Explaining the impact of mutations on quantification of SARS-CoV-2 in wastewater.

Wastewater surveillance is an effective tool for monitoring community spread of COVID-19 and other diseases. Quantitative PCR (qPCR) analysis for wastewater surveillance is more susceptible to mutations in target genome regions than binary PCR analysis for clinical surveillance. The SARS-CoV-2 concentrations in wastewater estimated by N1 and N2 qPCR assays started to diverge around July 2022 in data from different sampling sites, analytical methods, and analytical laboratories in Japan. On the basis of clinical genomic surveillance data and experimental data, we demonstrate that the divergence is due to two mutations in the N1 probe region, which can cause underestimation of viral concentrations. We further show that this inaccuracy can be alleviated if the qPCR data are analyzed with the second derivative method or the Cy0 method instead of the crossing point method.

A quantitative assay system for protein C activity, the regulator of blood coagulation, based on a chromogenic method mimicking the blood coagulation cascade.

Background and aims: Protein C is a plasma protein, and its active form regulates blood coagulation. The recommended unit of protein C activity is IU/mL; however, some laboratories use percentage. Some deficiencies cannot be detected owing to measurement principles. This study sought to quantify protein C activity levels and overcome the limitations of the current measurements. Materials and methods: Our protein C activity measurement method mimicked the blood coagulation cascade and used a thrombin-specific chromogenic reagent. The control was prepared by adding protein C to the protein C deficient plasma. The calibration curve was plotted as the increase in the absorbance per minute and the concentration of protein C in the control. Statistical tests were performed to compare our method with the current chromogenic method. Results: A calibration curve was constructed (y = -0.0132x + 0.14, R2 = 0.9987, n = 10). The statistical results of our method suggested non-inferiority when compared to the current chromogenic method (α = 0.05). Conclusion: The quantitative measurement was performed using plasma samples. Our method provides the possibility of expressing protein C activity quantitatively and detecting deficiencies that cannot be detected using the current chromogenic method.

Genetic Study of Four Candidate Holliday Junction Processing Proteins in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius.

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.

Manipulation of Intraday Durations of Blue- and Red-Light Irradiation to Improve Cos Lettuce Growth.

The morphology of plants growing under combined blue- and red-light irradiation is affected by the presence or absence of time slots of blue- and red-light mono-irradiation. The purposes of this study were to investigate the morphology and growth of cos lettuce grown under light irradiation combining several durations of blue and red light simultaneously and independent mono-irradiations of blue and red light during the day, and to clarify the effects of the durations of blue-light mono-irradiation and blue-light irradiation. Young cos lettuce seedlings were grown under 24-h blue-light irradiation with a photosynthetic photon flux density (PPFD) of 110µmol m-2 s-1 (B+0R) or under 24-h blue-light irradiation with a PPFD of 100µmol m-2 s-1 supplemented with 8 (B+8R), 16 (B+16R), and 24-h (B+24R) red-light irradiation with PPFDs of 30, 15, and 10µmol m-2 s-1, respectively (Experiment 1). The daily light integral was 9.50mol m-2 in all treatments. In Experiment 1, leaf elongation was promoted as the duration of red-light irradiation decreased and the duration of blue-light mono-irradiation increased. The maximum shoot dry weight was observed under the B+8R treatment. Growth was likely promoted by the expansion of the light-receptive area caused by moderate leaf elongation without tilting. In Experiment 2, young cos lettuce seedlings were grown as for Experiment 1, but blue- and red-light irradiation intensities were reversed (R+0B, R+8B, R+16B, and R+24B). Leaf elongation was promoted by the absence of blue-light irradiation (R+0B). The leaf surface was increasingly flattened, and the shoot dry weight was enhanced, as the duration of blue-light irradiation increased. Thus, cos lettuce leaf morphology may be manipulated by adjusting each duration of blue-light mono-irradiation, red-light mono-irradiation, and blue- and red-light simultaneous irradiation, which can, in turn, promote cos lettuce growth.

Binaural-centered mode-matching method for enhanced reproduction accuracy at listener's both ears in sound field reproduction.

This paper proposes a binaural-centered mode-matching (BCMM) method that performs sound field reproduction with two reproduction points, one at each ear. A sound field reproduced by higher-order Ambisonics converges to the target field around a sweet spot whose size is inversely proportional to the frequency when the truncated order of the spherical harmonic expansion is constant. By contrast, BCMM translates the spherical harmonic coefficients on the basis of the addition theorem to realize two reproduction points at both ear positions. The BCMM method thereby avoids degradation of reproduction due to a smaller sweet spot at higher frequencies, as occurs with the conventional method, and thereby, leads to an accurate reproduction at higher frequencies. A comparison of numerical simulations of the binaural signals obtained when rendering with BCMM and conventional methods shows that, compared with the conventional method, the BCMM method improves the reproduction accuracy.

Complete Genome and Plasmid Sequences of the Psychrotolerant Aureimonas Strain SA4125, Isolated from Antarctic Moss Vegetation.

The complete genome sequences of Aureimonas sp. strain SA4125 and its native plasmid pSA4125 were determined. The genome sequence comprises 4,968,066 bp, with a GC content of 66.0%, and contains 4,691 coding DNA sequences (CDSs), 3 rRNA operons, and 50 tRNAs. The native plasmid comprises 131,777 bp, with a GC content of 62.3%, and contains 138 CDSs.

Effects of Artificially Reproduced Fluctuations in Sunlight Spectral Distribution on the Net Photosynthetic Rate of Cucumber Leaves.

