Traditionally, proposed molecular mechanisms of fundamental biological processes have been tested against experiment. However, owing to a plethora of reasons-difficulty in designing, carrying out, and interpreting key experiments, use of different experimental models and systems, conduct of studies under widely varying experimental conditions, fineness in distinctions between competing mechanisms, complexity of the scientific issues, and the resistance of some scientists to discoveries that are contrary to popularly held beliefs-this has not solved the problem despite decades of work in the field/s. The author would like to prescribe an alternative way: that of testing competing models/mechanisms for their adherence to scientific laws and principles, and checking for errors in logic. Such tests are fairly commonly carried out in the mathematics, physics, and engineering literature. Further, reported experimental measurements should not be smaller than minimum detectable values for the measurement technique employed and should truly reflect function of the actual system without inapplicable extrapolation. Progress in the biological fields would be greatly accelerated, and considerable scientific acrimony avoided by adopting this approach. Some examples from the fundamental field of ATP synthesis in oxidative phosphorylation (OXPHOS) have been reviewed that also serve to illustrate the approach. The approach has never let the author down in his 35-yr-long experience on biological mechanisms. This change in thinking should lead to a considerable saving of both time and resources, help channel research efforts toward solution of the right problems, and hopefully provide new vistas to a younger generation of open-minded biological scientists.

Folding of the mitochondrial inner membrane (IM) into cristae greatly increases the ATP-generating surface area, S IM, per unit volume but also creates diffusional bottlenecks that could limit reaction rates inside mitochondria. This study explores possible effects of inner membrane folding on mitochondrial ATP output, using a mathematical model for energy metabolism developed by the Jafri group and two- and three-dimensional spatial models for mitochondria, implemented on the Virtual Cell platform. Simulations demonstrate that cristae are micro-compartments functionally distinct from the cytosol. At physiological steady states, standing gradients of ADP form inside cristae that depend on the size and shape of the compartments, and reduce local flux (rate per unit area) of the adenine nucleotide translocase. This causes matrix ADP levels to drop, which in turn reduces the flux of ATP synthase. The adverse effects of membrane folding on reaction fluxes increase with crista length and are greater for lamellar than tubular crista. However, total ATP output per mitochondrion is the product of flux of ATP synthase and S IM which can be two-fold greater for mitochondria with lamellar than tubular cristae, resulting in greater ATP output for the former. The simulations also demonstrate the crucial role played by intracristal kinases (adenylate kinase, creatine kinase) in maintaining the energy advantage of IM folding.

BACKGROUND: The Kai Yu Zhong Yu recipe (KYZY) is a classic herbal formula in traditional Chinese medicine (TCM) that has been used to treat infertility associated with psychological stress for more than three hundred years.
OBJECTIVE: Psychological stress has major impacts on fertility, with variable outcomes depending on the nature, strength, and duration of the stress. Stress can directly disturb ovulation, oocyte quality, maturation, and embryo development. The aim of this study is to investigate the molecular mechanism by which KYZY improves oocyte developmental potential under psychological stress.
METHODS: ICR female mice aged 4-5 weeks were randomly divided into five groups: control, stressed in the chronic unpredictable stress model (CUSM), and stressed plus KYZY treatment at 38.2 g/kg (KYZYH), 19.1 g/kg (KYZYM), or 9.6 g/kg (KYZYL). Ovary function was assessed by measuring serum levels of estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH). Oocyte quality was evaluated in terms of reactive oxygen species (ROS) levels, apoptotic DNA fragmentation, and mitochondria distribution. We used RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) between groups and then further analyzed the DEGs for gene ontology (GO) term enrichment and protein-protein interactions.
RESULTS: Mice in the stressed group had reduced serum E2, LH, and FSH as well as increased ROS levels, increased apoptosis, and disturbed mitochondria distribution in oocytes. Treatment with KYZY at all three doses reversed or ameliorated these negative effects of stress. DEG analysis identified 187 common genes between the two comparisons (stressed vs. control and KYZYM vs. stressed), 33 of which were annotated with six gene ontology (GO)\'s biological process (BP) terms: cell differentiation, apoptosis, ATP synthesis, protein homo-oligomerization, neuron migration, and negative regulation of peptidase activity. Protein-protein interaction network analysis of DEGs identified key hub genes. Notably, the genes Atp5o and Cyc1 were both involved in the ATP synthesis and among the top three hub genes, suggesting that regulation of oocyte mitochondrial electron transport and ATP synthesis is important in the response to stress and also is a possible mechanism of action for KYZY.
CONCLUSIONS: KYZY was effective in ameliorating the adverse effects of stress on oocyte competence, possibly by targeting the mitochondrial respiratory chain and ATP synthase.

Adenylate kinase plays an important role in cellular energy homeostasis by catalysing the interconversion of adenine nucleotides. The goal of present study was to evaluate the contribution of the adenylate kinase reaction to oxidative ATP synthesis by direct measurements of ATP using (31) P NMR spectroscopy. Results show that AMP can stimulate ATP synthesis in the presence or absence of ADP. In particular, addition of 1 mM AMP to the 0.6 mM ADP superfusion system of isolated superfused mitochondria (contained and maintained in agarose beads) led to a 25% increase in ATP synthesis as measured by the increase in βATP signal. More importantly, we show that AMP can support ATP synthesis in the absence of ADP, demonstrated as follows. Superfusion of mitochondria without ADP led to the disappearance of ATP γ, α and β signals and the increase of Pi . Addition of AMP to the medium restored the production of ATP, as demonstrated by the reappearance of γ, α and β ATP signals, in conjunction with a decrease in Pi , which is being used for ATP synthesis. Polarographic studies showed Mg(2+) dependence of this process, confirming the specificity of the adenylate kinase reaction. Furthermore, data obtained from this study demonstrate, for the first time, that different aspects of the adenylate kinase reaction can be evaluated with (31) P NMR spectroscopy. SIGNIFICANCE OF RESEARCH PARAGRAPH: The data generated in the present study indicate that (31) P NMR spectroscopy can effectively be used to study the adenylate kinase reaction under a variety of conditions. This is important because understanding of adenylate kinase function and/or malfunction is essential to understanding its role in health and disease. The data obtained with (31) P NMR were confirmed by polarographic studies, which further strengthens the robustness of the NMR findings. In summary, (31) P NMR spectroscopy provides a sensitive tool to study adenylate kinase activity in different physiological and pathophysiological conditions, including but not exclusive of, cancer, ischemic injury, hemolytic anemia and neurological problems such as sensorineural deafness.