Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding.

CRISPR/Cas9 is a popular genome editing technology. Although widely used, little is known about how this prokaryotic system behaves in humans. An unwanted consequence of eukaryotic Cas9 expression is off-target DNA binding leading to mutagenesis. Safer clinical implementation of CRISPR/Cas9 necessitates a finer understanding of the regulatory mechanisms governing Cas9 behavior in humans. Here, we report our discovery of Cas9 sumoylation and ubiquitylation, the first post-translational modifications to be described on this enzyme. We found that the major SUMO2/3 conjugation site on Cas9 is K848, a key positively charged residue in the HNH nuclease domain that is known to interact with target DNA and contribute to off-target DNA binding. Our results suggest that Cas9 ubiquitylation leads to decreased stability via proteasomal degradation. Preventing Cas9 sumoylation through conversion of K848 into arginine or pharmacologic inhibition of cellular sumoylation enhances the enzyme's turnover and diminishes guide RNA-directed DNA binding efficacy, suggesting that sumoylation at this site regulates Cas9 stability and DNA binding. More research is needed to fully understand the implications of these modifications for Cas9 specificity.

Daptomycin versus teicoplanin in the treatment of osteomyelitis: Results of the Göztepe retrospective cohort study.

OBJECTIVES: Daptomycin is highly effective against Gram-positive multidrug-resistant bacteria. Publications on daptomycin in osteomyelitis treatment are limited. PATIENTS AND METHODS: In this multicenter retrospective cohort study, the aim was to evaluate the outcomes of osteomyelitis cases having received daptomycin or teicoplanin. This multicenter retrospective cohort study gathered data from seven centers located in five cities of Turkey. Study inclusion criteria were as follows: (a) magnetic resonance imaging and/or direct X-ray revealed osteomyelitis or biopsy pathologic examination results concomitant with osteomyelitis. Chi-squareand Student t-tests were used for statistical comparison. RESULTS: A total of 72 patients, 38 cases in the daptomycin group and 34 cases in the teicoplanin group diagnosed with osteomyelitis fulfilling the study inclusion criteria, were included in the study. Clinical success at the end of induction therapy was achieved in 32/38 cases in the daptomycin cohort vs. 30/34 cases in the teicoplanin cohort (p: 0.73). CONCLUSION: Although this is a limited experience in a small but well-defined cohort, our data suggest that daptomycin may be a safe alternative to glycopeptides in osteomyelitis treatment. A randomized controlled clinical study involving larger cohorts may increase the available evidence.

Analysis of the Conditions That Affect the Selective Processing of Endogenous Notch1 by ADAM10 and ADAM17.

Notch signaling is critical for controlling a variety of cell fate decisions during metazoan development and homeostasis. This unique, highly conserved signaling pathway relies on cell-to-cell contact, which triggers the proteolytic release of the cytoplasmic domain of the membrane-anchored transcription factor Notch from the membrane. A disintegrin and metalloproteinase (ADAM) proteins are crucial for Notch activation by processing its S2 site. While ADAM10 cleaves Notch1 under physiological, ligand-dependent conditions, ADAM17 mainly cleaves Notch1 under ligand-independent conditions. However, the mechanism(s) that regulate the distinct contributions of these ADAMs in Notch processing remain unclear. Using cell-based assays in mouse embryonic fibroblasts (mEFs) lacking ADAM10 and/or ADAM17, we aimed to clarify what determines the relative contributions of ADAM10 and ADAM17 to ligand-dependent or ligand-independent Notch processing. We found that EDTA-stimulated ADAM17-dependent Notch1 processing is rapid and requires the ADAM17-regulators iRhom1 and iRhom2, whereas the Delta-like 4-induced ligand-dependent Notch1 processing is slower and requires ADAM10. The selectivity of ADAM17 for EDTA-induced Notch1 processing can most likely be explained by a preference for ADAM17 over ADAM10 for the Notch1 cleavage site and by the stronger inhibition of ADAM10 by EDTA. The physiological ADAM10-dependent processing of Notch1 cannot be compensated for by ADAM17 in Adam10-/- mEFs, or by other ADAMs shown here to be able to cleave the Notch1 cleavage site, such as ADAMs9, 12, and 19. Collectively, these results provide new insights into the mechanisms underlying the substrate selectivity of ADAM10 and ADAM17 towards Notch1.

