Chromosome jumping from D4S10 (G8) toward the Huntington disease gene.

The gene for Huntington disease (HD) has been localized to the distal portion of the short arm of human chromosome 4 by linkage analysis. Currently, the two closest DNA markers are D4S10 (G8), located approximately equal to 3 centimorgans centromeric to HD, and D4S43 (C4H), positioned 0-1.5 centimorgans from HD. In an effort to move closer to the HD gene, with the eventual goal of identifying the gene itself, we have applied the technique of chromosome jumping to this region. A 200-kilobase jumping library has been constructed, and a jump from D4S10 has been obtained and its approximate distance verified by pulsed field gel electrophoresis. Two restriction fragment length polymorphisms have been identified at the jump locus, which is denoted D4S81. Linkage analysis of previously identified recombinants between D4S10 and HD or D4S10 and D4S43 shows that in two of five events the jump has crossed the recombination points. This unequivocally orients D4S10 and D4S81 on the chromosome, provides additional markers for HD, and suggests that recombination frequency in this region of chromosome 4 may be increased, so that the physical distance from D4S10 to HD may not be as large as originally suspected.     

Prognostic indicators in patients with the acquired immune deficiency syndrome (AIDS) and respiratory infection

The records of 63 patients with acquired immune deficiency syndrome (AIDS) and respiratory infection comprising 78 hospitalizations over a 36-month period were reviewed to ascertain the etiology of the respiratory infection and to identify the factors influencing short-term survival. Pneumocystis carinii pneumonia (PCP) was diagnosed on 56 occasions in 46 patients. Fifty percent of patients with PCP died with respiratory failure; of these, all but 1 were diagnosed using fiber-optic bronchoscopy. In 18 patients in whom PCP was not identified by bronchoscopy, the in-hospital mortality was 17%. Of the clinical and laboratory findings on admission, only the arterial PO2 and the alveolar-arterial PO2 (AaPO2) difference were significantly different between the survivors and nonsurvivors. In patients with PCP and a AaPO2 greater than 60 mm Hg, 92% died. The demonstration of P. carinii by fiber-optic bronchoscopy and the presence of markedly abnormal gas exchange are associated with high in-hospital mortality.     

Long-read sequencing revealed cooccurrence,host range,and potential mobility of antibiotic resistome in cow feces

While it is well recognized that the environmental resistome is global, diverse, and augmented by human activities, it has been difficult to assess risk because of the inability to culture many environmental organisms, and it is difficult to evaluate risk from current sequence-based environmental methods. The four most important criteria to determine risk are whether the antibiotic-resistance genes (ARGs) are a complete, potentially functional complement; if they are linked with other resistances; whether they are mobile; and the identity of their host. Long-read sequencing fills this important gap between culture and short sequence-based methods. To address these criteria, we collected feces from a ceftiofur-treated cow, enriched the samples in the presence of antibiotics to favor ARG functionality, and sequenced long reads using Nanopore and PacBio technologies. Multidrug-resistance genes comprised 58% of resistome abundance, but only 0.8% of them were plasmid associated; fluroquinolone-, aminoglycoside-, macrolide-lincosamide-streptogramin (MLS)-, and β-lactam–resistance genes accounted for 2.7 to 12.3% of resistome abundance but with 19 to 78% located on plasmids. A variety of plasmid types were assembled, some of which share low similarity to plasmids in current databases. Enterobacteriaceae were dominant hosts of antibiotic-resistant plasmids; physical linkage of extended-spectrum β-lactamase genes (CTX-M, TEM, CMY, and CARB) was largely found with aminoglycoside-, MLS-, tetracycline-, trimethoprim-, phenicol-, sulfonamide-, and mercury-resistance genes. A draft circular chromosome of Vagococcus lutrae was assembled; it carries MLS-, tetracycline- (including tetM and tetL on an integrative conjugative element), and trimethoprim-resistance genes flanked by many transposase genes and insertion sequences, implying that they remain transferrable.

