Structural basis of direct and inverted DNA sequence repeat recognition by helix-turn-helix transcription factors.

Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix-turn-helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies.

Tips and tricks for a correct interpretation of contrast-enhanced ultrasound.

Although contrast-enhanced ultrasound (CEUS) is a widespread and easily manageable technique, image interpretation errors can occur due to the operator's inexperience and/or lack of knowledge of the frequent pitfalls, which may cause uncertain diagnosis and misdiagnosis. Indeed, knowledge of the basic physical and technical principles of ultrasound is needed both to understand sonographic image findings and to evaluate the potential and limits of the method. Like the B-mode ultrasound, the quality of the CEUS examination is also subject not only to the adequate manual skill of the operator but also to his/her deep knowledge of the technique which improves the quality of the image helping avoid misleading artifacts. In this review, the main parameters influencing a CEUS examination will be described by taking into account the most common errors and pitfalls and their possible solutions.

ArdC, a ssDNA-binding protein with a metalloprotease domain, overpasses the recipient hsdRMS restriction system broadening conjugation host range.

Plasmids, when transferred by conjugation in natural environments, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida. Expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. ardC was also required if the hsdRMS system was present in E. coli recipient cells. Thus, ArdC has antirestriction activity against the HsdRMS system and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity was not needed for the antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggered an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies and settle into new hosts.

The Underrated Role of Ultrasound in Peritoneal Dialysis.

Ultrasound is very effective in performing procedures and assessment of complications in peritoneal dialysis. The ultrasound examination can be applied for preoperative assessment, during the peritoneal catheter placement, for the detection and monitoring of infection, as well as for the evaluation of the catheter malfunction. Despite being not only a cost- and time-saving technique but also a bedside procedure, ultrasonography remains an underrated clinical tool in the field of peritoneal dialysis. This publication wants to explain and reinforce the clinical utility of US in PD and to expand the diagnostic equipment for the clinician.

Magnetic resonance urography of congenital abnormalities - what the radiologist needs to know.

Congenital abnormalities of the kidney and urinary tract include a wide range of malformations ranging from asymptomatic to life-threatening conditions. Although pediatric urogenital system imaging is based on the use of US (pre- and postnatal), voiding cystourethrography and scintigraphic study, magnetic resonance (MR) urography plays a fundamental role in the classification and management of congenital abnormalities of the kidney and urinary tract, giving an overview of the different clinical pictures, thanks to its panoramicity and high anatomical detail. In fact the anomalies of the urinary tract are phenotypically variable because they can affect simultaneously several segments of different embryonic derivation, with complex clinical pictures; they can appear both as isolated phenotypes or as complex malformative conditions, involving renal parenchyma, collecting system and bladder. A deep knowledge of this complex embryogenesis and its possible phenotypic patterns allows a correct interpretation of MR urography images. We describe the embryology and pathophysiology of congenital abnormalities of the kidney and urinary tract as well as MR urography technique and findings. Congenital abnormalities of the kidney and urinary tract are classified into four groups: (1) obstruction (proximal, middle and distal), (2) budding with respect to the Wolffian duct (site and number of ureter), (3) ascent and rotation (ectopia, malrotation and fusion of kidney) and (4) anomaly of metanephric differentiation (dysplasia, megapolicalycosis).

Dissemination of NDM-producing Klebsiella pneumoniae and Escherichia coli high-risk clones in Catalan healthcare institutions.

OBJECTIVES: To characterize the clonal spread of carbapenem-resistant Klebsiella pneumoniae and Escherichia coli isolates between different healthcare institutions in Catalonia, Spain. METHODS: Antimicrobial susceptibility was tested by disc diffusion. MICs were determined by gradient diffusion or broth microdilution. Carbapenemase production was confirmed by lateral flow. PCR and Sanger sequencing were used to identify the allelic variants of resistance genes. Clonality studies were performed by PFGE and MLST. Plasmid typing, conjugation assays, S1-PFGE plus Southern blotting and MinION Oxford Nanopore sequencing were used to characterize resistance plasmids. RESULTS: Twenty-nine carbapenem-resistant isolates recovered from three healthcare institutions between January and November 2016 were included: 14 K. pneumoniae isolates from a tertiary hospital in the south of Catalonia (hospital A); 2 K. pneumoniae isolates from a nearby healthcare centre; and 12 K. pneumoniae isolates and 1 E. coli isolate from a tertiary hospital in Barcelona (hospital B). The majority of isolates were resistant to all antimicrobial agents, except colistin, and all were NDM producers. PFGE identified a major K. pneumoniae clone (n = 27) belonging to ST147 and co-producing NDM-1 and CTX-M-15, with a few isolates also harbouring blaOXA-48. Two sporadic isolates of K. pneumoniae ST307 and E. coli ST167 producing NDM-7 were also identified. blaNDM-1 was carried in two related IncR plasmid populations and blaNDM-7 in a conjugative 50 kb IncX3 plasmid. CONCLUSIONS: We report the inter-hospital dissemination of XDR high-risk clones of K. pneumoniae and E. coli associated with the carriage of small, transferable plasmids harbouring blaNDM genes.