Sumoylation on its 25th anniversary: mechanisms, pathology, and emerging concepts.

Sumoylation is an essential post-translational modification intimately involved in a diverse range of eukaryotic cellular mechanisms. Small ubiquitin-like modifier (SUMO) protein isoforms can be reversibly linked to lysine residues that reside within specific motifs on thousands of target substrates, leading to modulations in stability, solubility, localization, and interactor profile. Since its initial discovery almost 25 years ago, SUMO has been described as a key regulator of genomic stability, cell proliferation, and infection among other processes. In this review, we trace the exciting developments in the history of this critical modifier, highlighting SUMO's roles in pathogenesis as well as its potential for the development of targeted therapies for numerous diseases.

Loss of odor-induced c-Fos expression of juxtaglomerular activity following maintenance of mice on fatty diets.

Diet-induced obesity (DIO) decreases the number of OMP+ olfactory sensory neurons (OSN) in the olfactory epithelium by 25% and reduces correlate axonal projections to the olfactory bulb (OB). Whether surviving OSNs have equivalent odor responsivity is largely unknown. Herein, we utilized c-fos immediate-early gene expression to map neuronal activity and determine whether mice weaned to control (CF), moderately-high fat (MHF), or high-fat (HF) diet for a period of 6 months had changes in odor activation. Diet-challenged M72-IRES-tau-GFP mice were exposed to either a preferred M72 (Olfr160) ligand, isopropyl tiglate, or clean air in a custom-made Bell-jar infusion chamber using an alternating odor exposure pattern generated by a picosprizer™. Mice maintained on fatty diets weighed significantly more and cleared glucose less efficiently as determined by an intraperitoneal glucose tolerance test (IPGTT). The number of juxtaglomerular cells (JGs) decreased following maintenance of the mice on the MHF diet for cells surrounding the medial but not lateral M72 glomerulus within a 4 cell-column distance. The percentage of c-fos + JGs surrounding the lateral M72 glomerulus decreased in fat-challenged mice whereas those surrounding the medial glomerulus were not affected by diet. Altogether, these results show an asymmetry in the responsiveness of the 'mirror image' glomerular map for the M72 receptor that shows greater sensitivity of the lateral vs. medial glomerulus upon exposure to fatty diet.

Impact of Prophylaxis vs Pre-emptive Approach for Cytomegalovirus Infection in Kidney Transplant Recipients.

Cytomegalovirus (CMV) is the most common viral infection during the post-transplant period, and it is one of the major causes of morbidity and mortality in kidney transplantation. In this study, the incidence and impact of pre-emptive and prophylactic approaches and long-term effects on graft and patient survival of CMV infection were investigated. Among 493 adult kidney transplant recipients, pretransplant CMV IgG-negative patients and patients with a follow-up shorter than a month were excluded. The patients were divided into 2 groups: pre-emptive group (n = 187, regular screening and acyclovir 400 mg twice daily for 6 months), and prophylaxis group (n = 275, valganciclovir 450 mg/d for 3 months). The pre-emptive group was screened for CMV with either pp65 antigenemia or CMV DNA. There were 462 patients, and mean follow-up was 37.7 months. There were more CMV infections in the pre-emptive group than in the prophylaxis group (n = 56, 30.1% vs n = 12, 4.4%, respectively; P < .001). Late CMV infections were significantly more frequent in the prophylaxis group (10 of 12, 83.3%) than in the pre-emptive group (8 of 56, 14.3%, P < .001). In multivariate analysis, valganciclovir prophylaxis was associated with a lower CMV infection (relative risk [RR]: 0.18, 95% confidence interval [CI] 0.08 to 0.39, P < .001). Delayed graft function was the only independent risk factor for graft loss during the follow-up on multivariate Cox regression analysis (RR: 2.66, 95% GA 1.17 to 6.04, P = .02). Valganciclovir prophylaxis was more protective against CMV infection than the pre-emptive approach. Neither prophylaxis/pre-emptive approaches nor CMV infection had negative effect on graft and patient survival.

