Quantum Dot-Peptide Conjugates as Energy Transfer Probes for Sensing the Proteolytic Activity of Matrix Metalloproteinase-14.

We detail the assembly and characterization of quantum dot (QD)-dye conjugates constructed using a peptide bridge specifically designed to recognize and interact with a breast cancer biomarker─matrix metalloproteinase-14 (MMP-14). The assembled QD conjugates are then used as optically addressable probes, relying on Förster resonance energy transfer (FRET) interactions as a transduction mechanism to detect the activity of MMP-14 in solution phase. The QDs were first coated with dithiolane poly(ethylene glycol) (PEG) bearing a carboxyl group that allows coupling via amide bond formation with different dye-labeled peptides. The analytical capability of the conjugates is enabled by correlating changes in the FRET efficiency with the conjugate valence and/or QD-to-dye separation distance, triggered and modulated by enzymatic proteolysis of surface-tethered peptides. The FRET probe exhibits great sensitivity to enzyme digestion with sub-nanomolar limit of detection. We further analyze the proteolysis data within the framework of the Michaelis-Menten model, which considers the fact that surface-attached peptides have a slower diffusion coefficient than free peptides. This results in reduced collision frequency and lower catalytic efficiency, kcat/KM. Our results suggest that our conjugate design is promising, effective, and potentially useful for in vivo analysis.

Targeting the perinatal diet to modulate the gut microbiota increases dietary variety and prebiotic and probiotic food intakes: results from a randomised controlled trial.

OBJECTIVE: To evaluate the hypothesis that a perinatal educational dietary intervention focused on 'eating for the gut microbiota' improves diet quality of pregnant women pre- and postnatally. DESIGN: The Healthy Parents, Healthy Kids study is a prospectively registered randomised controlled trial designed to evaluate the efficacy of a dietary intervention in altering the maternal and infant gut microbiota and improving perinatal diet quality. Eligible pregnant women were randomised to receive dietary advice from their healthcare provider or to additionally receive a three session dietary intervention. Dietary data were collected at gestation weeks 26, 31, 36 and postnatal week 4. Outcome measures were diet quality, dietary variety, prebiotic and probiotic food intakes, energy, fibre, saturated fat and discretionary food intakes. Between-group differential changes from baseline before and after birth in these dietary measures were assessed using generalised estimating equations. SETTING: Melbourne, Australia. PARTICIPANTS: Healthy pregnant women from gestation week 26. RESULTS: Forty-five women were randomised (twenty-two control, twenty-three intervention). Compared with the control group, the intervention group improved diet quality prior to birth (5·66 (95 % CI 1·65, 9·67), Cohen's d: 0·82 (se 0·33)). The intervention improved dietary variety (1·05 (95 % CI 0·17, 1·94), d: 0·66 (se 0·32)) and increased intakes of prebiotic (0·8 (95 % CI 0·27, 1·33), d: 0·91 (se 0·33)) and probiotic foods (1·05 (95 % CI 0·57, 1·53), d: 1·3(se 0·35)) over the whole study period compared with the control group. CONCLUSION: A dietary intervention focused on 'eating for the gut microbiota' can improve aspects of perinatal diet quality during and after pregnancy.

Immune tolerance negatively regulates B cells in knock-in mice expressing broadly neutralizing HIV antibody 4E10.

A major goal of HIV research is to develop vaccines reproducibly eliciting broadly neutralizing Abs (bNAbs); however, this has proved to be challenging. One suggested explanation for this difficulty is that epitopes seen by bNAbs mimic self, leading to immune tolerance. We generated knock-in mice expressing bNAb 4E10, which recognizes the membrane proximal external region of gp41. Unlike b12 knock-in mice, described in the companion article (Ota et al. 2013. J. Immunol. 191: 3179-3185), 4E10HL mice were found to undergo profound negative selection of B cells, indicating that 4E10 is, to a physiologically significant extent, autoreactive. Negative selection occurred by various mechanisms, including receptor editing, clonal deletion, and receptor downregulation. Despite significant deletion, small amounts of IgM and IgG anti-gp41 were found in the sera of 4E10HL mice. On a Rag1⁻/⁻ background, 4E10HL mice had virtually no serum Ig of any kind. These results are consistent with a model in which B cells with 4E10 specificity are counterselected, raising the question of how 4E10 was generated in the patient from whom it was isolated. This represents the second example of a membrane proximal external region-directed bNAb that is apparently autoreactive in a physiological setting. The relative conservation in HIV of the 4E10 epitope might reflect the fact that it is under less intense immunological selection as a result of B cell self-tolerance. The safety and desirability of targeting this epitope by a vaccine is discussed in light of the newly described bNAb 10E8.

Qualitative synthesis and systematic review in health professions education.

