Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation.

Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or 'antispacer', PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.

Altering Fish Behavior by Sensing Swarm Patterns of Fish in an Artificial Aquatic Environment Using an Interactive Robotic Fish.

Numerous studies have been conducted to prove the calming and stress-reducing effects on humans of visiting aquatic environments. As a result, many institutions have utilized fish to provide entertainment and treat patients. The most common issue in this approach is controlling the movement of fish to facilitate human interaction. This study proposed an interactive robot, a robotic fish, to alter fish swarm behaviors by performing an effective, unobstructed, yet necessary, defined set of actions to enhance human interaction. The approach incorporated a minimalistic but futuristic physical design of the robotic fish with cameras and infrared (IR) sensors, and developed a fish-detecting and swarm pattern-recognizing algorithm. The fish-detecting algorithm was implemented using background subtraction and moving average algorithms with an accuracy of 78%, while the swarm pattern detection implemented with a Convolutional Neural Network (CNN) resulted in a 77.32% accuracy rate. By effectively controlling the behavior and swimming patterns of fish through the smooth movements of the robotic fish, we evaluated the success through repeated trials. Feedback from a randomly selected unbiased group of subjects revealed that the robotic fish improved human interaction with fish by using the proposed set of maneuvers and behavior.

Peptide nucleic acids and their role in gene regulation and editing.

The unique properties of peptide nucleic acid (PNA) makes it a desirable candidate to be used in therapeutic and biotechnological interventions. It has been broadly utilized for numerous applications, with a major focus in regulation of gene expression, and more recently in gene editing. While the classic PNA design has mainly been employed to date, chemical modifications of the PNA backbone and nucleobases provide an avenue to advance the technology further. This review aims to discuss the recent developments in PNA based gene manipulation techniques and the use of novel chemical modifications to improve the current state of PNA mediated gene targeting.

Synthesis of ( R)- and ( S)-Fmoc-Protected Diethylene Glycol Gamma PNA Monomers with High Optical Purity.

A robust synthetic route has been developed for preparing optically pure, Fmoc-protected diethylene glycol-containing ( R)- and ( S)-γPNA monomers. The strategy involves the application of 9-(4-bromophenyl)-9-fluorenyl as a temporary, safety-catch protecting group for the suppression of epimerization in the O-alkylation and reductive amination steps. The optical purities of the final monomers were determined to be greater than 99.5% ee, as assessed by 19F-NMR and HPLC. The new synthetic methodology is well-suited for large-scale monomer production, with most synthetic steps providing excellent chemical yields without the need for chromatographic purification other than a simple workup and precipitation.

Design of Bivalent Nucleic Acid Ligands for Recognition of RNA-Repeated Expansion Associated with Huntington's Disease.

We report the development of a new class of nucleic acid ligands that is comprised of Janus bases and the MPγPNA backbone and is capable of binding rCAG repeats in a sequence-specific and selective manner via, inference, bivalent H-bonding interactions. Individually, the interactions between ligands and RNA are weak and transient. However, upon the installation of a C-terminal thioester and an N-terminal cystine and the reduction of disulfide bond, they undergo template-directed native chemical ligation to form concatenated oligomeric products that bind tightly to the RNA template. In the absence of an RNA target, they self-deactivate by undergoing an intramolecular reaction to form cyclic products, rendering them inactive for further binding. The work has implications for the design of ultrashort nucleic acid ligands for targeting rCAG-repeat expansion associated with Huntington's disease and a number of other related neuromuscular and neurodegenerative disorders.

Intimate partner violence in Sri Lanka: a scoping review.

South Asia is considered to have a high prevalence of intimate partner violence (IPV) against women. Therefore the World Health Organisation has called for context-specific information about IPV from different regions. A scoping review of published and gray literature over the last 35 years was conducted using Arksey and O'Malley's framework. Reported prevalence of IPV in Sri Lanka ranged from 20-72%, with recent reports of rates ranging from 25- 35%. Most research about IPV has been conducted in a few provinces and is based on the experience of legally married women. Individual, family, and societal risk factors for IPV have been studied, but their complex relationships have not been comprehensively investigated. Health consequences of IPV have been reported, with particular attention to physical health, but women are likely to underreport sexual violence. Women seek support mainly from informal networks, with only a few visiting agencies to obtain help. Little research has focused on health sector responses to IPV and their effectiveness. More research is needed on how to challenge gendered perceptions about IPV. Researchers should capture the experience of women in dating/cohabiting relationships and women in vulnerable sectors (post-conflict areas and rural areas), and assess how to effectively provide services to them. A critical evaluation of existing services and programmes is also needed to advance evidence informed programme and policy changes in Sri Lanka.