Piperacillin/tazobactam vs. cefoperazone/sulbactam in adult low-risk febrile neutropenia cases.

AIM: The aim of this study was to compare the efficacy of piperacillin/tazobactam (P/T) and cefoperazone/sulbactam (C/S) in the empirical treatment of adult neutropenic fever. METHODS: Data and outcomes of low-risk adult cases with neutropenic fever and treated with P/T (4.5 g q6h) or C/S (2 g q8h) between 2005 and 2011 June were extracted from our database. Risk evaluation was made according to criteria of Multinational Association for Supportive Care in Cancer (MASCC) and a score of ≥ 21 was considered as low risk. Data were collected prospectively by daily visits and evaluated retrospectively. Primary outcome was - fever defervescence at 72 h in combination with success without modification (referring to episodes where the patient recovered from fever with disappearance of signs of infection without modification to initial empirical treatment). All-cause mortality referred to death resulting from a documented or presumed infection or unidentified reason during the treatment and 30-day follow-up period. RESULTS: A total of 172 patients (113 cases P/T and 59 cases C/S) fulfilled the study inclusion criteria. Persistent response in P/T arm was 73.5%, whereas it was 64.5% in C/S arm (p > 0.05). Rates of any modification were also similar in both treatment arms. All-cause mortality during the treatment and 30-day follow-up period was not significantly different (P/T: 4/113 vs. C/S: 2/59, p > 0.05). There was no severe adverse effect requiring antibiotic cessation in both cohorts. CONCLUSION: In conclusion, our data suggest that C/S may be a safe alternative to P/T in the empirical treatment of adult low-risk febrile neutropenia cases.

Effectiveness of a multidimensional approach for prevention of ventilator-associated pneumonia in 11 adult intensive care units from 10 cities of Turkey: findings of the International Nosocomial Infection Control Consortium (INICC).

PURPOSE: To evaluate the impact of the International Nosocomial Infection Control Consortium (INICC) multidimensional approach on the reduction of ventilator-associated pneumonia (VAP) in adult patients hospitalized in 11 intensive care units (ICUs), from 10 hospitals, members of the INICC, in 10 cities of Turkey. METHODS: A prospective active before-after surveillance study was conducted to determine the effect of the INICC multidimensional approach in the VAP rate. The study was divided into two phases. In phase 1, active prospective surveillance of VAP was conducted using the definitions of the Centers for Disease Control and Prevention National Health Safety Network, and the INICC methods. In phase 2, we implemented the multidimensional approach for VAP. The INICC multidimensional approach included the following measures: (1) bundle of infection control interventions, (2) education, (3) outcome surveillance, (4) process surveillance, (5) feedback of VAP rates, and (6) performance feedback of infection control practices. We compared the rates of VAP obtained in each phase. A time series analysis was performed to assess the impact of our approach. RESULTS: In phase 1, we recorded 2,376 mechanical ventilator (MV)-days, and in phase 2, after implementing the multidimensional approach, we recorded 28,181 MV-days. The rate of VAP was 31.14 per 1,000 MV-days during phase 1, and 16.82 per 1,000 MV-days during phase 2, amounting to a 46 % VAP rate reduction (RR, 0.54; 95 % CI, 0.42-0.7; P value, 0.0001.) CONCLUSIONS: The INICC multidimensional approach was associated with a significant reduction in the VAP rate in these adult ICUs of Turkey.