Imaging in scrotal trauma: a European Society of Urogenital Radiology Scrotal and Penile Imaging Working Group (ESUR-SPIWG) position statement.

Imaging plays a crucial role in the evaluation of scrotal trauma. Among the imaging modalities, greyscale ultrasound and Colour Doppler ultrasound (CDUS) are the primary techniques with the selective utilisation of advanced techniques such as contrast-enhanced ultrasound (CEUS) and elastography. Despite ultrasound being the mainstay of imaging scrotal trauma, its diagnostic performance is not fully established. Considering these difficulties and their impact on clinical practice, the Scrotal and Penile Imaging Working Group of the European Society of Urogenital Radiology (ESUR-SPIWG) established an expert task force to review the current literature and consolidate their expertise on examination standards and imaging appearances of various entities in scrotal trauma. This paper provides the position statements agreed on by the task force with the aim of providing guidance for the use of imaging especially multiparametric US in scrotal trauma.Key Points• Greyscale and Colour Doppler ultrasound are the mainstay of imaging in patients with scrotal trauma.• Contrast-enhanced ultrasound and elastography are the advanced techniques useful as a problem-solving modality in equivocal cases.• This paper summarises the position statements of the ESUR-SPIWG on the appropriate utilisation of multiparametric ultrasound and other imaging modalities in the evaluation of scrotal trauma.

Nonscrotal Causes of Acute Scrotum.

Acute scrotum is characterized by intense acute scrotal pain, which may be associated with other symptoms and signs such as abdominal pain, inflammation, and fever. Many pathologic conditions can present in this way, most which involve the scrotal contents. Nonscrotal conditions, however, can rarely present clinically only as acute scrotum: among them, renal colic, aneurysm rupture or other causes of retroperitoneal hemorrhage, primary abdominal or pelvic tumors and metastases, pancreatitis, pelvic inflammation, and muscle injuries. The pathophysiologic characteristics of the clinical presentation, clues for diagnosis, and imaging features of a series of nonscrotal lesions presenting clinically with acute scrotal pain are herein reported and illustrated. In patients presenting with acute scrotal symptoms and normal scrotal ultrasound findings, nonscrotal causes of acute scrotal pain should be considered in the differential diagnosis. Therefore, an ultrasound investigation of the abdomen, groin, and thighs is indicated.

Emergency Ultrasound in Trauma Patients: Beware of Pitfalls and Artifacts!

BACKGROUND: Ultrasonography (US) is highly dependent on operators' skills. It is not only a matter of correct scan techniques; there are anatomical structures and variants, as well as artifacts, which can produce images difficult to interpret and which, if not properly understood, can be causes of errors. OBJECTIVES: This paper will review relatively common US pitfalls and artifacts that can be encountered in trauma patients and will offer tips to recognize and avoid them. DISCUSSION: Normal anatomical structures and anatomical variants can mimic fluid collections or perisplenic lesions. Examination along multiple scan planes, real-time observation of movements or repetition of the study after the patient has drunk some fluid or after placing a finger on her/his body wall can help proper identification. The term artifact in US imaging refers to display phenomena not properly representing the imaged structures. This can result in images suggesting fracture lines within organs or at their borders, lung consolidations, or pleural effusions, and abdominal fluid collections. Their knowledge is the first step to recognize them; then, use of multiple scan planes or repetition of the study after voiding or changes of equipment setting can make them disappear or clarify their nature. CONCLUSION: We present possible anatomic pitfalls and artifacts that may affect correct interpretation of US images in patients with abdominal trauma and suggest how to avoid or to clarify them during the examination. Knowing their existence, their appearances, and the reasons why they are produced is important for proper use of this diagnostic technique.

