Low-Cost Clamp-On Photometers (ClampOD) and Tube Photometers (TubeOD) for Online Cell Density Determination

Optical density (OD) measurement is the gold standard to estimate microbial cell density in aqueous systems. Recording microbial growth curves is essential to assess substrate utilization, gauge sensitivity to inhibitors or toxins, or determine the perfect sampling point. Manual sampling for cuvette-photometer-based measurements can cause disturbances and impact growth, especially for strictly anaerobic or thermophilic microbes. For slow growing microbes, manual sampling can cause data gaps that complicate analysis. Online OD measurement systems provide a solution, but are often expensive and ill-suited for applications such as monitoring microbial growth in custom or larger anaerobic vessels. Furthermore, growth measurements of thermophilic cultures are limited by the heat sensitivity of complex electronics. Here, we present two simple, low-cost, self-assembled photometers—a “TubeOD” for online measurement of anaerobic and thermophilic cultures in Hungate tubes and a “ClampOD” that can be attached to virtually any transparent growth vessel. Both OD-meters can be calibrated in minutes. We detail the manufacturing and calibration procedure and demonstrate continuous acquisition of high quality cell density data of a variety of microbes, including strict anaerobes, a thermophile, and gas-utilizing strains in various glassware. When calibrated and operated within their detection limits (ca. 0.3–90% of the photosensor voltage range), these self-build OD-meters can be used for continuous measurement of microbial growth in a variety of applications, thereby, simplifying and enhancing everyday lab operations.

Heat-Dependent Hairpin Melting Drives TRPV1 Opening

The capsaicin receptor TRPV1 can be activated by heat and thus serves as a thermometer in a primary afferent sensory neuron for noxious heat detection. However, the underlying molecular mechanism is unclear. Here, a hairpin topological structural model, together with graph theory, was developed to examine a role of temperature-dependent hairpin melting in controlling non-covalent interactions along the heat-evoked gating pathway of TRPV1. The results showed that heat-dependent hairpin melting rearranges non-covalent interactions, releases the resident lipid, and induces TRPV1 gating. A larger hairpin in the outer pore initiates a temperature threshold as a heat starter for channel opening while some smaller hairpins in the S4-S5 linker and the outer pore stabilize the heat efficacy and avoid heat denaturation as a heat fuse. The heat-induced global gating rearrangement may be responsible for the high heat sensitivity. This hairpin model may provide a broad structural basis for the thermo-gated ion channels.

Sprout Caffeoylquinic Acid Profiles as Affected by Variety, Cooking, and Storage

Various health-promoting properties inherent to plant-based foods have been attributed to their rich bioactive compounds, including caffeoylquinic acids (CQAs). The potential health benefits of CQAs have been well-documented. While sprouts are widely recognized as health-promoting foods owing to their high phytonutrient content, our knowledge regarding the effect of cooking and storage, commonly practiced by consumers, on the CQA content remains limited. First, sunflower sprouts were found to have the highest total CQA content (~ 22 mg/g dry weight) out of 11 commonly available sprouts. Then, the effect of variety, cooking, and low-temperature storage on the CQA profile of sunflower sprouts was investigated. Among the four different varieties of sunflower sprouts, variety 1 harbored the highest total CQA content. Notably, cooking adversely affected the CQA content of sunflower sprouts relative to the uncooked samples in a time-dependent manner, possibly due to the heat sensitivity of CQAs. Under simulated home-refrigeration storage conditions, we observed a significant decline in the content of major CQA compounds (5-monoCQA and 3,5-diCQA) at days 10 and 13 of storage. The results obtained herein provide consumers and food industrialists with increased insight into the effect of cooking and refrigeration on the CQA content of sunflower sprouts.

The Human SCN10AG1662S Point Mutation Established in Mice Impacts on Mechanical, Heat, and Cool Sensitivity