A randomised controlled trial of ribavirin in Crimean Congo haemorrhagic fever: ethical considerations.

The randomised controlled trial (RCT) constitutes a quantitative, comparative, controlled study of a particular treatment, and provides invaluable evidence regarding its pharmacotherapeutic efficacy. These studies are generally predicated upon the ethical principle of clinical equipoise. However, this may be insufficient to justify withholding treatment from a control group while assessing drug therapy in a potentially fatal disease. Thus, the criteria for randomisation, informed consent methodology and timing, and consideration of treatment options in such a scenario remain the province of medical ethics. This paper addresses the need for an RCT of ribavirin in the treatment of Crimean Congo haemorrhagic fever, and highlights underlying ethical concerns in light of the current medical, virological and ethical literature.

Carbapenem versus fosfomycin tromethanol in the treatment of extended-spectrum beta-lactamase-producing Escherichia coli-related complicated lower urinary tract infection.

The aim of this observational prospective study was to compare the effect of fosfomycin tromethanol (FT) and carbapenems (meropenem or imipenem cilastatin) in the treatment of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli-related complicated lower urinary tract infection (CLUTI). Inclusion criteria were: patients who were aged >18 yr with dysuria or problems with frequency or urgency in passing urine; those with >20 leukocytes/mm³ in urine microscopy and culture-proven ESBL-producing carbapenem or FT-sensitive E. coli in the urine (>105 cfu/mm³); no leukocytosis or fever; and who were treated with ft (oral 3 g sachet x 1 every other night, three times) or carbapenems between march 2005 and January 2006 in our outpatient clinic and hospital. A total of 47 CLUTI attacks in 47 patients (27 FT group, 20 carbapenem group) were observed prospectively. Clinical and microbiological success in the carbapenem and ft groups was similar (19/20 vs 21/27 and 16/20 vs 16/27 p>0.05). Drug acquisition costs were significantly lower in the FT group (p<0.001). Although it is not a randomized controlled study, these data show that ft may be a suitable, effective and cheap alternative in the treatment of ESBL-producing E. coli-related CLUTI.

Ethical and legal aspects of stem cell practices in Turkey: where are we?

Advances in medical technology and information have facilitated clinical practices that favourably affect the success rates of treatment for diseases. Regenerative medicine has been the focus of the recent medical agenda, to the extent of fundamentally changing treatment paradigms. Stem cell practices, their efficacy, and associated ethical concerns have been debated intensively in many countries. Stem cell research is carried out along with the treatment of patients. Thus, various groups affected by the practices inevitably participate in the discussions. In addition to discussions based on avoiding any harm, providing benefits and respecting personal autonomy and justice, problems arise owing to the lack of legal regulations for stem cell research and practice. The dimensions of the problems vary in the developing countries, with widespread use of advanced medical technology but with lack of sources allocated for healthcare, dominance of paternalistic physician-patient relationships and failure to achieve a sufficient level of awareness of patients' rights. This article discusses the current situation of stem cell practices within the context of regenerative medicine in Turkey and ethical concerns about some of the legal regulations, such as the Regulation for Umblical Cord Blood Banking and Guidelines for Non-embryonic Stem Cell Study for Non-clinical Purposes directing the research on this issue.

Eugenics concept: from Plato to present.

