The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis.

In order to validate a gel free quantitative proteomics assay for the model methylotrophic bacterium Methylobacterium extorquens AM1, we examined the M. extorquens AM1 proteome under single carbon (methanol) and multi-carbon (succinate) growth, conditions that have been studied for decades and for which extensive corroborative data have been compiled. In total, 4,447 proteins from a database containing 7,556 putative ORFs from M. extorquens AM1 could be identified with two or more peptide sequences, corresponding to a qualitative proteome coverage of 58%. Statistically significant non-zero (log2 scale) differential abundance ratios of methanol/succinate could be detected for 317 proteins using summed ion intensity measurements and 585 proteins using spectral counting, at a q-value cut-off of 0.01, a measure of false discovery rate. The results were compared to recent microarray studies performed under equivalent chemostat conditions. The M. extorquens AM1 studies demonstrated the feasibility of scaling up the multidimensional capillary HPLC tandem mass spectrometry approach to a prokaryotic organism with a proteome more than three times the size of microbes we have investigated previously, while maintaining a high degree of proteome coverage and reliable quantitative abundance ratios.

There is a growing realization that graduate education in the biomedical sciences is successful at teaching students how to conduct research but falls short in preparing them for a diverse job market, communicating with the public, and remaining versatile scientists throughout their careers. Major problems with graduate level education today include overspecialization in a narrow area of science without a proper grounding in essential critical thinking skills. Shortcomings in education may also contribute to some of the problems of the biomedical sciences, such as poor reproducibility, shoddy literature, and the rise in retracted publications. The challenge is to modify graduate programs such that they continue to generate individuals capable of conducting deep research while at the same time producing more broadly trained scientists without lengthening the time to a degree. Here we describe our first experiences at Johns Hopkins and propose a manifesto for reforming graduate science education.

To study the in vivo recruitment of different fiber types and their metabolic properties, 31P-nuclear magnetic resonance spectroscopy (31P-NMRS) of the human calf muscle was performed in seven normal sedentary subjects. In the exhaustive exercise protocol used, the work load was increased every minute during 5 min. This resulted in a prominent split of the Pi resonance in all subjects, indicating pH compartmentation in the muscles studied. From the chemical shift of the Pi peaks relative to phosphocreatine (PCr) at the end of the exercise, intracellular pH (pHi) averaged 6.92 +/- 0.05 (SD) in compartment 1 and 6.23 +/- 0.15 in compartment 2. The recovery of both Pi resonances after exercise could be followed easily in five of these subjects. The recovery rate of the Pi peak is a good estimate of the oxidative metabolism at the end of the exercise. A monoexponential regression analysis showed that the mean initial recovery rate S0 was 2.49 +/- 0.17%/s in compartment 1 and only 0.87 +/- 0.12%/s in compartment 2, indicating aerobic function three times higher in compartment 1 at the end of exercise. The mean relative ATP fraction dropped significantly (P less than 0.001), from 20.0 +/- 1.0% of the total 31P signal integral before exercise to 14.0 +/- 1.6% at the end of exercise. The simultaneous visualization of two compartments, in good order, one with high pHi and fast recovery and another with low pHi and slow recovery, is rationalized by the different metabolic behavior of type I and II fibers in human calf muscle in response to exhaustive exercise. This study demonstrates that 31P-NMRS is an excellent noninvasive procedure to quantify aerobic metabolism in both fiber types simultaneously.

Many doctoral curricula aim to produce narrowly focused researchers rather than critical thinkers. That can and must change, says Gundula Bosch. U nder pressure to turn out productive lab members quickly, many PhD programmes in the biomedical sciences have shortened their courses, squeezing out opportunities for putting research into its wider context. Consequently, most PhD curricula are unlikely to nurture the big thinkers and creative problem-solvers that society needs. That means students are taught every detail of a microbe's life cycle but little about the life scientific. They need to be taught to recognize how errors can occur. Trainees should evaluate case studies derived from flawed real research, or use interdisciplinary detective games to find logical fallacies in the literature. Above all, students must be shown the scientific process as it is-with its limitations and potential pitfalls as well as its fun side, such as serendipitous discoveries and hilarious blunders. This is exactly the gap that I am trying to fill at Johns Hopkins University in Baltimore, Maryland, where a new graduate science programme is entering its second year. Microbiologist Arturo Casadevall and I began pushing for reform in early 2015, citing the need to put the philosophy back into the doctorate of philosophy: that is, the 'Ph' back into the PhD. We call our programme R3, which means that our students learn to apply rigour to their design and conduct of experiments; view their work through the lens of social responsibility; and to think critically, communicate better, and thus improve reproducibility. Although we are aware of many innovative individual courses developed along these lines, we are striving for more-comprehensive reform. Our offerings are different from others at the graduate level. We have critical-thinking assignments in which students analyse errors in reasoning in a New York Times opinion piece about 'big sugar' , and the ethical implications of the arguments made in a New Yorker piece by surgeon Atul Gawande entitled 'The Mistrust of Science'. Our courses on rigorous research, scientific integrity, logic, and mathematical and programming skills are integrated into students' laboratory and fieldwork. Those studying the influenza virus, for example, work with reallife patient data sets and wrestle with the challenges of applied statistics. A new curriculum starts by winning allies. Both students and faculty members must see value in moving off the standard track. We used informal interviews and focus groups to identify areas in which students and faculty members saw gaps in their training. Recurring themes included the inability to apply theoretical knowledge in statistical tests in the laboratory, frequent mistakes in choosing an appropriate set of experimental controls, and significant difficulty in explaining work to non-experts. Introducing our programme to colleagues in the Johns Hopkins life-sciences departments was even more sensitive. I was startled by the oft-expressed opinion that scientific produ...