Next generation triplex-forming PNAs for site-specific genome editing of the F508del CFTR mutation.

BACKGROUND: Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein for which there is no cure. One approach to cure CF is to correct the underlying mutations in the CFTR gene. We have used triplex-forming peptide nucleic acids (PNAs) loaded into biodegradable nanoparticles (NPs) in combination with donor DNAs as reagents for correcting mutations associated with genetic diseases including CF. Previously, we demonstrated that PNAs induce recombination between a donor DNA and the CFTR gene, correcting the F508del CFTR mutation in human cystic fibrosis bronchial epithelial cells (CFBE cells) and in a CF murine model leading to improved CFTR function with low off-target effects, however the level of correction was still below the threshold for therapeutic cure. METHODS: Here, we report the use of next generation, chemically modified gamma PNAs (γPNAs) containing a diethylene glycol substitution at the gamma position for enhanced DNA binding. These modified γPNAs yield enhanced gene correction of F508del mutation in human bronchial epithelial cells (CFBE cells) and in primary nasal epithelial cells from CF mice (NECF cells). RESULTS: Treatment of CFBE cells and NECF cells grown at air-liquid interface (ALI) by NPs containing γtcPNAs and donor DNA resulted in increased CFTR function measured by short circuit current and improved gene editing (up to 32 %) on analysis of genomic DNA. CONCLUSIONS: These findings provide the basis for further development of PNA and NP technology for editing of the CFTR gene.

Association of levofloxacin resistance with mortality in adult patients with invasive pneumococcal diseases: a post hoc analysis of a prospective cohort.

OBJECTIVE: This study was conducted to identify risk factors for mortality and to evaluate the impact of antimicrobial resistance on outcome in adult patients with invasive pneumococcal disease (IPD). METHODS: A post hoc analysis of an observational cohort study on community-acquired pneumococcal infections was conducted and a total of 136 adult patients with IPD were analyzed in this study. RESULTS: Pneumonia was the most common type of infection (n = 84, 61.8 %), followed by primary bacteremia (n = 15, 11.0 %) and meningitis (n = 15, 11.0 %). One hundred and three patients (75.7 %) had concomitant pneumococcal bacteremia. The overall 30-day mortality rate was 26.5 % (36/136), and factors associated with 30-day mortality were corticosteroid use, presentation with septic shock, and development of acute respiratory distress syndrome (ARDS) (all P < 0.05). While penicillin and erythromycin resistance were associated with a lower mortality, an association between levofloxacin resistance and increased mortality was found in the univariate analysis; however, statistical significance was not reached (P = 0.083). Multivariable analysis showed that presentation with septic shock, corticosteroid use, development of ARDS, and levofloxacin resistance were independent factors associated with 30-day mortality. Of the five patients with IPD caused by levofloxacin-resistant Streptococcus pneumoniae, three (60 %) died within 30 days of diagnosis. CONCLUSION: Levofloxacin resistance was associated with increased mortality, along with septic shock, prior use of corticosteroids, and development of ARDS, in adult patients with IPD. Our data suggest that the emergence of levofloxacin resistance among invasive pneumococcal isolates is now becoming a challenge for clinicians managing community-acquired bacterial infections.

Bacterial phenylalanine and phenylacetate catabolic pathway revealed.

