ABSTRACT Repression of the memory suppressor gene h istone d e ac etylase 2 ( Hdac2 ) in mice elicits cognitive enhancement, and drugs that block HDAC2 catalytic activity are being investigated for treating disorders affecting memory. Currently available compounds that target HDAC2 are not specific to the HDAC2 isoform, and have short half-lives. Antisense oligonucleotides (ASOs) are a class of drugs that base pair with RNA targets and exhibit extremely long-lasting, specific inhibition relative to small molecule drugs. We utilized an ASO to reduce Hdac2 messenger RNA (mRNA) quantities, and explored its longevity, specificity, and mechanism of repression. A single injection of the Hdac2 -targeted ASO in the central nervous system diminished Hdac2 mRNA levels for at least 4 months in the brain, and knockdown of this factor resulted in significant memory enhancement for 8 weeks in mice. RNA-seq analysis of brain tissues revealed that the ASO repression was specific to the Hdac2 isoform relative to other classical Hdac genes, and caused alterations in levels of other memory-associated mRNAs. In cultured neurons, we observed that the Hdac2 -targeted ASO suppressed Hdac2 mRNA and an Hdac2 non-coding regulatory extra-coding RNA (ecRNA). The ASO not only triggered a reduction in mRNA levels, but also elicited direct transcriptional suppression of the Hdac2 gene through blocking RNA polymerase II elongation. These findings suggest transcriptional suppression of the target gene as a novel mechanism of action of ASOs, and opens up the possibility of using ASOs to achieve lasting gene silencing in the brain without altering the nucleotide sequence of a gene.

Lyme disease is a tick-borne infection caused by the bacteria Borrelia burgdorferi. Current diagnosis of early Lyme disease relies heavily on clinical criteria, including the presence of an erythema migrans rash. The sensitivity of current gold-standard diagnostic tests relies upon antibody formation, which is typically delayed and thus of limited utility in early infection. We conducted a study of blood and skin biopsy specimens from 57 patients with a clinical diagnosis of erythema migrans. Samples collected at the time of diagnosis were analyzed using an ultrasensitive, PCR-based assay employing an isothermal amplification step and multiple primers. In 75.4% of patients, we directly detected one or more B. burgdorferi genotypes in the skin. Two-tier testing showed that 20 (46.5%) of those found to be PCR positive remained serologically negative at both acute and convalescent time points. Multiple genotypes were found in three (8%) of those where a specific genotype could be identified. The 13 participants who lacked PCR and serologic evidence for exposure to B. burgdorferi could be differentiated as a group from PCR-positive participants by their levels of several immune markers as well as by clinical descriptors such as the number of acute symptoms and the pattern of their erythema migrans rash. These results suggest that within a Mid-Atlantic cohort, patient subgroups can be identified using PCR-based direct detection approaches. This may be particularly useful in future research such as vaccine trials and public health surveillance of tick-borne disease patterns.

Borrelia burgdorferi is the etiological agent of Lyme disease. In the current study, we used direct-detection PCR and electrospray ionization mass spectrometry to monitor and genotype B. burgdorferi isolates from serially collected whole-blood specimens from patients clinically diagnosed with early Lyme disease before and during 21 days of antibiotic therapy. B. burgdorferi isolates were detected up to 3 weeks after the initiation of antibiotic treatment, with ratios of coinfecting B. burgdorferi genotypes changing over time.