All prospective studies and purposes to improve cure and create a race that would be exempt of various diseases and disabilities are generally defined as eugenic procedures. They aim to create the "perfect" and "higher" human being by eliminating the "unhealthy" prospective persons. All of the supporting actions taken in order to enable the desired properties are called positive eugenic actions; the elimination of undesired properties are defined as negative eugenics. In addition, if such applications and approaches target the public as a whole, they are defined as macro-eugenics. On the other hand, if they only aim at individuals and/or families, they are called micro-eugenics. As generally acknowledged, Galton re-introduced eugenic proposals, but their roots stretch as far back as Plato. Eugenic thoughts and developments were widely accepted in many different countries beginning with the end of the 19th to the first half of the 20th centuries. Initially, the view of negative eugenics that included compulsory sterilizations of handicapped, diseased and "lower" classes, resulted in tens of thousands being exterminated especially in the period of Nazi Germany. In the 1930s, the type of micro positive eugenics movement found a place within the pro-natalist policies of a number of countries. However, it was unsuccessful since the policy was not able to become effective enough and totally disappeared in the 1960s. It was no longer a fashionable movement and left a deep impression on public opinion after the long years of war. However, developments in genetics and its related fields have now enabled eugenic thoughts to reappear under the spotlight and this is creating new moral dilemmas from an ethical perspective.

Laqve (wry mouth) considered in Avicenna's renowned treatise the Canon of Medicine.

Hemi-facial spasm, facial paralysis, and trigeminal neuralgia are prevailing signs and symptoms with which physicians have been coping for thousands of years. Ibn Sina (known as Avicenna in the West), who was among the leading figures during medieval ages and influenced the upcoming periods in the Eastern and Western hemispheres for long time, focused also on these crucial problems. In his principal medical work, the Canon of Medicine, Avicenna underlined the significance of wry mouth-related disorders and wrote a precise chapter over this topic with the heading "Laqve." However, the term "laqve" is usually accepted only as facial paralysis in most of historical texts. Further detailed analysis of the text reveals that, all the above-mentioned signs and symptoms are considered under the same heading. Therefore, the descriptions articulated by famous physician Avicenna pose great merit from the point of historical view of neurological sciences. The main aim of this article is to reintroduce essential parts of the text by adding comments over specific descriptions, and consequently, to make the text more comprehensible for today's scientists.

Laqve (wry mouth) considered in Avicenna’s renowned treatise the Canon of Medicine

Hemiespasmo facial, parálisis facial y neuralgia del trigémino son signos y síntomas a los cuales se han enfrentado los médicos durante milenios. Ibn Sina, (conocido como Avicena en Occidente), que fue una de las figuras más importantes de la Edad Media e influyó tanto en Oriente como en Occidente a lo largo de muchos siglos, también prestó gran atención a estos problemas. En su principal trabajo médico, el “Canon de la Medicina”, destacó el significado de la “cara torcida” y escribió un capítulo especial sobre estos procesos con el título de “Laqve”. Sin embargo, el término “laqve” sólo se acepta como tal, en la mayoría de los textos históricos, cuando se refiere a la parálisis facial. El análisis detallado del texto revela que todos los signos y síntomas mencionados se incluyen en el mismo título. Por consiguiente, las descripciones articuladas por el famoso médico Avicena poseen un gran mérito desde el punto de vista histórico de las ciencias neurológicas. La finalidad principal de este artículo es reintroducir partes esenciales del texto, añadiendo comentarios sobre descripciones específicas y, como consecuencia, hacer el texto más comprensible para los científicos de hoy

Hemi-facial spasm, facial paralysis, and trigeminal neuralgia are prevailing signs and symptoms with which physicians have been coping for thousands of years. Ibn Sina (known as Avicenna in the West), who was among the leading figures during medieval ages and influenced the upcoming periods in the Eastern and Western hemispheres for long time, focused also on these crucial problems. In his principal medical work, the Canon of Medicine, Avicenna underlined the significance of wry mouth-related disorders and wrote a precise chapter over this topic with the heading “Laqve.” However, the term “laqve” is usually accepted only as facial paralysis in most of historical texts. Further detailed analysis of the text reveals that, all the above mentioned signs and symptoms are considered under the same heading. Therefore, the descriptions articulated by famous physician Avicenna pose great merit from the point of historical view of neurological sciences. The main aim of this article is to reintroduce essential parts of the text by adding comments over specific descriptions, and consequently, to make the text more comprehensible for today’s scientists