Aromatic compounds constitute the second most abundant class of organic substrates and environmental pollutants, a substantial part of which (e.g., phenylalanine or styrene) is metabolized by bacteria via phenylacetate. Surprisingly, the bacterial catabolism of phenylalanine and phenylacetate remained an unsolved problem. Although a phenylacetate metabolic gene cluster had been identified, the underlying biochemistry remained largely unknown. Here we elucidate the catabolic pathway functioning in 16% of all bacteria whose genome has been sequenced, including Escherichia coli and Pseudomonas putida. This strategy is exceptional in several aspects. Intermediates are processed as CoA thioesters, and the aromatic ring of phenylacetyl-CoA becomes activated to a ring 1,2-epoxide by a distinct multicomponent oxygenase. The reactive nonaromatic epoxide is isomerized to a seven-member O-heterocyclic enol ether, an oxepin. This isomerization is followed by hydrolytic ring cleavage and beta-oxidation steps, leading to acetyl-CoA and succinyl-CoA. This widespread paradigm differs significantly from the established chemistry of aerobic aromatic catabolism, thus widening our view of how organisms exploit such inert substrates. It provides insight into the natural remediation of man-made environmental contaminants such as styrene. Furthermore, this pathway occurs in various pathogens, where its reactive early intermediates may contribute to virulence.

DNA recognition and induced genome modification by a hydroxymethyl-γ tail-clamp peptide nucleic acid.

Peptide nucleic acids (PNAs) can target and stimulate recombination reactions in genomic DNA. We have reported that γPNA oligomers possessing the diethylene glycol γ-substituent show improved efficacy over unmodified PNAs in stimulating recombination-induced gene modification. However, this structural modification poses a challenge because of the inherent racemization risk in O-alkylation of the precursory serine side chain. To circumvent this risk and improve γPNA accessibility, we explore the utility of γPNA oligomers possessing the hydroxymethyl-γ moiety for gene-editing applications. We demonstrate that a γPNA oligomer possessing the hydroxymethyl modification, despite weaker preorganization, retains the ability to form a hybrid with the double-stranded DNA target of comparable stability and with higher affinity than that of the diethylene glycol-γPNA. When formulated into poly(lactic-co-glycolic acid) nanoparticles, the hydroxymethyl-γPNA stimulates higher frequencies (≥ 1.5-fold) of gene modification than the diethylene glycol γPNA in mouse bone marrow cells.

Cholesterol degradation by Gordonia cholesterolivorans.

This paper reports physiological and genetic data about the type strain Gordonia cholesterolivorans, a strain that is able to degrade steroid compounds containing a long carbon side chain such as cholesterol (C(27)), cholestenone (C(27)), ergosterol (C(28)), and stigmasterol (C(29)). The length of the carbon side chain appears to be of great importance for this bacterium, as the strain is unable to grow using steroids with a shorter or nonaliphatic carbon side chain such as cholic acid (C(24)), progesterone (C(21)), testosterone, androsterone, 4-androstene-3,17-dione (all C(19)), and further steroids. This study also demonstrates that the degradation of cholesterol is a quite common feature of the genus Gordonia by comparing Gordonia cholesterolivorans with some other species of this genus (e.g., G. sihwensis, G. hydrophobica, G. australis, and G. neofelifaecis). Pyrosequencing of the genome of G. cholesterolivorans led to the identification of two conventional cholesterol oxidase genes on an 8-kb and a 12.8-kb genomic fragment with genetic organizations that are quite unique as compared to the genomes of other cholesterol-degrading bacteria sequenced so far. The identified two putative cholesterol oxidases of G. cholesterolivorans are both intracellularly acting enzymes of the class I type. Whereas one of these two cholesterol oxidases (ChoOx-1) shows high identity with an oxidoreductase of the opportunistic pathogen G. bronchialis and is not transcribed during growth with cholesterol, the other one (ChoOx-2) appears phylogenetically closer to cholesterol oxidases from members of the genus Rhodococcus and is transcribed constitutively. By using targeted gene disruption, a G. cholesterolivorans ChoOx-2 gene mutant strain that was unable to grow with steroids was obtained.

Adjuvant Therapy for High-risk Endometrial Carcinoma.

