From prediction to experimental validation: desmoglein 2 is a functionally relevant substrate of matriptase in epithelial cells and their reciprocal relationship is important for cell adhesion

Accurate identification of substrates of a protease is critical in defining its physiological functions. We previously predicted that Dsg-2 (desmoglein-2), a desmosomal protein, is a candidate substrate of the transmembrane serine protease matriptase. The present study is an experimental validation of this prediction. As demanded by our published method PNSAS [Prediction of Natural Substrates from Artificial Substrate of Proteases; Venkatraman, Balakrishnan, Rao, Hooda and Pol (2009) PLoS ONE 4, e5700], this enzyme-substrate pair shares a common subcellular distribution and the predicted cleavage site is accessible to the protease. Matriptase knock-down cells showed enhanced immunoreactive Dsg-2 at the cell surface and formed larger cell clusters. When matriptase was mobilized from intracellular storage deposits to the cell surface there was a decrease in the band intensity of Dsg-2?in the plasma membrane fractions with a concomitant accumulation of a cleaved product in the conditioned medium. The exogenous addition of pure active recombinant matriptase decreased the surface levels of immunoreactive Dsg-2, whereas the levels of CD44 and E-cadherin were unaltered. Dsg-2 with a mutation at the predicted cleavage site is resistant to cleavage by matriptase. Thus Dsg-2 seems to be a functionally relevant physiological substrate of matriptase. Since breakdown of cell-cell contact is the first major event in invasion, this reciprocal relationship is likely to have a profound role in cancers of epithelial origin. Our algorithm has the potential to become an integral tool for discovering new protease-substrate pairs.     

MicroRNA-Mediated Suppression of the TGF-β Pathway Confers Transmissible and Reversible CDK4/6 Inhibitor Resistance

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Extensive duplication of the Wolbachia DNA in chromosome four of Drosophila ananassae

Background

Lateral gene transfer (LGT) from bacterial Wolbachia endosymbionts has been detected in ~20% of arthropod and nematode genome sequencing projects. Many of these transfers are large and contain a substantial part of the Wolbachia genome.

Results

Here, we re-sequenced three D. ananassae genomes from Asia and the Pacific that contain large LGTs from Wolbachia. We find that multiple copies of the Wolbachia genome are transferred to the Drosophila nuclear genome in all three lines. In the D. ananassae line from Indonesia, the copies of Wolbachia DNA in the nuclear genome are nearly identical in size and sequence yielding an even coverage of mapped reads over the Wolbachia genome. In contrast, the D. ananassae lines from Hawaii and India show an uneven coverage of mapped reads over the Wolbachia genome suggesting that different parts of these LGTs are present in different copy numbers. In the Hawaii line, we find that this LGT is underrepresented in third instar larvae indicative of being heterochromatic. Fluorescence in situ hybridization of mitotic chromosomes confirms that the LGT in the Hawaii line is heterochromatic and represents ~20% of the sequence on chromosome 4 (dot chromosome, Muller element F).

Conclusions

This collection of related lines contain large lateral gene transfers composed of multiple Wolbachia genomes that constitute >2% of the D. ananassae genome (~5 Mbp) and partially explain the abnormally large size of chromosome 4 in D. ananassae.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1097) contains supplementary material, which is available to authorized users.     

Calyceal Diverticula: A Comprehensive Review

Calyceal diverticula are rare outpouchings of the upper collecting system that likely have a congenital origin. Stones can be found in up to 50% of calyceal diverticula, although, over the combined reported series, 96% of patients presented with stones. Diagnosis is best made by intravenous urography or computed tomography urogram. Shock wave lithotripsy (SWL) is an option for first-line therapy in patients with stone-bearing diverticula that have radiologically patent necks in mid- to upper-pole diverticula and small stone burdens. Stone-free rates are the lowest with SWL, although patients report being asymptomatic following therapy in up to 75% of cases with extended follow-up. Ureteroscopy (URS) is best suited for management of anteriorly located mid- to upperpole diverticular stones. Drawbacks to URS include difficulty in identifying the ostium and low rate of obliteration. Percutaneous management is best used in posteriorly located mid- to lower-pole stones, and offers the ability to directly ablate the diverticulum. Percutaneous nephrolithotomy remains effective in the management of upperpole diverticula, but carries the risk of pulmonary complications unless subcostal access strategies such as triangulation or renal displacement are used. Laparoscopic surgery provides definitive management, but should be reserved for cases with large stones in anteriorly located diverticula with thin overlying parenchyma, and cases that are refractory to other treatment. This article reviews the current theories on the pathogenesis of calyceal diverticula. The current classification is examined in addition to the current diagnostic methods. Here we summarize an extensive review of the literature on the outcomes of the different treatment approaches.Key words: Calyceal diverticula, Percutaneous nephrostolithotomy, Laparoscopic surgery, Shock wave lithotripsy, UreterorenoscopyCalyceal diverticula are eventrations of the upper collecting system lying within the renal parenchyma.¹ These nonsecretory outpouchings are lined by transitional cell epithelium and communicate with the main collecting system via a narrow channel, allowing for passive filling with urine. They were first described in 1841 by Rayer in “Traitements des maladies des reins.”² Thought to be either cysts or localized hydronephrosis, he used the term kyste urinaire to describe his finding of intrarenal urine-containing cavities that communicate with calyces. Other investigators reported similar findings and—depending on location and postulated etiology—described them as pelvic cysts,³ peripelvic cysts,⁴ pyelorenal cysts,⁵ pyelosynaptic cysts,⁶ pyelogenous cysts,⁷ hydrocalicosis,⁸ cystic dilatations of the calyx,⁹ congenital cortical cysts,¹⁰ congenital cystic dysplasia,^4,11 calyceal pseudocysts,¹² juxta-calyceal cysts,¹³ pelvic diverticula, ¹⁴ congenital diverticula of the calyx,¹⁵ and finally, calyceal diverticula.^16–18 Prather is credited with coining the term and the definition of calyceal diverticulum that we use today.     

