A high-throughput screening fluorescence polarization assay for fatty acid adenylating enzymes in Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), encodes for an astonishing 34 fatty acid adenylating enzymes (FadDs), which play key roles in lipid metabolism. FadDs involved in lipid biosynthesis are functionally nonredundant and serve to link fatty acid and polyketide synthesis to produce some of the most architecturally complex natural lipids including the essential mycolic acids as well as the virulence-conferring phthiocerol dimycocerosates, phenolic glycolipids, and mycobactins. Here we describe the systematic development and optimization of a fluorescence polarization assay to identify small molecule inhibitors as potential antitubercular agents. We fluorescently labeled a bisubstrate inhibitor to generate a fluorescent probe/tracer, which bound with a K_D of 245 nM to FadD28. Next, we evaluated assay performance by competitive binding experiments with a series of known ligands and assessed the impact of control parameters including incubation time, stability of the signal, temperature, and DMSO concentration. As a final level of validation the LOPAC1280 library was screened in a 384-well plate format and the assay performed with a Z-factor of 0.75, demonstrating its readiness for high-throughput screening.     

Making secular ‘French’ medical ethics: transcendent morality and patient subjectivities in southern French hospitals

Drawing on ethnographic fieldwork with physicians and nurses working in two state-funded southern French hospitals, this article explores why and how medical care providers connected their everyday deliberations about patient care to what they considered to be distinctively French forms of medical responsibility. Many healthcare professionals saw French medical morality in opposition to ‘Anglo-Saxon’ discourses of individual autonomy and transactional choice. In contrast to such ‘transactionalism’, they insisted that ‘French’ ethics required limits that transcended particular circumstances. And yet it was difficult for doctors and nurses working in secular and increasingly neoliberal hospitals to argue against individual transactionalism in an overtly moral register, one that might appear religious and paternalist. Through a close look at two different cases – one in assisted reproduction and one in palliative care – I show how the language of folk psychoanalysis provided some health professionals with a way out of this impasse. Care providers used pseudo-psychoanalytic accounts of patient subjectivities to depict individuals as incapable of knowing, let alone ‘owning’ or rationally mastering, themselves. This, in turn, suggests that some aspects of French secularity may be far less Protestant and liberal than contemporary anthropological work tends to assume.     

Pelagibacter metabolism of diatom-derived volatile organic compounds imposes an energetic tax on photosynthetic carbon fixation

Volatile organic compounds (VOCs) produced by phytoplankton are molecules with high vapor pressures that can diffuse across cell membranes into the environment, where they become public goods. VOCs likely comprise a significant component of the marine dissolved organic carbon (DOC) pool utilized by microorganisms, but they are often overlooked as growth substrates because their diffusivity imposes analytical challenges. The roles of VOCs in the growth of the photoautotrophic diatom Thalassiosira pseudonana and heterotrophic bacterium Pelagibacter sp. HTCC1062 (SAR11) were examined using co-cultures and proton-transfer reaction time-of-flight mass spectrometry. VOCs at 82 m/z values were produced in the cultures, and the concentrations of 9 of these m/z values changed in co-culture relative to the diatom monoculture. Several of the m/z values were putatively identified, and their metabolism by HTCC1062 was confirmed by measuring ATP production. Diatom carbon fixation rates in co-culture with HTCC1062 were 20.3% higher than the diatom monoculture. Removal of VOCs from the T. pseudonana monoculture using a hydrocarbon trap caused a similar increase in carbon fixation (18.1%). These results show that a wide range of VOCs are cycled in the environment, and the flux of VOCs from phytoplankton to bacterioplankton imposes a large and unexpected tax on phytoplankton photosynthesis.     

Sexual Healthcare Preferences among Gay and Bisexual Men: A Qualitative Study in San Francisco,California

Background

Research on gay and other men who have sex with men''s (G/MSM) preferences for sexual healthcare services focuses largely on HIV testing and to some extent on sexually transmitted infections (STI). This research illustrates the frequency and location of where G/MSM interface with the healthcare system, but it does not speak to why men seek care in those locations. As HIV and STI prevention strategies evolve, evidence about G/MSM''s motivations and decision-making can inform future plans to optimize models of HIV/STI prevention and primary care.

Methods

We conducted a phenomenological study of gay men''s sexual health seeking experiences, which included 32 in-depth interviews with gay and bisexual men. Interviews were transcribed verbatim and entered into Atlas.ti. We conducted a Framework Analysis.

Findings

We identified a continuum of sexual healthcare seeking practices and their associated drivers. Men differed in their preferences for separating sexual healthcare from other forms of healthcare (“fragmentation”) versus combining all care into one location (“consolidation”). Fragmentation drivers included: fear of being monitored by insurance companies, a desire to seek non-judgmental providers with expertise in sexual health, a desire for rapid HIV testing, perceiving sexual health services as more convenient than primary care services, and a lack of healthcare coverage. Consolidation drivers included: a comfortable and trusting relationship with a provider, a desire for one provider to oversee overall health and those with access to public or private health insurance.

Conclusions

Men in this study were likely to separate sexual healthcare from primary care. Based on this finding, we recommend placing new combination HIV/STI prevention interventions within sexual health clinics. Furthermore, given the evolution of the financing and delivery of healthcare services and in HIV prevention, policymakers and clinicians should consider including more primary care services within sexual healthcare settings.     

A FRET-Based Real-Time PCR Assay to Identify the Main Causal Agents of New World Tegumentary Leishmaniasis

In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America.     

The impact of sex on brain responses to smoking cues: a perfusion fMRI study

Background

Anecdotal and clinical theories purport that females are more responsive to smoking cues, yet few objective, neurophysiological examinations of these theories have been conducted. The current study examines the impact of sex on brain responses to smoking cues.

Methods

Fifty-one (31 males) cigarette-dependent sated smokers underwent pseudo- continuous arterial spin-labeled perfusion functional magnetic resonance imaging during exposure to visual smoking cues and non-smoking cues. Brain responses to smoking cues relative to non-smoking cues were examined within males and females separately and then compared between males and females. Cigarettes smoked per day was included in analyses as a covariate.

Results

Both males and females showed increased responses to smoking cues compared to non-smoking cues with males exhibiting increased medial orbitofrontal cortex and ventral striatum/ventral pallidum responses, and females showing increased medial orbitofrontal cortex responses. Direct comparisons between male and female brain responses revealed that males showed greater bilateral hippocampal/amygdala activation to smoking cues relative to non-smoking cues.

Conclusions

Males and females exhibit similar responses to smoking cues relative to non-smoking cues in a priori reward-related regions; however, direct comparisons between sexes indicate that smoking cues evoke greater bilateral hippocampal/amygdalar activation among males. Given the current literature on sex differences in smoking cue neural activity is sparse and incomplete, these results contribute to our knowledge of the neurobiological underpinnings of drug cue reactivity.