About one-fifth of endometrial cancers are 'high risk', which carries a poorer prognosis. Management strategies to optimise their survival have been under investigation for many years. Despite recent advances, their overall survival remains relatively poor. The definition of high risk in endometrial cancers has been based on clinicopathological factors until recently, when molecular profiling has shown greater discrimination. There is, however, poor correlation between traditional clinicopathological factors and their molecular profile. This is the subject of ongoing trials to better individualise adjuvant post-hysterectomy treatment. The management of high-risk tumours is traditionally based on surgery followed by radiotherapy, despite no proven overall survival benefit in early stages. The place of chemotherapy remains under investigation, with recent trials showing benefit in more advanced stages. The Post Operative Radiation Therapy in Endometrial Carcinoma (PORTEC) and Gynecologic Oncology Group trials support the use of chemoradiation and chemotherapy for stage III and adverse histological subgroups. In addition, there is now early evidence of correlation between benefit from adjuvant chemotherapy based on molecular alterations in the tumour cells. In this review, we look at the current evidence on management strategies in the evolving era of molecular diagnosis and stratification.

Functional magnetic resonance studies in eating behavior disorders.

Eating behavior disorders (EBD) constitute a serious somatic and psychiatric condition that occurs mainly in adolescent and young adult women and is characterized by a persistent desire to be extremely thin, pathologic fear of gaining weight and distortion of body perception. From a neurobiological vantage point, it has been suggested that alterations in some neural systems of these patients may exist, either as a cause or effect of their condition. In recent years various research studies have been conducted with the aim of identifying underlying brain disorders in EBD. The purpose of this article was to review the main findings obtained in neuroimaging studies, including PET, SPECT, magnetic resonance spectroscopy (MRS), focusing mainly on functional magnetic resonance imaging (fMRI). Some alterations and changes in metabolism and blood perfusion that accompany the neuroimaging findings will be reviewed, as well as studies designed to determine whether these alterations persist after recovery from the disease.

Prospective multinational serosurveillance study of Bordetella pertussis infection among 10- to 18-year-old Asian children and adolescents.

OBJECTIVES: Bordetella pertussis continues to cause outbreaks worldwide. To assess the role of children and adolescent in transmission of pertussis in Asia, we performed a multinational serosurveillance study. METHODS: From July 2013 to June 2016, individuals aged 10 to 18 years who had not received any pertussis-containing vaccine within the prior year were recruited in 10 centres in Asia. Serum anti-pertussis toxin (PT) IgG was measured by ELISA. Demographic data and medical histories were obtained. In the absence of pertussis immunization, anti-PT IgG ≥62.5 IU/mL was interpreted as B. pertussis infection within 12 months prior, among them levels ≥125 IU/mL were further identified as infection within 6 months. RESULTS: A total of 1802 individuals were enrolled. Anti-PT IgG geometric mean concentration was 4.5, and 87 (4.8%) individuals had levels ≥62.5 IU/mL; among them, 73 (83.9%) had received three or more doses of pertussis vaccine before age 6 years. Of 30 participants with persistent cough during the past 6 months, one (3.3%) had level ≥125 IU/mL. There was no significant difference in proportions with anti-PT IgG ≥62.5 IU/mL among age groups (13-15 vs. 10-12 years, 16-18 vs. 10-12 years), between types of diphtheria, pertussis and tetanus (DTP; whole cell vs. acellular), number of doses before age 6 years within the DTP whole-cell pertussis vaccine (five vs. four doses) or acellular pertussis vaccine (five vs. four doses) and history of persistent cough during the past 6 months (yes vs. no). CONCLUSIONS: There is significant circulation of B. pertussis amongst Asian children and adolescents, with one in 20 having serologic evidence of recent infection regardless of vaccination background.

Low cost in-house PCR for the routine diagnosis of extra-pulmonary tuberculosis.

SETTING: Conventional methods for the identification of mycobacteria are slow and labour intensive. DNA amplification methods offer rapid sensitive and specific diagnosis. OBJECTIVE: To determine the feasibility of an in-house polymerase chain reaction (PCR) method to detect Mycobacterium tuberculosis in clinical samples. DESIGN: The present study focused mainly on diagnosing extra-pulmonary tuberculosis (EPTB) using an in-house PCR method in 465 clinical samples. This study also compared the efficacy of a standard phenol-chloroform (PC) extraction procedure and the guanidine thiocyanate with diatomaceous silica (GTCS) method of DNA extraction and purification. A subsample of patients was used for the validation of results based on the final diagnosis. RESULTS: Among 373 patients with suspected EPTB, 75 specimens were positive by PCR, four by microscopy and six by culture. Of the 25 PCR-positive patients, 95% had a final diagnosis of TB. Globally, the GTCS method was found to be superior to the PC method for DNA extraction and removal of inhibitors from clinical specimens. CONCLUSION: The DNA amplification method was found to be significantly more sensitive and rapid compared to culture and microscopy for a reliable final diagnosis of EPTB.