Antibiotic resistance is one of the greatest global public health crises, as more than 700,000 people die from antibiotic resistance-associated disease every year, and the number is predicted to rise dramatically if radical actions are not taken (1). Studies have shown that many clinical antibiotic-resistant genes (ARGs), such as the extended-spectrum β-lactamase (ESBL) CTX-M and the quinolone-resistance gene qnr, have direct links to the environmental resistome (2, 3). The rapid emergence of ARGs in Acinetobacter, making it one of the most difficult gram-negative bacteria to treat, likely acquired its resistance from Pseudomonas, Salmonella, or Escherichia coli mediated by environmental reservoirs (4). In accord with the “One Health” concept, it’s critical to understand the mobility risk of environmental ARGs, as well as their transmission routes to clinical settings (5).The diversity and abundance of ARGs have been well documented in various contexts, such as soils (6), animal feces (7, 8), wastewater sludge (9), and aquatic ecosystems (10), and so forth. ARGs in livestock and poultry manures are particularly noticeable due to the use of antibiotics in animal farming industry (11). Studies have demonstrated that antibiotics and some metals can increase abundance of antibiotic-resistant bacteria (ARB), ARGs, and metal-resistance genes (MRGs) (12). For example, oxytetracycline treatment significantly boosted tetracycline-resistant coliform bacteria in pig manure (13). The long-term presence of antibiotics in animal manure imposes selection on microbes, which likely means functional antibiotic resistance and higher risk of horizontal gene transfer (HGT) compared to other contexts (14). Moreover, large populations of human commensals in animal manure also increases the possibilities for ARG transfer to human-associated pathogens (15).In recent years valuable insights have been gained about environmental resistomes with qPCR and the next-generation sequencing. For example, diverse and high abundance of ARGs were identified in pig manure from three distantly located farms in China with high-throughput qPCR, and further analysis showed positive correlation between ARG abundance and heavy metal concentrations (16). Furthermore, metagenomic sequencing improved our understanding of the antibiotic resistome, mobile genetic elements (MGEs), and their microbial contexts in environmental samples. Widespread cooccurrence patterns of different classes of ARGs and MRGs have been revealed by metagenomic and network analyses (17, 18). However, most studies have estimated ARG risk by abundance quantification of ARG fragments and not the whole gene, nor whether other resistance genes are linked or their potential for transfer, features central to risk.It is now well recognized that risk of ARGs relies on their mobility and host contexts rather than simply their high diversity or abundance (19). MGEs—including plasmids, transposons, and integrative conjugative elements (ICEs)—play key roles in ARG propagation through HGT. ARGs on plasmids suggests higher transfer potential to pathogens, thus posing a larger threat to public health (20). Besides antibiotics, heavy metals, and biocides can also facilitate the enrichment and spread of ARGs and ARBs (21), hence their cooccurrence pattern can provide insights into coselection risks. The third-generation sequencing technologies, such as PacBio and Nanopore, can produce up to hundreds of kilobase reads and provide new opportunities for estimating ARG cooccurrence, mobility, and host range. For example, the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58 was resolved using Nanopore sequencing (22). The transfer of a tetracycline-resistance–carrying plasmid was observed from Staphylococcus haemolyticus to Staphylococcus aureus by PacBio sequencing (23). Most studies on ARG mobility, however, are conducted on cultured isolates. An approach is needed to assess the mobility of environmental resistomes and their potential risk.Ceftiofur is a third-generation cephalosporin antibiotic approved by the US Food and Drug Administration for therapeutic use in food animals and is widely used in cattle to treat respiratory diseases, foot rot, metritis, and mastitis (24). Resistance to ceftiofur also causes ineffectiveness to many clinically important antibiotics, which are critical to human health (24). Cattle manure is usually recycled to agricultural fields, representing a potential path of ARGs from manure to soils, ground water, and food systems (25, 26). Here, we collected feces from a dairy cow treated with ceftiofur for a uterine infection after calving, a standard treatment for metritis and known to increase the proportion of ceftiofur-resistant E. coli in the manure (27). Our objectives were to: 1) determine the genetic location (chromosomal, ICE-associated, and plasmidic) and hosts of different classes of ARGs; 2) define complete/near-complete plasmids in manure and infer transferability; 3) determine the linkage pattern of ARGs, MRGs, and biocide-resistance genes (BRGs); and 4) define a long-read sequencing method for environmental resistome evaluation. This approach fills the gap between culture-based isolate analyses and sequencing-based surveys and hence improves the ability to assess risk of environmental resistomes.     

Conversion of a 3-desoxysteroid to 3-desoxyestrogen by human placental aromatase.