We thank Dr. Elder for his comments and for the opportunity to continue a dialogue on the potential consequences of eliminating performance-based assessments as a requirement for medical licensure in the United States. We agree that careful consideration should be given to the impact such a decision would have on international medical graduates (IMGs). Interestingly, the United Kingdom is engaged in similar conversations. The General Medical Council (GMC) revealed plans to implement a national licensing examination (NLE) for all UK graduates and IMGs seeking to enter the medical register.1 The GMC has published reports of its engagement with UK medical schools2 and the public.3 As a result of the United Kingdom’s European Union membership, physicians from the European Economic Area (EEA) are not required to demonstrate competence by an NLE to gain registration in the United Kingdom, yet most physicians outside of the EEA are required to pass the United Kingdom’s Professional and Linguistic Assessments Board. The Medical Licensing Assessment (MLA) will be a two-part assessment of applied knowledge and clinical skills. Inevitable issues surrounding exam cost and burden have been raised. As a result, the GMC currently states that the clinical skills assessment will occur at individual universities for UK medical students as part of their degree program, and “we will check that this meets our requirements.”4 It will be interesting to see how the GMC balances cost, standardization, and oversight within a decentralized system, especially when concerns about variability in medical schools’ finals have already been expressed.5 Furthermore, the GMC plans to administer the clinical skills assessment for IMGs, which raises the question of whether candidates will be held to a common standard regardless of the country in which they were trained. The United Kingdom offers a unique case study of the various ways medical graduates gain registration, and as the United Kingdom withdraws from the European Union, we can learn lessons from the implementation of the MLA. Ultimately, assessment drives learning and demonstrates that our professional values align with public concerns. We do not support forfeiting a national clinical skills competency standard in the United States. Rather, we support further evolution of the United States Medical Licensing Examination Step 2 Clinical Skills to reflect the needs of students, medical schools, training programs, and patients as well as the desired outcomes for physicians of the future. David J. Ecker, MDAssistant professor of medicine, assistant director of education, Hospital Medicine Group, and director, Foundations of Doctoring Curriculum, Integrated Clinicians Course, and Clinical Practice Exam, University of Colorado School of Medicine, Aurora, Colorado; [email protected] Michelle Daniel, MD, MHPEAssistant dean for curriculum, University of Michigan Medical School, Ann Arbor, Michigan. Felise B. Milan, MDProfessor of medicine and director, Ruth L. Gottesman Clinical Skills Center and Introduction to Clinical Medicine Program, Albert Einstein College of Medicine, Bronx, New York.

Recently, a student-initiated movement to end the United States Medical Licensing Examination Step 2 Clinical Skills and the Comprehensive Osteopathic Medical Licensing Examination Level 2-Performance Evaluation has gained momentum. These are the only national licensing examinations designed to assess clinical skills competence in the stepwise process through which physicians gain licensure and certification. Therefore, the movement to end these examinations and the ensuing debate merit careful consideration. The authors, elected representatives of the Directors of Clinical Skills Courses, an organization comprising clinical skills educators in the United States and beyond, believe abolishing the national clinical skills examinations would have a major negative impact on the clinical skills training of medical students, and that forfeiting a national clinical skills competency standard has the potential to diminish the quality of care provided to patients. In this Perspective, the authors offer important additional background information, outline key concerns regarding the consequences of ending these national clinical skills examinations, and provide recommendations for moving forward: reducing the costs for students, exploring alternatives, increasing the value and transparency of the current examinations, recognizing and enhancing the strengths of the current examinations, and engaging in a national dialogue about the issue.

ObjectivesBlood culture results inadequately stratify the mortality risk in critically ill patients with sepsis. We sought to establish the prognostic significance of the presence of microbial DNA in the bloodstream of patients hospitalized with suspected sepsis.MethodsWe analysed the data collected during the Rapid Diagnosis of Infections in the Critically Ill (RADICAL) study, which compared a novel culture-independent PCR/electrospray ionization-mass spectrometry (ESI-MS) assay with standard microbiological testing. Patients were eligible for the study if they had suspected sepsis and were either hospitalized or were referred to one of nine intensive care units from six European countries. The blood specimen for PCR/ESI-MS assay was taken along with initial blood culture taken for clinical indications.ResultsOf the 616 patients recruited to the RADICAL study, 439 patients had data on outcome, results of the blood culture and PCR/ESI-MS assay available for analysis. Positive blood culture and PCR/ESI-MSI result was found in 13% (56/439) and 40% (177/439) of patients, respectively. Either a positive blood culture (p 0.01) or a positive PCR/ESI-MS (p 0.005) was associated with higher SOFA scores on enrolment to the study. There was no difference in 28-day mortality observed in patients who had either positive or negative blood cultures (35% versus 32%, p 0.74). However, in patients with a positive PCR/ESI-MS assay, mortality was significantly higher in comparison to those with a negative result (42% versus 26%, p 0.001).ConclusionsPresence of microbial DNA in patients with suspected sepsis might define a patient group at higher risk of death.

