RESUMEN
Despite an increasingly detailed picture of the molecular mechanisms of bacteriophage (phage)-bacterial interactions, we lack an understanding of how these interactions evolve and impact disease within patients. In this work, we report a year-long, nationwide study of diarrheal disease patients in Bangladesh. Among cholera patients, we quantified Vibrio cholerae (prey) and its virulent phages (predators) using metagenomics and quantitative polymerase chain reaction while accounting for antibiotic exposure using quantitative mass spectrometry. Virulent phage (ICP1) and antibiotics suppressed V. cholerae to varying degrees and were inversely associated with severe dehydration depending on resistance mechanisms. In the absence of antiphage defenses, predation was "effective," with a high predator:prey ratio that correlated with increased genetic diversity among the prey. In the presence of antiphage defenses, predation was "ineffective," with a lower predator:prey ratio that correlated with increased genetic diversity among the predators. Phage-bacteria coevolution within patients should therefore be considered in the deployment of phage-based therapies and diagnostics.
Asunto(s)
Bacteriófagos , Cólera , Variación Genética , Vibrio cholerae , Cólera/microbiología , Vibrio cholerae/genética , Vibrio cholerae/virología , Bacteriófagos/genética , Bacteriófagos/fisiología , Humanos , Bangladesh , Antibacterianos/uso terapéutico , Índice de Severidad de la Enfermedad , Adulto , MetagenómicaRESUMEN
Despite an increasingly detailed picture of the molecular mechanisms of phage-bacterial interactions, we lack an understanding of how these interactions evolve and impact disease within patients. Here we report a year-long, nation-wide study of diarrheal disease patients in Bangladesh. Among cholera patients, we quantified Vibrio cholerae (prey) and its virulent phages (predators) using metagenomics and quantitative PCR, while accounting for antibiotic exposure using quantitative mass spectrometry. Virulent phage (ICP1) and antibiotics suppressed V. cholerae to varying degrees and were inversely associated with severe dehydration depending on resistance mechanisms. In the absence of anti-phage defenses, predation was 'effective,' with a high predator:prey ratio that correlated with increased genetic diversity among the prey. In the presence of anti-phage defenses, predation was 'ineffective,' with a lower predator:prey ratio that correlated with increased genetic diversity among the predators. Phage-bacteria coevolution within patients should therefore be considered in the deployment of phage-based therapies and diagnostics.
RESUMEN
The application of genetic and biochemical techniques in yeast has informed our knowledge of transcription in mammalian cells. Such systems have allowed investigators to determine whether a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in the nature of the transcription factors essential for transcription by Pol I in yeast and mammalian cells, and yeast RNA polymerase I contains 14 subunits while mammalian polymerase contains 13 subunits. We previously reported the adaptation of the auxin-dependent degron that enabled a combination of a "genetics-like" approach and biochemistry to study mammalian rDNA transcription. Using this system, we studied the mammalian orthologue of yeast RPA34.5, PAF49, and found that it is essential for rDNA transcription and cell division. The auxin-induced degradation of PAF49 induced nucleolar stress and the accumulation of P53. Interestingly, the auxin-induced degradation of AID-tagged PAF49 led to the degradation of its binding partner, PAF53, but not vice versa. A similar pattern of co-dependent expression was also found when we studied the non-essential, yeast orthologues. An analysis of the domains of PAF49 that are essential for rDNA transcription demonstrated a requirement for both the dimerization domain and an "arm" of PAF49 that interacts with PolR1B. Further, we demonstrate this interaction can be disrupted to inhibit Pol I transcription in normal and cancer cells which leads to the arrest of normal cells and cancer cell death. In summary, we have shown that both PAF53 and PAF49 are necessary for rDNA transcription and cell growth.
Asunto(s)
Proteínas Portadoras , Proteínas Nucleares , ARN Polimerasa I , Saccharomyces cerevisiae , Animales , Humanos , Ratones , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Ácidos Indolacéticos/metabolismo , Mamíferos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , ARN Polimerasa I/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Centromeres are known to cluster around nucleoli in Drosophila and mammalian cells, but the significance of the nucleoli-centromere interaction remains underexplored. To determine whether the interaction is dynamic under different physiological and pathological conditions, we examined nucleolar structure and centromeres at various differentiation stages using cell culture models and the results showed dynamic changes in nucleolar characteristics and nucleoli-centromere interactions through differentiation and in cancer cells. Embryonic stem cells usually have a single large nucleolus, which is clustered with a high percentage of centromeres. As cells differentiate into intermediate states, the nucleolar number increases and the centromere association decreases. In terminally differentiated cells, including myotubes, neurons, and keratinocytes, the number of nucleoli and their association with centromeres are at the lowest. Cancer cells demonstrate the pattern of nucleoli number and nucleoli-centromere association that is akin to proliferative cell types, suggesting that nucleolar reorganization and changes in nucleoli-centromere interactions may play a role in facilitating malignant transformation. This idea is supported in a case of pediatric rhabdomyosarcoma, in which induced differentiation reduces the nucleolar number and centromere association. These findings suggest active roles of nucleolar structure in centromere function and genome organization critical for cellular function in both normal development and cancer.
