Orbitally Shaken Single-Use Bioreactor for Animal Cell Cultivation: Fed-Batch and Perfusion Mode.

Increasing the cultivation volume from small to large scale can be a rather complex and challenging process when the method of aeration and mixing is different between scales. Orbitally shaken bioreactors (OSBs) utilize the same hydrodynamic principles that define the success of smaller-scale cultures, which are developed on an orbitally shaken platform, and can simplify scale-up. Here we describe the basic working principles of scale-up in terms of the volumetric oxygen transfer coefficient (ka) and mixing time and how to define these parameters experimentally.

HEK293 Cell-Based Bioprocess Development at Bench Scale by Means of Online Monitoring in Shake Flasks (RAMOS and SFR).

The platforms for bioprocess development have been developed in parallel to the needs of the manufacturing industry of biopharmaceuticals, aiming to ensure the quality and safety of their products. In this sense, Quality by Design (QbD) and Process Analytical Technology (PAT) have become the pillars for quality control and quality assurance.A new combination of Shake Flask Reader (SFR) and Respiration Activity Monitoring System for online determination of OTR and CTR (RAMOS) allows online monitoring of main culture parameters needed for bioprocess development (pH, pO, OTR, CTR, and QR) as presented below.

Endogenous signalling pathways and caged IP evoke Ca puffs at the same abundant immobile intracellular sites.

The building blocks of intracellular Ca signals evoked by inositol 1,4,5-trisphosphate receptors (IPRs) are Ca puffs, transient focal increases in Ca concentration that reflect the opening of small clusters of IPRs. We use total internal reflection fluorescence microscopy and automated analyses to detect Ca puffs evoked by photolysis of caged IP or activation of endogenous muscarinic receptors with carbachol in human embryonic kidney 293 cells. Ca puffs evoked by carbachol initiated at an estimated 65±7 sites/cell, and the sites remained immobile for many minutes.

The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation.

The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca(2+) homeostasis, trafficking, and membrane organization.

Spatial organization of intracellular Ca2+ signals.

The ability of Ca(2+), the simplest of all intracellular messengers, selectively to regulate so many cellular behaviours is due largely to the complex spatiotemporal organization of intracellular Ca(2+) signals. Most signalling pathways, including those that culminate in Ca(2+) signals, comprise sequences of protein-protein interactions linked by diffusible messengers. Using specific examples to illustrate key principles, we consider the roles of both components in defining the spatial organization of Ca(2+) signals.

Pitch perception beyond the traditional existence region of pitch.

Humans' ability to recognize musical melodies is generally limited to pure-tone frequencies below 4 or 5 kHz. This limit coincides with the highest notes on modern musical instruments and is widely believed to reflect the upper limit of precise stimulus-driven spike timing in the auditory nerve. We tested the upper limits of pitch and melody perception in humans using pure and harmonic complex tones, such as those produced by the human voice and musical instruments, in melody recognition and pitch-matching tasks.

Musical intervals and relative pitch: frequency resolution, not interval resolution, is special.

Pitch intervals are central to most musical systems, which utilize pitch at the expense of other acoustic dimensions. It seemed plausible that pitch might uniquely permit precise perception of the interval separating two sounds, as this could help explain its importance in music. To explore this notion, a simple discrimination task was used to measure the precision of interval perception for the auditory dimensions of pitch, brightness, and loudness.

Pitch perception for mixtures of spectrally overlapping harmonic complex tones.

This study measured difference limens for fundamental frequency (DLF0s) for a target harmonic complex in the presence of a simultaneous spectrally overlapping harmonic masker. The resolvability of the target harmonics was manipulated by bandpass filtering the stimuli into a low (800-2400 Hz) or high (1600-3200 Hz) spectral region, using different nominal F0s for the targets (100, 200, and 400 Hz), and different masker F0s (0, +9, or -9 semitones) relative to the target. Three different modes of masker presentation, relative to the target, were tested: ipsilateral, contralateral, and dichotic, with a higher masker level in the contralateral ear.

An ancestral deuterostome family of two-pore channels mediates nicotinic acid adenine dinucleotide phosphate-dependent calcium release from acidic organelles.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent and widespread calcium-mobilizing messenger, the properties of which have been most extensively described in sea urchin eggs. The molecular basis for calcium release by NAADP, however, is not clear and subject to controversy. Recent studies have provided evidence that members of the two-pore channel (TPC) family in mammals are the long sought after target channels for NAADP.

Can temporal fine structure represent the fundamental frequency of unresolved harmonics?

At least two modes of pitch perception exist: in one, the fundamental frequency (F0) of harmonic complex tones is estimated using the temporal fine structure (TFS) of individual low-order resolved harmonics; in the other, F0 is derived from the temporal envelope of high-order unresolved harmonics that interact in the auditory periphery. Pitch is typically more accurate in the former than in the latter mode. Another possibility is that pitch can sometimes be coded via the TFS from unresolved harmonics.