

Results

The mean of the TEE-derived maximum defect diameter was significantly lower than the mean of the BOD (17.8 ± 4.5 vs 22.1 ± 5.1 mm; p < 0.001) and the mean size of the implanted occluder device (17.8 ± 4.5 vs 23.3 ± 5.1 mm; p < 0.001). However, a good correlation was found between the TEE-derived defect size and the BOD (BOD = 0.898 × TEE defect size + 6.212, R = 0.824; p < 0.001) and between the TEE measurement and the final size of the implanted Amplatzer (device size = 0.928 × TEE defect size + 6.853, R = 0.822; p < 0.001).

Conclusions

2D TEE may provide a good equation to predict the BOD or the size of the occluder device; however, further studies are needed to investigate whether it is feasible to perform transcatheter ASD occlusion without balloon sizing.     

Plasma Volume Status and Its Association With In-Hospital and Postdischarge Outcomes in Decompensated Heart Failure

BackgroundPrior analyses suggest an association between formula-based plasma volume (PV) estimates and outcomes in heart failure (HF). We assessed the association between estimated PV status by the Duarte-ePV and Kaplan Hakim (KH-ePVS) formulas, and in-hospital and postdischarge clinical outcomes, in the ASCEND-HF trial.Methods and ResultsThe KH-ePVS and Duarte-ePV were calculated on admission. We assessed associations with in-hospital worsening HF, 30-day composite cardiovascular mortality or HF rehospitalization and 180-day all-cause mortality. There were 6373 (89.2%), and 6354 (89.0%) patients who had necessary characteristics to calculate KH-ePVS and Duarte-ePV, respectively. There was no association between PV by either formula with in-hospital worsening HF. KH-ePVS showed a weak correlation with N-terminal prohormone BNP, and with measures of decongestion such as body weight change and urine output (r < 0.3 for all). Duarte-ePV was trending toward an association with worse 30-day (adjusted odds ratio 1.07, 95% confidence interval [CI] 1.00–1.15, P = .058), but not 180-day outcomes (adjusted hazard ratio 1.03, 95% CI 0.97–1.09, P = .289). A continuous KH-ePVS of >0 (per 10-unit increase) was associated with improved 30-day outcomes (adjusted odds ratio 0.75, 95% CI 0.62–0.91, P = .004). The continuous KH-ePVS was not associated with 180-day outcomes (adjusted hazard ratio 1.05, 95% CI 0.98–1.12, P = .139).ConclusionsBaseline PV estimates had a weak association with in-hospital measures of decongestion. The Duarte-ePV trended toward an association with early clinical outcomes in decompensated HF, and may improve risk stratification in HF.