Ixodes ricinus ticks are vectors of numerous human and animal pathogens. They are host generalists able to feed on more than 300 vertebrate species. The prevalence of tick-borne pathogens is influenced by host-vector-pathogen interactions that results in spatial distribution of infection risk. Broad-range polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was used to analyze 435 I. ricinus nymphs from four localities in the south of the Czech Republic for the species identification of tick-borne pathogens. Borrelia burgdorferi sensu lato spirochetes were the most common pathogen detected in the ticks; 21% of ticks were positive for a single genospecies and 2% were co-infected with two genospecies. Other tick-borne pathogens detected included Rickettsia helvetica (3.9%), R. monacensis (0.2%), Anaplasma phagocytophilum (2.8%), Babesia venatorum (0.9%), and Ba. microti (0.5%). The vertebrate host of the ticks was determined using PCR followed by reverse line blot hybridization from the tick's blood-meal remnants. The host was identified for 61% of ticks. DNA of two hosts was detected in 16% of samples with successful host identification. The majority of ticks had fed on artiodactyls (50.7%) followed by rodents (28.6%) and birds (7.8%). Other host species were wild boar, deer, squirrels, field mice and voles.

Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. Disclaimer: The IRIDICA BAC BSI Assay is not available in the United States.

BACKGROUND:Rapid molecular diagnostic (RMD) platforms may lead to better antibiotic use. Our objective was to develop analytical strategies to enhance the interpretation of RMDs for clinicians. METHODS:We compared the performance characteristics of 4 RMD platforms for detecting resistance against β-lactams in 72 highly resistant isolates of Escherichia coli and Klebsiella pneumoniae (PRIMERS I). Subsequently, 2 platforms were used in a blinded study in which a heterogeneous collection of 196 isolates of E. coli and K. pneumoniae (PRIMERS II) were examined. We evaluated the genotypic results as predictors of resistance or susceptibility against β-lactam antibiotics. We designed analytical strategies and graphical representations of platform performance, including discrimination summary plots and susceptibility and resistance predictive values, that are readily interpretable by practitioners to inform decision-making. RESULTS:In PRIMERS I, the 4 RMD platforms detected β-lactamase (bla) genes and identified susceptibility or resistance in >95% of cases. In PRIMERS II, the 2 platforms identified susceptibility against extended-spectrum cephalosporins and carbapenems in >90% of cases; however, against piperacillin/tazobactam, susceptibility was identified in <80% of cases. Applying the analytical strategies to a population with 15% prevalence of ceftazidime-resistance and 5% imipenem-resistance, RMD platforms predicted susceptibility in >95% of cases, while prediction of resistance was 69%-73% for ceftazidime and 41%-50% for imipenem. CONCLUSIONS:RMD platforms can help inform empiric β-lactam therapy in cases where bla genes are not detected and the prevalence of resistance is known. Our analysis is a first step in bridging the gap between RMDs and empiric treatment decisions.