Post-Radical Prostatectomy Incontinence: Etiology and Prevention

All patients undergoing a radical prostatectomy (RP) using any surgical approach, be it open, laparoscopic, or robotic, are at risk of developing postprostatectomy urinary incontinence. This side effect of RP has an effect on the patient’s quality of life and can be associated with moderate to severe postoperative morbidity. The authors present a review of the etiology and prevention strategies of postprostatectomy urinary incontinence. Based on the current literature, the authors conclude that there is a paucity of studies that can accurately answer the exact anatomic and physiologic etiologies of postprostatectomy urinary incontinence. The aim of urologic surgeons performing RP should be to reduce the rate of postoperative incontinence rather than attempting to treat it once it has occurred. Further studies aimed at providing a detailed anatomic map of the pelvic anatomy related to continence will help to improve surgical techniques and reduce postoperative urinary incontinence following RP.Key words: Radical prostatectomy, Urinary incontinence, Urethral lengthProstate cancer is the most common cancer in men over age 50 years.¹ The most common treatment for organ-confined disease in a suitably selected patient is a radical prostatectomy (RP); however, one of the major morbidities of this procedure is urinary incontinence. Rates of postoperative incontinence range from 4% to 8%^1,2; however, rates may be much higher depending on definitions used and whether validated questionnaires of incontinence were used. Most studies quantify postprostatectomy urinary incontinence accurately as the number of pads being used as a marker of the degree of urinary incontinence.³ Examining the potential causes of postprostatectomy incontinence is important for prevention, but, at present, our understanding is limited, due in part to the lack of anatomic and functional knowledge of continence, as well as the lack of postoperative studies.     

The Physiologic and Anesthetic Considerations in Elderly Patients Undergoing Robotic Renal Surgery

A number of patients are diagnosed with renal malignancies incidentally worldwide. Once a diagnosis of a renal malignancy is established, after a careful evaluation, patients can be offered a robotic nephrectomy or partial nephrectomy. We present a review of the physiologic and anesthetic considerations in elderly patients who are being considered for robotic renal surgery.Key words: Robotic partial nephrectomy, Robotic radical nephrectomy, Physiologic considerations, Anesthetic considerationsFrom the mid-1970s through the mid-1990s, the incidence of renal cell cancer (RCC) has risen by approximately 3% per annum in the United States¹ and 2.5% per annum in northern England.² The main reason for an increase in the incidence of RCC is the increased detection of early and pre-symptomatic tumors by routine radiologic imaging.² Urologists are now seeing more patients with RCC at early stages (T1) and offering these patients a robotic partial nephrectomy (RPN) or robotic radical nephrectomy (RRN) if the surgical expertise is available.³ A minimally invasive partial nephrectomy for small renal masses has been reported to show excellent functional and oncologic outcomes, with 5- to 10-year cancer-specific survival rates of 95% to 100%.⁴Many of the patients with newly diagnosed RCC are of advanced age and/or have some major comorbidity that often results in their poor performance status. It is envisaged that with an increase in life expectancy, urologists and urologic oncologists will see an increase in new referrals of RCC.⁵With the introduction of robotic renal surgery in the United Kingdom, it is likely that more patients will undergo an RRN/RPN over the next decade. Current literature supports the use of RPN in patients versus laparoscopic partial nephrectomy (LPN) due to a reduction in the warm ischemia time (WIT).⁶ This factor is of crucial importance in patients with a solitary kidney or patients with renal impairment undergoing a partial nephrectomy. The WIT reduces with RPN when compared with LPN.⁷We present a review of the important physiologic and anesthetic considerations in patients being considered for an RPN or RRN.     