CONTEXT: Formal qualitative synthesis is the process of pooling qualitative and mixed-method research data, and then drawing conclusions regarding the collective meaning of the research. Qualitative synthesis is regularly used within systematic reviews in the health professions literature, although such use has been heavily debated in the general literature. This controversy arises in part from the inherent tensions found when generalisations are derived from in-depth studies that are heavily context-dependent. METHODS: We explore three representative qualitative synthesis methodologies: thematic analysis; meta-ethnography, and realist synthesis. These can be understood across two dimensions: integrative to interpretative, and idealist to realist. Three examples are used to illustrate the relative strengths and limitations of these approaches. DISCUSSION: Against a backdrop of controversy and diverse methodologies, readers must take a critical stand when reading literature reviews that use qualitative synthesis to derive their findings. We argue that notions of qualitative rigour such as transparency and acknowledgment of the researchers' stance should be applied to qualitative synthesis.

Targeting the Infant Gut Microbiota Through a Perinatal Educational Dietary Intervention: Protocol for a Randomized Controlled Trial.

BACKGROUND: The early life gut microbiota are an important regulator of the biological pathways contributing toward the pathogenesis of noncommunicable disease. It is unclear whether improvements to perinatal diet quality could alter the infant gut microbiota. OBJECTIVE: The aim of this study is to assess the efficacy of a perinatal educational dietary intervention in influencing gut microbiota in mothers and infants 4 weeks after birth. METHODS: The Healthy Parents, Healthy Kids randomized controlled trial aimed to recruit 90 pregnant women from Melbourne, Victoria, Australia. At week 26 of gestation, women were randomized to receive dietary advice from their doctor (n=45), or additionally receive a dietary intervention (n=45). The intervention included an educational workshop and 2 support calls aiming to align participants' diets with the Australian Dietary Guidelines and increase intakes of prebiotic and probiotic foods. The educational design focused on active learning and self-assessment. Behavior change techniques were used to support dietary adherence, and the target behavior was eating for the gut microbiota. Exclusion criteria were age under 18 years, diagnosed mental illnesses, obesity, diabetes mellitus, diagnosed bowel conditions, exclusion diets, illicit drug use, antibiotic use, prebiotic or probiotic supplementation, and those lacking dietary autonomy. The primary outcome measure is a between-group difference in alpha diversity in infant stool collected 4 weeks after birth. Secondary outcomes include evaluating the efficacy of the intervention in influencing infant and maternal stool microbial composition and short chain fatty acid concentrations, epigenetic profile, and markers of inflammation and stress, as well as changes in maternal dietary intake and well-being. The study and intervention feasibility and acceptance will also be evaluated as secondary outcomes. RESULTS: The study results are yet to be written. The first participant was enrolled on July 28, 2016, and the final follow-up assessment was completed on October 11, 2017. CONCLUSIONS: Data from this study will provide new insights regarding the ability of interventions targeting the perinatal diet to alter the maternal and infant gut microbiota. If this intervention is proven, our findings will support larger studies aiming to guide the assembly of gut microbiota in early life. TRIAL REGISTRATION: Australian Clinical Trials Registration Number ACTRN12616000936426; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=370939. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/14771.

Carbon-deuterium bonds as probes of protein thermal unfolding.

We report a residue-specific characterization of the thermal unfolding mechanism of ferric horse heart cytochrome c using C-D bonds site-specifically incorporated at residues dispersed throughout three different structural elements within the protein. As the temperature increases, Met80 first dissociates from the heme center, and the protein populates a folding intermediate before transitioning to a solvent exposed state. With further increases in temperature, the C-terminal helix frays and then loses structure along with the core of the protein. Interestingly, the data also reveal that the state populated at high temperature retains some structure and possibly represents a molten globule. Elucidation of the detailed unfolding mechanism and the structure of the associated molten globule, both of which represent challenges to conventional techniques, highlights the utility of the C-D technique.

Evaluating the potential for halogen bonding in the oxyanion hole of ketosteroid isomerase using unnatural amino acid mutagenesis.

There has recently been an increasing interest in controlling macromolecular conformations and interactions through halogen bonding. Halogen bonds are favorable electrostatic interactions between polarized, electropositive chlorine, bromine, or iodine atoms and electronegative atoms such as oxygen or nitrogen. These interactions have been likened to hydrogen bonds in terms of their favored acceptor molecules, their geometries, and their energetics. We asked whether a halogen bond could replace a hydrogen bond in the oxyanion hole of ketosteroid isomerase, using semisynthetic enzymes containing para-halogenated phenylalanine derivatives to replace the tyrosine hydrogen bond donor. Formation of a halogen bond to the oxyanion in the transition state would be expected to rescue the effects of mutation to phenylalanine, but all of the halogenated enzymes were comparable in activity to the phenylalanine mutant. We conclude that, at least in this active site, a halogen bond cannot functionally replace a hydrogen bond.