Human haploinsufficiency of the transcription factor Tcf4 leads to a rare autism spectrum disorder called Pitt-Hopkins syndrome (PTHS), which is associated with severe language impairment and development delay. Here, we demonstrate that Tcf4 haploin-sufficient mice have deficits in social interaction, ultrasonic vocalization, prepulse inhibition, and spatial and associative learning and memory. Despite learning deficits, Tcf4(+/-) mice have enhanced long-term potentiation in the CA1 area of the hippocampus. In translationally oriented studies, we found that small-molecule HDAC inhibitors normalized hippocampal LTP and memory recall. A comprehensive set of next-generation sequencing experiments of hippocampal mRNA and methylated DNA isolated from Tcf4-deficient and WT mice before or shortly after experiential learning, with or without administration of vorinostat, identified ''memory-associated'' genes modulated by HDAC inhibition and dysregulated by Tcf4 haploinsufficiency. Finally, we observed that Hdac2 isoform-selective knockdown was sufficient to rescue memory deficits in Tcf4(+/-) mice.

Objective: Early identification of causative microorganism(s) in patients with severe infection is crucial to optimize antimicrobial use and patient survival. However, current culture-based pathogen identification is slow and unreliable such that broad-spectrum antibiotics are often used to insure coverage of all potential organisms, carrying risks of overtreatment, toxicity, and selection of multidrug-resistant bacteria. We compared the results obtained using a novel, culture-independent polymerase chain reaction/electrospray ionization-mass spectrometry technology with those obtained by standard microbiological testing and evaluated the potential clinical implications of this technique. Design: Observational study. Setting: Nine ICUs in six European countries. Patients: Patients admitted between October 2013 and June 2014 with suspected or proven bloodstream infection, pneumonia, or sterile fluid and tissue infection were considered for inclusion. Interventions: None. Measurements and Main Results: We tested 616 bloodstream infection, 185 pneumonia, and 110 sterile fluid and tissue specimens from 529 patients. From the 616 bloodstream infection samples, polymerase chain reaction/electrospray ionization-mass spectrometry identified a pathogen in 228 cases (37%) and culture in just 68 (11%). Culture was positive and polymerase chain reaction/electrospray ionization-mass spectrometry negative in 13 cases, and both were negative in 384 cases, giving polymerase chain reaction/electrospray ionization-mass spectrometry a sensitivity of 81%, specificity of 69%, and negative predictive value of 97% at 6 hours from sample acquisition. The distribution of organisms was similar with both techniques. Similar observations were made for pneumonia and sterile fluid and tissue specimens. Independent clinical analysis of results suggested that polymerase chain reaction/electrospray ionization-mass spectrometry technology could potentially have resulted in altered treatment in up to 57% of patients. Conclusions: Polymerase chain reaction/electrospray ionization-mass spectrometry provides rapid pathogen identification in critically ill patients. The ability to rule out infection within 6 hours has potential clinical and economic benefits.

The cornerstone of sepsis management requires identifying the causative pathogen and initiating appropriate antimicrobial therapy. Current pathogen detection relies on culture techniques, a technology that is over 100 years old, slow and unreliably. It is not unusual for only 10% of critical care blood cultures to be positive. Due to the low yield and time taken to obtain a result, they rarely alter patient management. a novel molecular pathogen detection system, known as IRIDICA, employs polymerase chain reaction and electro spray ionisation mass spectroscopy (PCR/ESI-MS) to identify over 1000 pathogens, direct from sample without culture and within 8 hours. The RADICAL study was created to assess this technology in a real world critical care environment.

Ixodes pacificus ticks can harbor a wide range of human and animal pathogens. To survey the prevalence of tick-borne known and putative pathogens, we tested 982 individual adult and nymphal I. pacificus ticks collected throughout California between 2007 and 2009 using a broad-range PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to detect a wide range of tick-borne microorganisms. Overall, 1.4% of the ticks were found to be infected with Borrelia burgdorferi, 2.0% were infected with Borrelia miyamotoi and 0.3% were infected with Anaplasma phagocytophilum. In addition, 3.0% were infected with Babesia odocoilei. About 1.2% of the ticks were co-infected with more than one pathogen or putative pathogen. In addition, we identified a novel Anaplasmataceae species that we characterized by sequencing of its 16S rRNA, groEL, gltA, and rpoB genes. Sequence analysis indicated that this organism is phylogenetically distinct from known Anaplasma species with its closest genetic near neighbors coming from Asia. The prevalence of this novel Anaplasmataceae species was as high as 21% at one site, and it was detected in 4.9% of ticks tested statewide. Based upon this genetic characterization we propose that this organism be called 'Candidatus Cryptoplasma californiense'. Knowledge of this novel microbe will provide awareness for the community about the breadth of the I. pacificus microbiome, the concept that this bacterium could be more widely spread; and an opportunity to explore whether this bacterium also contributes to human or animal disease burden.

