Barlow-Hall in vitro Evolution Protocol

The Barlow-Hall method for in vitro evolution is a simple alternative to methods such as DNA shuffling and is particularly applicable to predicting the evolution of target genes in nature. This detailed protocol includes the procedure itself and, as Appendices, describes particularly effective protocols for electroporation and for preparation of electrocompetent cells

Designing antibiotic cycling strategies by determining and understanding local adaptive landscapes

The evolution of antibiotic resistance among bacteria threatens our continued ability to treat infectious diseases. The need for sustainable strategies to cure bacterial infections has never been greater. So far, all attempts to restore susceptibility after resistance has arisen have been unsuccessful, including restrictions on prescribing [1] and antibiotic cycling [2,3]. Part of the problem may be that those efforts have implemented different classes of unrelated antibiotics, and relied on removal of resistance by random loss of resistance genes from bacterial populations (drift). Here, we show that alternating structurally similar antibiotics can restore susceptibility to antibiotics after resistance has evolved. We found that the resistance phenotypes conferred by variant alleles of the resistance gene encoding the TEM {\beta}-lactamase (blaTEM) varied greatly among 15 different {\beta}-lactam antibiotics. We captured those differences by characterizing complete adaptive landscapes for the resistance alleles blaTEM-50 and blaTEM-85, each of which differs from its ancestor blaTEM-1 by four mutations. We identified pathways through those landscapes where selection for increased resistance moved in a repeating cycle among a limited set of alleles as antibiotics were alternated. Our results showed that susceptibility to antibiotics can be sustainably renewed by cycling structurally similar antibiotics. We anticipate that these results may provide a conceptual framework for managing antibiotic resistance. This approach may also guide sustainable cycling of the drugs used to treat malaria and HIV

Ethanol and Reactive Species Increase Basal Sequence Heterogeneity of Hepatitis C Virus and Produce Variants with Reduced Susceptibility to Antivirals

Hepatitis C virus (HCV) exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. In addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism, modify signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence variability of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA (Con1, AJ242652) were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO) that decreases intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of HCV RNA by 4–18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Therefore, alcohol is likely to synergize with virus-induced oxidative/nitrosative stress to modulate the basal mutation rate of HCV. Positive selection induced by alcohol and reactive species may contribute to antiviral resistance

The Gut as Reservoir of Antibiotic Resistance: Microbial Diversity of Tetracycline Resistance in Mother and Infant

The microbiota in the human gastrointestinal tract (GIT) is highly exposed to antibiotics, and may be an important reservoir of resistant strains and transferable resistance genes. Maternal GIT strains can be transmitted to the offspring, and resistances could be acquired from birth. This is a case study using a metagenomic approach to determine the diversity of microorganisms conferring tetracycline resistance (Tcr) in the guts of a healthy mother-infant pair one month after childbirth, and to investigate the potential for horizontal transfer and maternal transmission of Tcr genes. Fecal fosmid libraries were functionally screened for Tcr, and further PCR-screened for specific Tcr genes. Tcr fosmid inserts were sequenced at both ends to establish bacterial diversity. Mother and infant libraries contained Tcr, although encoded by different genes and organisms. Tcr organisms in the mother consisted mainly of Firmicutes and Bacteroidetes, and the main gene detected was tet(O), although tet(W) and tet(X) were also found. Identical Tcr gene sequences were present in different bacterial families and even phyla, which may indicate horizontal transfer within the maternal GIT. In the infant library, Tcr was present exclusively in streptococci carrying tet(M), tet(L) and erm(T) within a novel composite transposon, Tn6079. This transposon belongs to a family of broad host range conjugative elements, implying a potential for the joint spread of tetracycline and erythromycin resistance within the infant's gut. In addition, although not found in the infant metagenomic library, tet(O) and tet(W) could be detected in the uncloned DNA purified from the infant fecal sample. This is the first study to reveal the diversity of Tcr bacteria in the human gut, to detect a likely transmission of antibiotic resistance from mother to infant GITs and to indicate the possible occurrence of gene transfers among distantly related bacteria coinhabiting the GIT of the same individual

Class 1 Integrons in Resistant Escherichia coli and Klebsiella spp., US Hospitals

We examined Escherichia coli and Klebsiella spp. from US hospitals for class 1 integrons. Of 320 isolates, 181 (57%) were positive; association of integrons with resistance varied by drug and organism. Thus, determining integron epidemiology will improve understanding of how antibacterial resistance determinants spread in the United States

High Rate of Mobilization for blaCTX-Ms

The blaCTX-Ms have been mobilized to plasmids more frequently than other class A β-lactamases

Shopping, sex, and lies: Mimong/Sweet Dreams (1936) and the disruptive process of colonial girlhood

In the early Korean film we follow the melodramatic life of an unfaithful housewife. Sweet Dreams situates itself at the heart of the Korean colonial experience with urban Seoul as the backdrop to a narrative of deceit, adultery and consumerism. This article will explore how Sweet Dreams functions both as a warning about the perils of modern womanhood and, simultaneous to this, a vision of consumerist pleasure and delight. This article examines how the actions of lead character Ae-soon constitute a process by which the adult women is rendered girl via her positioning at the locus of female visual pleasure. I use the term girl as a process rather than a static category since, as will be explored, the attributes of girlhood with relation to Sweet Dreams are both expansive and fluid. In this way girlhood can be appropriated for transgressive purposes, not only in terms of a visualization of a desiring femininity, but also as a marker of colonial dissent. I argue that Sweet Dreams uses the interplay between the categories of woman and girl to disrupt the colonial drive towards a productive body in favour of the delights of consumption

The Day-to-Day Impact of Urogenital Aging: Perspectives from Racially/Ethnically Diverse Women

Urogenital symptoms affect up to half of women after menopause, but their impact on women’s day-to-day functioning and wellbeing is poorly understood. Postmenopausal women aged 45 to 80 years reporting urogenital dryness, soreness, itching, or pain during sex were recruited to participate in in-depth focus groups to discuss the impact of their symptoms. Focus groups were homogenous with respect to race/ethnicity and stratified by age (for White or Black women) or language (for Latina women). Transcripts of sessions were analyzed according to grounded theory. Six focus groups were conducted, involving 44 women (16 White, 14 Black, 14 Latina). Five domains of functioning and wellbeing affected by symptoms were identified: sexual functioning, everyday activities, emotional wellbeing, body image, and interpersonal relations. For some participants, symptoms primarily affected their ability to have and enjoy sex, as well as be responsive to their partners. For others, symptoms interfered with everyday activities, such as exercising, toileting, or sleeping. Participants regarded their symptoms as a sign that they were getting old or their body was deteriorating; women also associated symptoms with a loss of womanhood or sexuality. Additionally, participants reported feeling depressed, embarrassed, and frustrated about their symptoms, and expressed reluctance to discuss them with friends, family, or health care providers. Urogenital symptoms can have a marked impact on sexual functioning, everyday activities, emotional wellbeing, body image, and interpersonal relations after menopause. Clinicians may need to question women actively about these symptoms, as many are reluctant to seek help for this problem