Little is known about the global structure of honey and the arrangement of its main macromolecules. We hypothesized that the conditions in ripened honeys resemble macromolecular crowding in the cell and affect the concentration, reactivity, and conformation of honey macromolecules. Combined results from UV spectroscopy, DLS and SEM showed that the concentration of macromolecules was a determining factor in honey structure. The UV spectral scans in 200–400 nm visualized and allowed quantification of UV-absorbing compounds in the following order: dark > medium > light honeys (p < 0.0001). The high concentration of macromolecules promoted their self-assembly to micron-size superstructures, visible in SEM as two-phase system consisting of dense globules distributed in sugar solution. These particles showed increased conformational stability upon dilution. At the threshold concentration, the system underwent phase transition with concomitant fragmentation of large micron-size particles to nanoparticles in hierarchical order. Honey two-phase conformation was an essential requirement for antibacterial activity and hydrogen peroxide production. These activities disappeared beyond the phase transition point. The realization that active macromolecules of honey are arranged into compact, stable multicomponent assemblies with colloidal properties reframes our view on global structure of honey and emerges as a key property to be considered in investigating its biological activity.
We have recently identified the bacterial cell wall as the cellular target for honey antibacterial compounds; however, the chemical nature of these compounds remained to be elucidated. Using Concavalin A- affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties. The glps possessed two distinct functionalities: (a) specific binding and agglutination of bacterial cells, but not rat erythrocytes and (b) non-specific membrane permeabilization of both bacterial cells and erythrocytes. The isolated glps induced concentration- and time-dependent changes in the cell shape of both E. coli and B. subtilis as visualized by light and SEM microscopy. The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively. The time-kill kinetics showed a rapid, >5-log10 reduction of viable cells within 15 min incubation at 1xMBC, indicating that the glps-induced damage of the cell wall was lethal. Unexpectedly, MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, (ESI-Q-TOF-MS/MS) analysis of glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2, and 4. The presence of high-mannose structures explained the lectin-like activity of MRJP1, while the presence of Jelleins in MRJP1 may explain cell wall disruptions. Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced. Antibacterial activity of MRJP1 glps directly correlated with the overall antibacterial activity of honey, suggesting that it is honey’s active principle responsible for this activity.
PurposeComorbid familial nonobstructive azoospermia (NOA) and congenital cataract (CC) have not been reported previously, and no single human gene has been associated with both diseases in humans. Our purpose was to uncover novel human mutations and genes causing familial NOA and CC.MethodsWe performed whole-exome sequencing for two brothers with both NOA and CC from a consanguineous family. Mutation screening of TDRD7 was performed in another similar consanguineous family and 176 patients with azoospermia or CC alone and 520 healthy controls. Histological analysis was performed for the biopsied testicle sample in one patient, and knockout mice were constructed to verify the phenotype of the mutation in TDRD7.ResultsTwo novel loss-of-function mutations (c.324_325insA (T110Nfs*30) and c.688_689insA (p.Y230X), respectively) of TDRD7 were found in the affected patients from the two unrelated consanguineous families. Histological analysis demonstrated a lack of mature sperm in the male patient's seminiferous tubules. The mutations were not detected in patients with CC or NOA alone. Mice with Tdrd7 gene disrupted at a similar position precisely replicated the human syndrome.ConclusionWe identified TDRD7 causing CC as a new pathogenic gene for male azoospermia in human, with an autosomal recessive mode of inheritance.GENETICS in MEDICINE advance online publication, 24 August 2017; doi:10.1038/gim.2017.130.
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