Selective arterial embolization for a high-flow priapism following perineal trauma in a young gymnast.

INTRODUCTION: High-flow priapism is a rare condition in children, usually due to a perineal trauma. MATERIALS AND METHODS: We present a case of traumatic highflow priapism investigated by Doppler ultrasound and managed by angiography and selective embolization of a branch of the internal pudendal artery. RESULTS: A 13-year-old gymnast underwent perineal trauma during training and developed a high-flow priapism. The first ultrasound (immediately after the trauma) showed the presence of an inhomogeneous area of 3 x 2 cm associated with an anechoic vascularized area (pseudoaneurysm) in the right corpus cavernosum. On the left side there was a similar finding, but of smaller size. After 3 weeks the pseudoaneurysm on the left was completely obliterated while the right one was still present. Angiography and superselective catheterization of a branch of the left pudendal artery and its embolization with microspheres and with metal microcoils were performed. After the procedure, ultrasound showed that the right pseudoaneurysm was completely obliterated and there were no more branches reaching it. The cavernous arteries were both pervious. CONCLUSIONS: Selective arterial embolization is a safe treatment that can also be used in pediatric patients.

Negative feedback and transcriptional overshooting in a regulatory network for horizontal gene transfer.

Horizontal gene transfer (HGT) is a major force driving bacterial evolution. Because of their ability to cross inter-species barriers, bacterial plasmids are essential agents for HGT. This ability, however, poses specific requisites on plasmid physiology, in particular the need to overcome a multilevel selection process with opposing demands. We analyzed the transcriptional network of plasmid R388, one of the most promiscuous plasmids in Proteobacteria. Transcriptional analysis by fluorescence expression profiling and quantitative PCR revealed a regulatory network controlled by six transcriptional repressors. The regulatory network relied on strong promoters, which were tightly repressed in negative feedback loops. Computational simulations and theoretical analysis indicated that this architecture would show a transcriptional burst after plasmid conjugation, linking the magnitude of the feedback gain with the intensity of the transcriptional burst. Experimental analysis showed that transcriptional overshooting occurred when the plasmid invaded a new population of susceptible cells. We propose that transcriptional overshooting allows genome rebooting after horizontal gene transfer, and might have an adaptive role in overcoming the opposing demands of multilevel selection.

Challenges and pitfalls in the management of endocrine toxicities from immune checkpoint inhibitors: a case presentation of synchronous thyrotoxicosis and primary adrenal insufficiency in a melanoma patient.

BACKGROUND: Immune checkpoint inhibitors have revolutionized the therapeutic approach to several solid tumors, becoming the standard of care for cancer treatment in different disease settings. Despite the fact that these agents are better tolerated than conventional chemotherapy, their use is associated with a specific toxicity profile, so-called immune-related adverse events (irAEs), that can involve several organs. Endocrine irAEs are among the most frequent toxicities (around 10 to 16%) and include hypophysitis, thyroid disorders, adrenalitis, and diabetes mellitus. Some of them may be life-threatening if not promptly recognized (such as diabetic ketoacidosis and acute adrenal crisis). CASE PRESENTATION: A 55-year-old woman with a personal history of euthyroid Hashimoto's thyroiditis was diagnosed with a metastatic melanoma, BRAF wild type. Under treatment with anti-PD-1 pembrolizumab, she developed thyrotoxicosis followed by hypothyroidism due to destructive thyroiditis and concurrent primary adrenal insufficiency due to adrenalitis. CONCLUSIONS: The simultaneous occurrence of adrenal and thyroid autoimmune diseases, resembling autoimmune polyendocrine syndrome type 2, may occur as a rare but serious side effect of ICI treatment. It often presents with abrupt onset and rapid evolution towards polyglandular insufficiency. Physicians should be aware of the potential association of two or more endocrine disorders and careful monitoring of endocrine function is needed during ICI therapy.

What Is the Malignant Potential of Small (<2 cm), Nonpalpable Testicular Incidentalomas in Adults? A Systematic Review.