Human placental aromatase is a cytochrome P-450 enzyme system which converts androgens to estrogens by three successive oxidative reactions. The first two steps have been shown to be hydroxylations at the androgen 19-carbon, but the third step remains unknown. A leading theory for the third step involves ferric peroxide attack on the 19-oxo group to produce a 19,19-hydroxyferric peroxide intermediate and subsequent collapse to estrogen. We had previously developed a nonenzymatic peroxide model reaction which was based on the above-mentioned theory, and we demonstrated the importance of 3-ketone enolization in facilitating aromatization. This study discusses the synthesis and nonenzymatic and enzymatic study of a 3-desoxy-2,4-diene-19-oxo androgen analogue. This compound was found to be a potent nonenzymatic model substrate and competitive inhibitor of aromatase (Ki = 73 nM). Furthermore, in an unprecedented event, this compound served as a substrate for aromatase, with conversion to the corresponding 3-desoxyestrogen.     

Controlled and behaviorally relevant levels of oral ethanol intake in rhesus macaques using a flavorant-fade procedure

BACKGROUND: Flavorant-fading procedures can initiate and maintain oral ethanol intake in rodents. The present study developed a similar procedure to achieve controlled and behaviorally relevant levels of ethanol intake in monkeys. METHODS: Male rhesus macaques (N = 13) were initially given the opportunity to consume 0.5 g/kg of a 1% (w/v) ethanol plus 4% (w/v) Tang solution in 1-hr limited-access sessions without the requirement of an operant response. Once consumption was stable at a particular concentration (%) and/or amount (g/kg), animals were given access to higher concentrations and/or amounts of ethanol. Animals were tested on a bimanual motor skill (BMS) task 20 and 90 min after consumption to assess behavioral impairment. Blood alcohol levels (BALs) were assessed after a session in which animals had the opportunity to consume up to 3.0 g/kg of 6% (w/v) ethanol. RESULTS: The gradual fading up of higher concentrations and amounts of ethanol resulted in controlled and robust levels (>2.0 g/kg) of ethanol intake in half of the subjects. Increasing the concentration of the sweetener from 4 to 6% (w/v) was effective in initiating consumption in three animals. Two monkeys required the additional step of presenting the increased-sweetener solutions after a meal (postprandial consumption) to initiate significant ethanol intake. Animals were significantly impaired on the BMS task after consumption of 2.0, 2.5, and 3.0 g/kg of ethanol. Individual consumption ranging from 0.8 to 3.0 g/kg of ethanol produced BALs of 18 to 269 mg/dl. CONCLUSIONS: The flavorant-fading procedure was effective in producing behaviorally relevant levels of ethanol consumption in rhesus macaques. This model facilitated a randomized-dose procedure to determine the behavioral effects of 0.5 to 3.0 g/kg of ethanol. This procedure therefore is of significant utility in determining behavioral or physiologic effects of specific doses of consumed ethanol in monkeys.     

Characterization of invasive Neisseria meningitidis from Atlantic Canada, 2009 to 2013: With special reference to the nonpolysaccharide vaccine targets (PorA,factor H binding protein,Neisseria heparin-binding antigen and Neisseria adhesin A)

BACKGROUND:Serogroup B Neisseria meningitidis (MenB) has always been a major cause of invasive meningococcal disease (IMD) in Canada. With the successful implementation of a meningitis C conjugate vaccine, the majority of IMD in Canada is now caused by MenB.OBJECTIVE:To investigate IMD case isolates in Atlantic Canada from 2009 to 2013. Data were analyzed to determine the potential coverage of the newly licensed MenB vaccine.METHODS:Serogroup, serotype and serosubtype antigens were determined from IMD case isolates. Clonal analysis was performed using multilocus sequence typing. The protein-based vaccine antigen genes were sequenced and the predicted peptides were investigated.RESULTS:The majority of the IMD isolates were MenB (82.5%, 33 of 40) and, in particular, sequence type (ST)-154 B:4:P1.4 was responsible for 47.5% (19 of 40) of all IMD case isolates in Atlantic Canada. Isolates of this clone expressed the PorA antigen P1.4 and possessed the nhba genes encoding for Neisseria heparin-binding antigen peptide 2, which together matched exactly with two of the four components of the new four-component meningococcal B vaccine. Nineteen MenB isolates had two antigenic matches, another five MenB and one meningitis Y isolate had one antigenic match. This provided 75.8% (25 of 33) potential coverage for MenB, or a 62.5% (25 of 40) overall potential coverage for IMD.CONCLUSION:From 2009 to 2013, IMD in Atlantic Canada was mainly caused by MenB and, in particular, the B:4:P1.4 ST-154 clone, which accounted for 47.5% of all IMD case isolates. The new four-component meningococcal B vaccine appeared to offer adequate coverage against MenB in Atlantic Canada.