Asunto(s)
Nucléolo Celular , Neoplasias , Animales , Nucléolo Celular/metabolismo , Centrómero , Núcleo Celular/metabolismo , Mamíferos , Neoplasias/metabolismoRESUMEN
BACKGROUND: Identifying and treating hearing loss can help improve communication skills, which often leads to improved quality of life. Many people do not seek medical treatment and, therefore, go undiagnosed for an extended period before realizing they have hearing loss. This study presents a self-administered, low-cost, smartphone-based hearing test application (HearTest) to quantify the pure-tone hearing thresholds of a user. The HearTest application can be used with commercially available smartphone devices and an earphone with the mentioned specification. METHODS: Air-conduction-based pure-tone audiometry for the smartphone application was designed and implemented to detect hearing thresholds using a traditional "10 dB down and 5 dB up" approach. Employed smartphone-earphone combination was calibrated with respect to a GSI-61 audiometer and insert earphone ER-3A to maintain clinical standards with the help of subjective testing on 20 normal-hearing (NH) participants. RESULTS: Further subjective testing on 14 participants with NH and retesting on five participants showed that HearTest achieves high-accuracy audiogram within clinically acceptable limits (≤10 dB HL mean difference) when compared with the reference clinical audiometer. Hardware challenges and limitations in air-conduction-based hearing tests through smartphones and ways to improve their accuracy and reliability are discussed. CONCLUSION: The proposed smartphone application provides a simple, affordable, and reliable means for people to learn more about their hearing health without needing access to a formal clinical facility.
Asunto(s)
Calidad de Vida , Teléfono Inteligente , Audiometría de Tonos Puros , Umbral Auditivo , Humanos , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: The global rise in the incidence of non-tuberculosis mycobacterial infections is of increasing concern due their high levels of intrinsic antibiotic resistance. Although integrated viral genomes, called prophage, are linked to increased antibiotic resistance in some bacterial species, we know little of their role in mycobacterial drug resistance. RESULTS: We present here for the first time, evidence of increased antibiotic resistance and expression of intrinsic antibiotic resistance genes in a strain of Mycobacterium chelonae carrying prophage. Strains carrying the prophage McProf demonstrated increased resistance to amikacin. Resistance in these strains was further enhanced by exposure to sub-inhibitory concentrations of the antibiotic, acivicin, or by the presence of a second prophage, BPs. Increased expression of the virulence gene, whiB7, was observed in strains carrying both prophages, BPs and McProf, relative to strains carrying a single prophage or no prophages. CONCLUSIONS: This study provides evidence that prophage alter expression of important mycobacterial intrinsic antibiotic resistance genes and additionally offers insight into the role prophage may play in mycobacterial adaptation to stress.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana , Mycobacterium chelonae/metabolismo , Mycobacterium chelonae/virología , Profagos/fisiología , Factores de Virulencia/metabolismo , Proteínas Bacterianas/genética , Mycobacterium chelonae/efectos de los fármacos , Mycobacterium chelonae/genética , Factores de Virulencia/genéticaRESUMEN
Aqueous microdroplets with a volume of a few femtoliters are an ideal sample size for single-molecule fluorescence experiments. In particular, they enable prolonged measurements to be made on individual molecules that can diffuse freely in the surrounding medium. However, the rapid production of monodisperse droplets in a hydrodynamic flow, such as microfluidic flow focusing, will often involve volumes that are typically too large (>0.5 pL) for single-molecule studies. Desired volumes of a few femtoliters, or smaller, can be produced by either tip streaming or step emulsification in a flow-focusing device; however, in both of these methods, the aqueous droplets are dispersed in a large volume of the continuous phase, where individual droplets can diffuse perpendicular to the flow direction, and the monodispersity of droplet size produced by tip streaming is difficult to sustain for more than transient time scales. We show here that the optimized design and fabrication of microfluidic devices with shallow channel depths can result in the reliable production of stable droplets of a few femtoliters at a high rate in the dripping regime of flow focusing. Furthermore, the generated microdroplets are localized in a two-dimensional plane to enable immediate analysis. We have demonstrated the fluorescence monitoring of single molecules of encapsulated green fluorescent protein. The apparatus is straightfoward, inexpensive, and readily assembled within an ordinary laboratory environment.