CONTEXT: Unlike palpable lumps, a large number of nonpalpable testicular lesions found incidentally at ultrasound in asymptomatic postpuberal males are either benign tumours or non-neoplastic lesions. The prevalence of malignancy, however, is appraised based on small case series. Dedicated studies report a large number of patients, and systematic review articles are lacking. OBJECTIVE: This systematic review is aimed to assess, from the analysis of the pooled data of the available literature, the incidence of benign tumours, malignant tumours, and non-neoplastic lesions, and to identify predictive characteristics for malignancy. EVIDENCE ACQUISITION: A systematic review of PubMed, Scopus, Google Scholar, Turning Research Into Practice (TRIP) database, and the Cochrane Library was conducted on January 6, 2022, according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement. Studies were retrieved reporting on adult asymptomatic men, with single, incidentally identified small testicular lesions, either fertile or infertile, with negative tumour markers and without specific risk factors for malignancy. Lesions ≤20 mm were considered small. Seventy-four studies were selected for inclusion in this analysis. Twenty-six additional publications have been retrieved by the bibliography quoted in the selected articles. EVIDENCE SYNTHESIS: Pooled data of 1348 lesions in 1348 patients were collected. Of these lesions, 408 could be retrieved individually, 44.6% were benign, 27.2% were malignant, and 20.8% were non-neoplastic. Virtually all lesions <3 mm and 86.6% of lesions <5 mm were benign. Lesions >10 mm have a 38.14% probability of being benign. Hyperechoic lesions are likely benign. Fertility status does not affect the risk of malignancy. CONCLUSIONS: Very small (<3 mm) and small (<5 mm) incidentally detected testicular lesions in asymptomatic postpuberal men with normal tumour markers could be frequently benign. More prospective studies are needed to better support this finding. Management strategies should be developed for these patients to reduce overtreatment. PATIENT SUMMARY: Small testicular lesions are incidentally founded at ultrasound. It is not easy to distinguish a benign lesion from a malignant one. Results of this study are reporting a higher incidence of benign lesions with a diameter of <5 mm. More studies are needed to better understand the biology and the management strategy for small testicular lesions.

Extraprostatic incidental findings on prostate mpMRI: A pictorial review from the ESUR junior network.

The role of multiparametric MRI (mpMRI) in prostate cancer setting is increasingly consolidated and, as a result, its usage in clinical practice is in exponential growth. However, beyond the prostate gland, several key structures are included in the field of view of mpMRI scans. Consequently, various extra-prostatic incidental findings (IFs) belonging to different anatomical systems can be accidentally recognized. Therefore, it is mandatory for a radiologist to be familiar with the wide range of pathologies potentially encountered, to guide management and avoid patient anxiety and costs due to additional work-up prompted by clinically insignificant extra-prostatic findings. With this pictorial review, we aim to illustrate a wide range of IFs that can be detected when performing mpMRI of the prostate, focusing on their imaging characteristics, differential diagnosis, and clinical relevance. Additionally, we propose the CheckDEEP, the Checklist for DEtection of ExtraProstatic findings, to be used for a thorough evaluation of target areas within each anatomical system.

Determination of conjugation rates on solid surfaces.

A cytometric method for the estimation of end-point conjugation rates is developed and adapted to surface conjugation. This method improves the through-put of conjugation assays based on replica-plating and results in less noisy experimental data. Although conjugation on solid surfaces deviates from ideal conditions in which cells are continuously mixed, results show that, within the limits of high initial population densities and short mating times, end-point estimates of the conjugation rates are robust measurements. They are independent of the donor/recipient ratios and, to some extent, of the sampling time. Remixing the mating population in the course of a conjugation experiment results in a boost in the frequency of transconjugants.

Numbers on the edges: a simplified and scalable method for quantifying the gene regulation function.

The gene regulation function (GRF) provides an operational description of a promoter behavior as a function of the concentration of one of its transcriptional regulators. Behind this apparently trivial definition lies a central concept in biological control: the GRF provides the input/output relationship of each edge in a transcriptional network, independently from the molecular interactions involved. Here we discuss how existing methods allow direct measurement of the GRF, and how several trade-offs between scalability and accuracy have hindered its application to relatively large networks. We discuss the theoretical and technical requirements for obtaining the GRF. Based on these requirements, we introduce a simplified and easily scalable method that is able to capture the significant parameters of the GRF. The GRF is able to predict the behavior of a simple genetic circuit, illustrating how addressing the quantitative nature of gene regulation substantially increases our comprehension on the mechanisms of gene control.