The voltage-gated sodium channel NAV1.8 is expressed in primary nociceptive neurons and is involved in pain transmission. Mutations in the SCN10A gene (encoding NAV1.8 channel) have been identified in patients with idiopathic painful small fiber neuropathy (SFN) including the SCN10AG1662S gain-of-function mutation. However, the role of this mutation in pain sensation remains unknown. We have generated the first mouse model for the G1662S mutation by using homologous recombination in embryonic stem cells. The corresponding Scn10aG1663S mouse line has been analyzed for Scn10a expression, intraepidermal nerve fiber density (IENFD), and nociception using behavioral tests for thermal and mechanical sensitivity. The Scn10aG1663S mutants had a similar Scn10a expression level in dorsal root ganglia (DRG) to their wild-type littermates and showed normal IENFD in hindpaw skin. Mutant mice were more sensitive to touch than wild types in the von Frey test. In addition, sexual dimorphism was observed for several pain tests, pointing to the relevance of performing the phenotypical assessment in both sexes. Female homozygous mutants tended to be more sensitive to cooling stimuli in the acetone test. For heat sensitivity, male homozygous mutants showed shorter latencies to radiant heat in the Hargreaves test while homozygous females had longer latencies in the tail flick test. In addition, mutant males displayed a shorter reaction latency on the 54°C hot plate. Collectively, Scn10aG1663S mutant mice show a moderate but consistent increased sensitivity in behavioral tests of nociception. This altered nociception found in Scn10aG1663S mice demonstrates that the corresponding G1662 mutation of SCN10A found in SFN patients with pain contributes to their pain symptoms.

A temperature sensitive mutation in the CstF77 subunit of the polyadenylation complex reveals the critical function of mRNA 3' end formation for a robust heat stress response in plants

Background: Heat Shock Protein 101 (HSP101) in plants and orthologs in bacteria (Caseinolytic peptidase B, ClpB) and yeast (Hsp104) are essential for thermotolerance. To investigate molecular mechanisms of thermotolerance involving HSP101, we performed a suppressor screen in Arabidopsis thaliana of a semi-dominant, missense HSP101 allele, hot1-4 (A499T). Plants carrying the hot1-4 mutation are more heat-sensitive than an HSP101 null mutant (hot1-3), indicating the toxicity of hot1-4 allele. Results: We report that one suppressor (shot2, suppressor of hot1-4 2) has a temperature-sensitive, missense mutation (E170K) in the CstF77 (Cleavage stimulation factor 77) subunit of the polyadenylation complex, which is critical for 3' end maturation of pre-mRNA. RNA-Seq analysis of total RNA depleted of ribosomes reveals that heat treatment causes transcriptional readthrough events in shot2, specifically in highly heat-induced genes, including the toxic hot1-4 gene. In addition, failure of correct transcript processing leads to reduced accumulation of many HSP RNAs and proteins, suppressing heat sensitivity of the hot1-4 mutant, due to reduction of the toxic mutant HSP101 protein. Notably, the shot2 mutation makes plants more sensitive to heat stress in the HSP101 null (hot1-3) and wild-type backgrounds correlated with the reduced expression of other heat-inducible genes required for thermotolerance. Conclusions: Our study reveals the critical function of CstF77 for 3' end formation of mRNA during heat stress, as well as the dominant role of HSP101 in dictating the outcome of severe heat stress in plants.

Impact of alanyl-tRNA synthetase editing deficiency in yeast

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that provide the ribosome with aminoacyl-tRNA substrates for protein synthesis. Mutations in aaRSs lead to various neurological disorders in humans. Many aaRSs utilize editing to prevent error propagation during translation. Editing defects in alanyl-tRNA synthetase (AlaRS) cause neurodegeneration and cardioproteinopathy in mice and are associated with microcephaly in human patients. The cellular impact of AlaRS editing deficiency in eukaryotes remains unclear. Here we use yeast as a model organism to systematically investigate the physiological role of AlaRS editing. Our RNA sequencing and quantitative proteomics results reveal that AlaRS editing defects surprisingly activate the general amino acid control pathway and attenuate the heatshock response. We have confirmed these results with reporter and growth assays. In addition, AlaRS editing defects downregulate carbon metabolism and attenuate protein synthesis. Supplying yeast cells with extra carbon source partially rescues the heat sensitivity caused by AlaRS editing deficiency. These findings are in stark contrast with the cellular effects caused by editing deficiency in other aaRSs. Our study therefore highlights the idiosyncratic role of AlaRS editing compared with other aaRSs and provides a model for the physiological impact caused by the lack of AlaRS editing.