Azo Dye Decolorization under Microaerophilic Conditions by a Bacterial Mixture Isolated from Anthropogenic Dye‐Contaminated Soil

Soil samples isolated from dye-contaminated sites were exploited for isolation of dye decolorizing microorganisms. A novel bacterial mixture, RkNb1, was selected based on its efficiency, showing maximum and faster decolorization of textile dyes. Seven bacterial strains were isolated and identified from the bacterial mixture as Ochrobactrum intermedium (HM480365), Ochrobactrum intermedium strain M16-10-4 (HM030758), Enterococcus faecalis (HM480367), Arthrobacter crystallopoietes (HM480368), Kocuria flavus (HM480369), Bacillus beijingensis (HM480370), and Citrobacter freundii (HM480371) by 16S rRNA gene sequence analysis. This bacterial mixture showed 98.17% decolorization of Reactive Violet 5 (400 mg L^?1) within 8 h. The culture exhibited good decolorization ability at pH 8 and at a temperature of 37°C. Malt extract and peptone was found to enhance the decolorization rate of Reactive Violet 5. Plackett-Burman experimental design was used for elucidation of medium components affecting Reactive Violet 5 decolorization. Dye degradation products obtained during the course of decolorization were analyzed by high-performance thin-layer chromatography (HPTLC), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR). The potential of this bacterial mixture to decolorize Reactive Violet 5 dye from manufacturing industry effluent is to be carried out using appropriate bioreactors.     

Pterostilbene-Isothiocyanate Conjugate Suppresses Growth of Prostate Cancer Cells Irrespective of Androgen Receptor Status

Chemotherapy and anti-hormonal therapies are the most common treatments for non-organ-confined prostate cancer (PCa). However, the effectiveness of these therapies is limited, thus necessitating the development of alternative approaches. The present study focused on analyzing the role of pterostilbene (PTER)-isothiocyanate (ITC) conjugate – a novel class of hybrid compound synthesized by appending an ITC moiety on PTER backbone – in regulating the functions of androgen receptor (AR), thereby causing apoptosis of PCa cells. The conjugate molecule caused 50% growth inhibition (IC₅₀) at 40±1.12 and 45±1.50 μM in AR positive (LNCaP) and negative (PC-3) cells, respectively. The reduced proliferation of PC-3 as well as LNCaP cells by conjugate correlated with accumulation of cells in G2/M phase and induction of caspase dependent apoptosis. Both PI3K/Akt and MAPK/ERK pathways played an important and differential role in conjugate-induced apoptosis of these PCa cells. While the inhibitor of Akt (A6730) or Akt-specific small interference RNA (siRNA) greatly sensitized PC-3 cells to conjugate-induced apoptosis, on the contrary, apoptosis was accelerated by inhibition of ERK (by PD98059 or ERK siRNA) in case of LNCaP cells, both ultimately culminating in the expression of cleaved caspase-3 protein. Moreover, anti-androgenic activity of the conjugate was mediated by decreased expression of AR and its co-activators (SRC-1, GRIP-1), thus interfering in their interactions with AR. All these data suggests that conjugate-induced inhibition of cell proliferation and induction of apoptosis are partly mediated by the down regulation of AR, Akt, and ERK signaling. These observations provide a rationale for devising novel therapeutic approaches for treating PCa by using conjugate alone or in combination with other therapeutics.     

Peripapillary Retinal Nerve Fiber Layer Assessment of Spectral Domain Optical Coherence Tomography and Scanning Laser Polarimetry to Diagnose Preperimetric Glaucoma

Purpose

To compare the abilities of peripapillary retinal nerve fiber layer (RNFL) parameters of spectral domain optical coherence tomograph (SDOCT) and scanning laser polarimeter (GDx enhanced corneal compensation; ECC) in detecting preperimetric glaucoma.

Methods

In a cross-sectional study, 35 preperimetric glaucoma eyes (32 subjects) and 94 control eyes (74 subjects) underwent digital optic disc photography and RNFL imaging with SDOCT and GDx ECC. Ability of RNFL parameters of SDOCT and GDx ECC to discriminate preperimetric glaucoma eyes from control eyes was compared using area under receiver operating characteristic curves (AUC), sensitivities at fixed specificities and likelihood ratios (LR).

Results

AUC of the global average RNFL thickness of SDOCT (0.786) was significantly greater (p<0.001) than that of GDx ECC (0.627). Sensitivities at 95% specificity of the corresponding parameters were 20% and 8.6% respectively. AUCs of the inferior, superior and temporal quadrant RNFL thickness parameters of SDOCT were also significantly (p<0.05) greater than the respective RNFL parameters of GDx ECC. LRs of outside normal limits category of SDOCT parameters ranged between 3.3 and 4.0 while the same of GDx ECC parameters ranged between 1.2 and 2.1. LRs of within normal limits category of SDOCT parameters ranged between 0.4 and 0.7 while the same of GDx ECC parameters ranged between 0.7 and 1.0.

Conclusions

Abilities of the RNFL parameters of SDOCT and GDx ECC to diagnose preperimetric glaucoma were only moderate. Diagnostic abilities of the RNFL parameters of SDOCT were significantly better than that of GDx ECC in preperimetric glaucoma.