Functional Magnetic Resonance Urography in Ureteropelvic Junction Obstruction: Proposal for a Pediatric Quantitative Score.

Background: Ureteropelvic junction obstruction (UPJO) is one of the most frequent causes of congenital hydronephrosis. It is essential to distinguish UPJO which needs surgical treatment. fMRU combines high quality morphological details of the kidney and excretory pathways with functional data. Objective: This study aims to introduce a new radiological score based on fMRU findings to be able to differentiate surgical from non-surgical kidneys. Materials and Methods: We retrospectively selected patients with hydronephrosis due to UPJO who underwent fMRU (January 2009-June 2018). A multidisciplinary team identified a list of fMRU morpho-functional predictive variables to be included in the analysis. To evaluate the role of different independent variables in predicting the outcome, a multivariable logistic regression model has been performed; the outcome variable was the surgical intervention. For each predictive variable, Odds Ratio and 95% Confidence Intervals were calculated. The likelihood ratio test was used to assess the significance of the variables. Using the regression model, we assigned a numerical value to each predictive variable, rounding up the beta-coefficients. The cut-off value of the total score was obtained from the ROC curve analysis. Results: A total of 192 patients were enrolled, corresponding to 200 pathological kidneys. All of them underwent fMRU; 135 were surgically treated, while 65 underwent ultrasound or MRU follow-up. Predictive variables significantly associated with surgery resulted to be the urographic phase, the presence of abnormal vessels, and a baseline anterior-posterior pelvic diameter >23 mm. Beta coefficients of the logistic regression model were then converted in scores. The ROC curve of the score showed high sensitivity (84.3%) and specificity (81.3%) with a cut-off > 2.5. Conclusions: We propose a new fMRU score able to identify surgical vs. non-surgical kidneys with UPJO.

Characterization of the DNA Binding Domain of StbA, A Key Protein of A New Type of DNA Segregation System.

Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 Å resolution. It folds into an HTH DNA-binding domain, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.

Role of Contrast-Enhanced Ultrasound (CEUS) in Native Kidney Pathology: Limits and Fields of Action.

Gray scale ultrasound has an important diagnostic role in native kidney disease. Low cost, absence of ionizing radiation and nephrotoxicity, short performance time, and repeatability even at the bedside, are the major advantages of this technique. The introduction of contrast enhancement ultrasound (CEUS) in daily clinical practice has significantly reduced the use of contrast enhancement computed tomography (CECT) and contrast enhancement magnetic resonance (CEMR), especially in patients with renal disease. Although there are many situations in which CECT and CEMRI are primarily indicated, their use may be limited by the administration of the contrast medium, which may involve a risk of renal function impairment, especially in the elderly, and in patients with acute kidney injury (AKI) and moderate to severe chronic kidney disease (CKD). In these cases, CEUS can be a valid diagnostic choice. To date, numerous publications have highlighted the role of CEUS in the study of parenchymal micro-vascularization and renal pathology by full integration with second level imaging methods (CECT and CEMRI) both in patients with normal renal function and with diseased kidneys. The aim of this review is to offer an updated overview of the limitations and potential applications of CEUS in native kidney disease.

Contrast-enhanced ultrasound after successful cryoablation of benign and malignant renal tumours: how long does tumour enhancement persist?

INTRODUCTION: To investigate how long successfully ablated tumours take to become completely avascular at CEUS after cryoablation. METHODS: Ninety-five patients had percutaneous cryoablation of 103 renal tumours investigated at CEUS on post-operative day one. If the lesion was avascular, a contrast-CT/MR was scheduled six months after the procedure, while CEUS was repeated if the lesion still displayed enhancement, until the disappearance of intralesional vascularity. Technical success was defined when the tumour was covered completely by the ablation zone. Technique efficacy was assessed at six months of follow-up. RESULTS: Technical efficacy was obtained for 101/103 cryoablations, 56% of which (57/101) were avascular on post-operative day one. After one week, two weeks, one month 83%, 91% and 100% of these 101 lesions, respectively, were avascular. Two tumours were unsuccessfully treated. They displayed persistent intralesional vascularity at CEUS one month after the procedure. CONCLUSIONS: After cryoablation, obtaining CEUS before one month may be misleading. When technical efficacy is obtained, disappearance of intralesional enhancement is observed within two weeks in the majority of cases (91%), but can persist until one month. Identification of tumour enhancement after one month may be concerning for residual viable tumour.