Heat-Stress Responses Differ among Species from Different ‘Bangia’ Clades of Bangiales (Rhodophyta)

The red alga ‘Bangia’ sp. ESS1, a ‘Bangia’ 2 clade member, responds to heat stress via accelerated asexual reproduction and acquires thermotolerance based on heat-stress memory. However, whether these strategies are specific to ‘Bangia’ 2, especially ‘Bangia’ sp. ESS1, or whether they are employed by all ‘Bangia’ species is currently unknown. Here, we examined the heat-stress responses of ‘Bangia’ sp. ESS2, a newly identified ‘Bangia’ clade 3 member, and Bangia atropurpurea. Intrinsic thermotolerance differed among species: Whereas ‘Bangia’ sp. ESS1 survived at 30 °C for 7 days, ‘Bangia’ sp. ESS2 and B. atropurpurea did not, with B. atropurpurea showing the highest heat sensitivity. Under sublethal heat stress, the release of asexual spores was highly repressed in ‘Bangia’ sp. ESS2 and completely repressed in B. atropurpurea, whereas it was enhanced in ‘Bangia’ sp. ESS1. ‘Bangia’ sp. ESS2 failed to acquire heat-stress tolerance under sublethal heat-stress conditions, whereas the acquisition of heat tolerance by priming with sublethal high temperatures was observed in both B. atropurpurea and ‘Bangia’ sp. ESS1. Finally, unlike ‘Bangia’ sp. ESS1, neither ‘Bangia’ sp. ESS2 nor B. atropurpurea acquired heat-stress memory. These findings provide insights into the diverse heat-stress response strategies among species from different clades of ‘Bangia’.

Alanine Represses γ-Aminobutyric Acid Utilization and Induces Alanine Transaminase Required for Mitochondrial Function in Saccharomyces cerevisiae

The γ-aminobutyric acid (GABA) shunt constitutes a conserved metabolic route generating nicotinamide adenine dinucleotide phosphate (NADPH) and regulating stress response in most organisms. Here we show that in the presence of GABA, Saccharomyces cerevisiae produces glutamate and alanine through the irreversible action of Uga1 transaminase. Alanine induces expression of alanine transaminase (ALT1) gene. In an alt1Δ mutant grown on GABA, alanine accumulation leads to repression of the GAD1, UGA1, and UGA2 genes, involved in the GABA shunt, which could result in growth impairment. Induced ALT1 expression and negative modulation of the GABA shunt by alanine constitute a novel regulatory circuit controlling both alanine biosynthesis and catabolism. Consistent with this, the GABA shunt and the production of NADPH are repressed in a wild-type strain grown in alanine, as compared to those detected in the wild-type strain grown on GABA. We also show that heat shock induces alanine biosynthesis and ALT1, UGA1, UGA2, and GAD1 gene expression, whereas an uga1Δ mutant shows heat sensitivity and reduced NADPH pools, as compared with those observed in the wild-type strain. Additionally, an alt1Δ mutant shows an unexpected alanine-independent phenotype, displaying null expression of mitochondrial COX2, COX3, and ATP6 genes and a notable decrease in mitochondrial/nuclear DNA ratio, as compared to a wild-type strain, which results in a petite phenotype. Our results uncover a new negative role of alanine in stress defense, repressing the transcription of the GABA shunt genes, and support a novel Alt1 moonlighting function related to the maintenance of mitochondrial DNA integrity and mitochondrial gene expression.

Disentangling ‘sciatica’ to understand and characterise somatosensory profiles and potential pain mechanisms

Objectives The study aimed to investigate if patients with lumbar radicular pain only and those with combined lumbar radicular pain + radiculopathy differ in their somatosensory profiles and pain experiences. Methods Quantitative sensory testing (QST) was performed in 26 patients (mean age 47 ± 10 years, 10 females) with unilateral leg pain in the L5 or S1 distribution in their main pain area (MPA) and contralateral mirror side, in the relevant foot dermatome on the symptomatic side and in the hand dorsum. Pain experience was captured on the painDETECT. Results Eight patients presented with lumbar radicular pain only and 18 patients with combined radicular pain + radiculopathy. Patients with radicular pain only demonstrated widespread loss of function (mechanical detection) bilaterally in the MPA (p<0.003) and hand (p=0.002), increased heat sensitivity in both legs (p<0.019) and cold/heat sensitivity in the hand (p<0.024). QST measurements in the dermatome did not differ compared to HCs and patients with radiculopathy. Patients with lumbar radiculopathy were characterised by a localised loss of function in the symptomatic leg in the MPA (warm, mechanical, vibration detection, mechanical pain threshold, mechanical pain sensitivity p<0.031) and dermatome (mechanical, vibration detection p<0.001), consistent with a nerve root lesion. Pain descriptors did not differ between the two groups with the exception of numbness (p<0.001). Patients with radicular pain did not report symptoms of numbness, while 78% of patients with radiculopathy did. Conclusions Distinct differences in somatosensory profiles and pain experiences were demonstrated for each patient group, suggesting differing underlying pain mechanisms.