Factors that predict survival in patients with cirrhosis considered for liver transplantation.

OBJECTIVE: To identify prognostic factors for survival at 6 and 12 months in patients evaluated for liver transplantation using Child-Pugh (CP) classification and the Model for End-Stage Liver Disease (MELD) score. METHODS: We evaluated 144 patients with cirrhosis who were candidates for liver transplantation. We excluded patients with hepatocellular carcinoma, recent liver recipients, and patients who died because of factors unrelated to liver disease. The studied variables were age and sex; prothrombin time; platelet count; albumin, cholesterol, bilirubin, creatinine, and serum sodium concentrations; CP classification and MELD score; and the presence of ascites, encephalopathy, hepatorenal syndrome, spontaneous bacterial peritonitis, and previous variceal bleeding. Data were processed using statistical software (SPSS version 13.0). RESULTS: Of the 144 patients, 27 (18.7%) did not survive because of complications of liver disease. Univariate analysis showed the most significant factors to be sex, prothrombin time, bilirubin and albumin levels; ascites, encephalopathy, CP classification, and MELD score. At Cox regression analysis, only CP classification proved to be a valid predictor of survival in our cohort. The lowest survival according to CP classification at 6 and 12 months corresponded to stage C and to MELD scores higher than 15. CONCLUSIONS: Child-Pugh classification is an independent prognostic factor for recipient survival. Stage C in the CP classification and a MELD score higher than 15 were strongly related to worse survival. Both scores must be taken into consideration for adequate evaluation of liver transplantation for candidates.

[n-3 and n-6 fatty acids in plasma at birth and one year of age and relationship with feeding]. / Acidos grasos n-3 y n-6 en plasma al nacer y al año de edad y relación con el tipo de alimentación.

AIM: Compare the variations of long-chain polyunsaturated fatty acids (LCPUFA) levels at birth and at the first year of age in children on extended breast-feeding, medium term breast-feeding and formula feeding. PATIENTS: 77 healthy term infants divided in three groups: A (N=25): extended breast-feeding (more than 6 months), B (N=26): medium term breast-feeding (more than 3 and less than 5 months) and C (N=26): exclusive formula feeding. Fatty acids in plasma were measured at birth and at the first year of age. RESULTS: There were no differences in the levels at birth. However, there is a significant decrease in the proportion of the main LCPUFA, docosahexaenoic acid (DHA) and arachidonic acid (AA), between birth and the first year of age. At one year of age, the percentage of DHA in Group A differs significantly between the other two: 2.46+/-0.84 vs. 1.80+/-0.48 and 1.89+/-0.75 (p<0.01). CONCLUSIONS: 1. At birth, there are no differences in LCPUFA. 2. A significant decrease in the main LCPUFA is observed with age. 3. The extended breast-feeding group shows higher DHA levels than the other two. Therefore, breast-feeding for more than 6 months is required to achieve higher plasma DHA values.

Shape selective bifacial recognition of double helical DNA.

An impressive array of antigene approaches has been developed for recognition of double helical DNA over the past three decades; however, few have exploited the 'Watson-Crick' base-pairing rules for establishing sequence-specific recognition. One approach employs peptide nucleic acid as a molecular reagent and strand invasion as a binding mode. However, even with integration of the latest conformationally-preorganized backbone design, such an approach is generally confined to sub-physiological conditions due to the lack of binding energy. Here we report the use of a class of shape-selective, bifacial nucleic acid recognition elements, namely Janus bases, for targeting double helical DNA or RNA. Binding occurs in a highly sequence-specific manner under physiologically relevant conditions. The work may provide a foundation for the design of oligonucleotides for targeting the secondary and tertiary structures of nucleic acid biopolymers.

Complexity of the early genetic response to growth factors in mouse fibroblasts.

Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.