Survival from sepsis and bloodstream infections (BSI) often depends upon rapid identification of the infecting pathogen and expeditious antimicrobial therapy. We report findings from the final analysis of RADICAL, a multicenter observational study that compared results from direct blood specimen testing using PCR/ESI-MS to standard microbiology in critically ill patients.

La presente invention concerne des compositions et des procedes pour l'alteration de la methylation par des ARNpi synthetiques ou par l'alteration de la fonction de ARNpi. De telles alterations sont utiles dans la regulation et la commande de l'expression genique (par exemple l'expression genique neurale) et des fonctions concomitantes (par exemple les fonctions neurales). La presente invention concerne en outre des systemes et des procedes pour l'identification de sites cibles pour la regulation par des ARNpi.

Background A limitation of both culture-based and molecular methods of screening for staphylococcal infection is that current tests determine only the presence or absence of colonization with no information on the colonizing strain type. A technique that couples polymerase chain reaction to mass spectrometry (PCR/ESI-MS) has recently been developed and an assay validated to identify and genotype S. aureus and coagulase-negative staphylococci (CoNS). Methods This study was conducted to determine the rates, risk factors, and molecular genotypes of colonizing Staphylococcus aureus in adult patients presenting to an inner-city academic emergency department. Participants completed a structured questionnaire to assess hospital and community risks for infection with methicillin-resistant S. aureus (MRSA). Nasal swabs were analyzed by PCR/ESI-MS to identify and genotype S. aureus and CoNS. Results Of 200 patients evaluated, 59 were colonized with S. aureus ; 27 of these were methicillin-resistant strains. Twenty-four of the 59 S. aureus carriers were co-colonized with a CoNS and 140 of the 200 patients were colonized exclusively with CoNS. The molecular genotypes of the 59 S. aureus strains were diverse; 21 unique molecular genotypes belonging to seven major clonal complexes were identified. Eighty-five of 200 patients carried strains with high-level mupirocin resistance. Of these eighty-five participants, 4 were colonized exclusively with S. aureus , 16 were co-colonized with S. aureus and CoNS, and 65 were colonized exclusively with CoNS. Conclusion The prevalence of S. aureus and methicillin-resistant S. aureus colonization in a random sample of patients seeking care in Emergency Department was 29.5% and 13.5%, respectively. A substantial fraction of the S. aureus -colonized patients were co-colonized with CoNS and high-level mupirocin-resistant CoNS. Determining the molecular genotype of S. aureus during intake screening may prove valuable in the future if certain molecular genotypes become associated with increased infection risk.

Abstract Ticks harbor numerous pathogens of significance to human and animal health. A better understanding of the pathogens carried by ticks in a given geographic area can alert health care providers of specific health risks leading to better diagnosis and treatments. In this study, we tested 226 Ixodes ricinis ticks from Southern Germany using a broad-range PCR and electrospray ionization mass spectrometry assay (PCR/ESI-MS) designed to identify tick-borne bacterial and protozoan pathogens in a single test. We found 21.2% of the ticks tested carried Borrelia burgdorferi sensu lato consisting of diverse genospecies; a surprisingly high percentage of ticks were infected with Babesia microti (3.5%). Other organisms found included Borrelia miyamotoi, Rickettsia helvetica, Rickettsia monacensis, and Anaplasma phagocytophilum. Of further significance was our finding that more than 7% of ticks were infected with more than one pathogen or putative pathogen.

ABSTRACT The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.