Efficient integration of transmembrane domains depends on the folding properties of the upstream sequences

The topology of most membrane proteins is defined by the successive integration of α-helical transmembrane domains at the Sec61 translocon. The translocon provides a pore for the transfer of polypeptide segments across the membrane while giving them lateral access to the lipid. For each polypeptide segment of ∼20 residues, the combined hydrophobicities of its constituent amino acids were previously shown to define the extent of membrane integration. Here, we discovered that different sequences preceding a potential transmembrane domain substantially affect its hydrophobicity requirement for integration. Rapidly folding domains, sequences that are intrinsically disordered or very short or capable of binding chaperones with high affinity, allow for efficient transmembrane integration with low-hydrophobicity thresholds for both orientations in the membrane. In contrast, long protein fragments, folding-deficient mutant domains, and artificial sequences not binding chaperones interfered with membrane integration, requiring higher hydrophobicity. We propose that the latter sequences, as they compact on their hydrophobic residues, partially folded but unable to reach a native state, expose hydrophobic surfaces that compete with the translocon for the emerging transmembrane segment, reducing integration efficiency. The results suggest that rapid folding or strong chaperone binding is required for efficient transmembrane integration.

Expansion of EasyClone-MarkerFree toolkit for Saccharomyces cerevisiae genome with new integration sites

Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

Common Microcirculatory Framework for Monitoring Integrated Microcirculation

Wide variation in magnitudes, units, and ranges of the microcirculatory variables brings hindrance in describing and evaluating the integrated microcirculatory function of tissues. We designed to establish common microcirculatory framework that contains microhemodynamic and microcirculatory oxygen parameters. To integrate microcirculatory information, demo microcirculatory permutations were generated by a computer algorithm based on microcirculatory characteristics. Four dimensionless methods (Z-score, Min-max, L2, and median scaling) were applied to transform microcirculatory data set into the dimensionless form. Three-dimensional (3-D) common microcirculatory framework was constructed and visualized by using Python and Apache ECharts. The performance of the four dimensionless methods in the pre-processing of multiple microcirculatory variables and the establishment of the common microcirculatory framework were compared. Microhemodynamic and microcirculatory oxygen parameters were embedded in the common microcirculatory framework. After processing by Min-max normalization, the transformed multiple microcirculatory values remained positive with fixed range mapping within [0, 1] and maintained the identity property of microcirculation both of microhemodynamic and microcirculatory oxygen variables in the common microcirculatory framework. Conclusively, Min-max normalization displays preferable integration efficiency, compatibility, and adaptability in the establishment of the 3-D visualized multiparametric common microcirculatory framework.

Sensitivity to Pulse Phase Duration as a Marker of Neural Health Across Cochlear Implant Recipients and Electrodes

In cochlear implants, loudness has been shown to grow more slowly with increasing pulse phase duration (PPD) than with pulse amplitude (PA), possibly due to “leaky” charge integration. This leakiness has been recently quantified in terms of “charge integration efficiency,” defined as the log difference between the PPD dynamic range and PA dynamic range (both expressed in charge units), relative to a common threshold anchor. Such leakiness may differ across electrodes and/or test ears, and may reflect underlying neural health. In this study, we examined the across-site variation of charge integration in recipients of Cochlear© devices. PPD and PA dynamic ranges were measured relative to two threshold anchors with either a 25- or 50-microsecond PPD. Strength-duration functions, previously shown to relate to survival of spiral ganglion cells and peripheral processes, were compared to charge integration efficiency on selected electrodes. Results showed no significant or systematic relationship between the across-site variation in charge integration efficiency and electrode position or threshold levels. Charge integration efficiency was poorer with the 50-μs threshold anchor, suggesting that greater leakiness was associated with larger PPD dynamic ranges. Poorer and more variable charge integration efficiency across electrodes was associated with longer duration of any hearing loss, consistent with the idea that poor integration is related to neural degeneration. More variable integration efficiency was also associated with poorer speech recognition performance across test ears. The slopes of the strength-duration functions at maximum acceptable loudness were significantly correlated with charge integration efficiency. However, the strength-duration slopes were not predictive of duration of any hearing loss or speech recognition performance in our participants. As such, charge integration efficiency may be a better candidate to measure leakiness in neural populations across the electrode array, as well as the general health of the auditory nerve in human cochlear implant recipients.