Characterization and modulation of endoplasmic reticulum stress response target genes in Kluyveromyces marxianus to improve secretory expressions of heterologous proteins

Background Kluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. Expressions of heterologous proteins usually lead to the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER) and then cause ER stress. To cope with this problem, a group of ER stress response target genes (ESRTs) are induced, mainly through a signaling network called unfolded protein response (UPR). Characterization and modulation of ESRTs direct the optimization of heterologous expressions. However, ESRTs in K. marxianus have not been identified so far. Results In this study, we characterized the ER stress response in K. marxianus for the first time, by using two ER stress-inducing reagents, dithiothreitol (DTT) and tunicamycin (TM). Results showed that the Kar2–Ire1–Hac1 pathway of UPR is well conserved in K. marxianus. About 15% and 6% of genes were upregulated during treatment of DTT and TM, respectively. A total of 115 upregulated genes were characterized as ESRTs, among which 97 genes were identified as UPR target genes and 37 UPR target genes contained UPR elements in their promoters. Genes related to carbohydrate metabolic process and actin filament organization were identified as new types of UPR target genes. A total of 102 ESRTs were overexpressed separately in plasmids and their effects on productions of two different lignocellulolytic enzymes were systematically evaluated. Overexpressing genes involved in carbohydrate metabolism, including PDC1, PGK and VID28, overexpressing a chaperone gene CAJ1 or overexpressing a reductase gene MET13 substantially improved secretion expressions of heterologous proteins. Meanwhile, overexpressing a novel gene, KLMA_50479 (named ESR1), as well as overexpressing genes involved in ER-associated protein degradation (ERAD), including HRD3, USA1 andYET3, reduced the secretory expressions. ESR1 and the aforementioned ERAD genes were deleted from the genome. Resultant mutants, except the yet3Δ mutant, substantially improved secretions of three different heterologous proteins. During the fed-batch fermentation, extracellular activities of an endoxylanase and a glucanase in hrd3Δ cells improved by 43% and 28%, respectively, compared to those in wild-type cells. Conclusions Our results unveil the transcriptional scope of the ER stress response in K. marxianus and suggest efficient ways to improve productions of heterologous proteins by manipulating expressions of ESRTs.

Effective Generalized Partial Response Target and Serial Detector for Two-Dimensional Bit-Patterned Media Recording Channel Including Track Mis-Registration

With the development of 5G technology, programs are gradually moving to cloud services. This leads to an increasing demand for storage. In the field of high-density data storage, bit-pattern media recording (BPMR) is considered a promising approach, as it can expand the data density to 4 Tb/in2. However, in high-density BPMR, bits or magnetic islands are very close to each other, leading to significant intertrack interference (ITI) from the cross-track direction and intersymbol interference (ISI) from the down-track direction. To minimize two-dimensional interference, including ITI and ISI, the serial detector method has been highly effective. However, in this method, the signal at the output of the first decoder is still a hard output. Therefore, we suggest methods to convert the output of the first detector into a soft output. Additionally, we have developed a new form of generalized partial response target to overcome the track mis-registration. The results show that our proposed methods apparently improve bit error rate performance.

Contextually-Based Social Attention Diverges across Covert and Overt Measures

Humans spontaneously attend to social cues like faces and eyes. However, recent data show that this behavior is significantly weakened when visual content, such as luminance and configuration of internal features, as well as visual context, such as background and facial expression, are controlled. Here, we investigated attentional biasing elicited in response to information presented within appropriate background contexts. Using a dot-probe task, participants were presented with a face–house cue pair, with a person sitting in a room and a house positioned within a picture hanging on a wall. A response target occurred at the previous location of the eyes, mouth, top of the house, or bottom of the house. Experiment 1 measured covert attention by assessing manual responses while participants maintained central fixation. Experiment 2 measured overt attention by assessing eye movements using an eye tracker. The data from both experiments indicated no evidence of spontaneous attentional biasing towards faces or facial features in manual responses; however, an infrequent, though reliable, overt bias towards the eyes of faces emerged. Together, these findings suggest that contextually-based social information does not determine spontaneous social attentional biasing in manual measures, although it may act to facilitate oculomotor behavior.

Placeholders dissociate two forms of inhibition of return

Decades of research using Posner’s classic spatial cueing paradigm has uncovered at least two forms of inhibition of return (IOR) in the aftermath of an exogenous, peripheral orienting cue. One prominent dissociation concerns the role of covert and overt orienting in generating IOR effects that relate to perception- and action-oriented processes, respectively. Another prominent dissociation concerns the role of covert and overt orienting in generating IOR effects that depend on object- and space-based representation, respectively. Our objective was to evaluate whether these dichotomies are functionally equivalent by manipulating placeholder object presence in the cueing paradigm. By discouraging eye movements throughout, Experiments 1A and 1B validated a perception-oriented form of IOR that depended critically on placeholders. Experiment 2A demonstrated that IOR was robust without placeholders when eye movements went to the cue and back to fixation before the manual response target. In Experiment 2B, we replicated Experiment 2A’s procedures except we discouraged eye movements. IOR was observed, albeit only weakly and significantly diminished relative to when eye movements were involved. We conclude that action-oriented IOR is robust against placeholders but that the magnitude of perception-oriented IOR is critically sensitive to placeholder presence when unwanted oculomotor activity can be ruled out.

Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system

The expression of the autism-related mutant R451C neuroligin3 activates the three branches of the unfolded protein response in neuronal-like PC12 cells and leads to up-regulation of the response target genes BiP and CHOP.