The effects of photosynthetic photon flux density (PPFD) fluctuations in sunlight have already been investigated; however, the spectral photon flux density distribution (SPD) has hardly been considered. Here, sunlight SPD fluctuations recorded for 200 min in October in Tokyo, Japan were artificially reproduced using an LED-artificial sunlight source system. The net photosynthetic rate (P n) of cucumber leaves under reproduced sunlight was measured and compared with the P n estimated from a steady-state PPFD-P n curve for the same leaves. The measured and estimated P n agreed except when the PPFD was low, where the measured P n was lower than the estimated P n. The ratio of measured P n to estimated P n was 0.94-0.95 for PPFD ranges of 300-700 µmol m-2 s-1, while the value was 0.98-0.99 for 900-1,300 µmol m-2 s-1, and the overall ratio was 0.97. This 3% reduction in the measured P n compared with the P n estimated from a steady-state PPFD-P n curve was significantly smaller than the approximately 20-30% reduction reported in previous experimental and simulation studies. This result suggests that the loss of integral net photosynthetic gain under fluctuating sunlight can vary among days with different fluctuation patterns or may be non-significant when fluctuations in both PPFD and relative SPD of sunlight are taken into consideration.

Repeated standing back extension exercise: Influence on muscle shear modulus change after lumbodorsal muscle fatigue.

BACKGROUND: In low back pain, multifidus muscle fibers reportedly exhibit increased stiffness. Low back pain was associated with lumbodorsal muscle fatigue. There is no report of using shear modulus to verify the mechanism of an immediate effect of exercise on low back pain. Here, temporary lumbodorsal muscle fatigue was created, simulating fatigue-related nonspecific low back pain. OBJECTIVE: To assess the effect of standing back extension exercise on fatigued lumbodorsal muscle based on the results of multifidus muscle elasticity measured using shear wave elastography. METHODS: Thirty-three healthy subjects were randomly divided into three groups. The subjects performed the Biering-Sorensen test as the fatigue-task of the lumbodorsal muscle before the standing back extension exercise. The fatigue-exercise group exercised five sets after completing the fatigue-task. The fatigue-non-exercise group remained standing for the same duration as the fatigue-exercise group without doing the exercise after the fatigue-task. The non-fatigue-exercise group exercised five sets of without performing the fatigue-task. As intra-group and inter-group factors, the shear modulus of the multifidus muscle was compared before and after the exercise. RESULTS: The shear modulus of the multifidus muscle after the standing back extension exercise was significantly lower in the fatigue-exercise group, and no significant decrease was observed in the fatigue-non-exercise and non-fatigue-exercise group. CONCLUSIONS: The standing back extension exercise improved the shear modulus of the fatigued multifidus muscle. Therefore, it was suggested that the change in the elasticity of fatigued muscle might lead to the prevention of low back pain caused by muscle fatigue.

Estimating Light Acclimation Parameters of Cucumber Leaves Using Time-Weighted Averages of Daily Photosynthetic Photon Flux Density.

Leaves acclimate to day-to-day fluctuating levels of photosynthetic photon flux density (PPFD) by adjusting their morphological and physiological parameters. Accurate estimation of these parameters under day-to-day fluctuating PPFD conditions benefits crop growth modeling and light environment management in greenhouses, although it remains challenging. We quantified the relationships between day-to-day PPFD changes over 6 days and light acclimation parameters for cucumber seedling leaves, including leaf mass per area (LMA), chlorophyll (Chl) a/b ratio, maximum net photosynthetic rate (P nmax), maximum rate of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (V cmax), and maximum rate of electron transport (J max). The last two parameters reflect the capacity of the photosynthetic partial reactions. We built linear regression models of these parameters based on average or time-weighted averages of daily PPFDs. For time-weighted averages of daily PPFDs, the influence of daily PPFD was given a specific weight. We employed three types of functions to calculate this weight, including linear, quadratic, and sigmoid derivative types. We then determined the trend of weights that estimated each parameter most accurately. Moreover, we introduced saturating functions to calibrate the average or time-weighted averages of daily PPFDs, considering that light acclimation parameters are usually saturated under high PPFDs. We found that time-weighted average PPFDs, in which recent PPFD levels had larger weights than earlier levels, better estimated LMA than average PPFDs. This suggests that recent PPFDs contribute more to LMA than earlier PPFDs. Except for the Chl a/b ratio, the average PPFDs estimated P nmax, V cmax, and J max with acceptable accuracy. In contrast, time-weighted averages of daily PPFDs did not improve the estimation accuracy of these four parameters, possibly due to their low response rates and plasticity. Calibrating functions generally improved estimation of Chl a/b ratio, V cmax, and J max because of their saturating tendencies under high PPFDs. Our findings provide a reasonable approach to quantifying the extent to which the leaves acclimate to day-to-day fluctuating PPFDs, especially the extent of LMA.

Reproducibility of elastic modulus measurement of the multifidus using the shear wave elastography function of an ultrasound diagnostic device.

[Purpose] This study aimed to obtain evidence of the musculo-physiological involvement in the effect of physiotherapy on low back pain by examining the reproducibility of elasticity measurements of the multifidus muscle at different trunk angles via the shear wave elastography function of an ultrasound diagnostic device. [Participants and Methods] This study included 11 healthy adults. Measurements were conducted with participants in the prone position, and the elasticity of the superficial and deeper layers of the multifidus muscle was measured under the following 3 conditions: trunk at neutral position, trunk flexed at 40°, and trunk extended at 20°. Next, intraclass correlation coefficients (1, 1) were calculated to examine the intrarater reliability. [Results] All intraclass correlation coefficients for the superficial and deeper layers of the multifidus muscle were ≥0.85 for all 3 conditions. [Conclusion] Regardless of the trunk position, the elastic modulus measurement of inner muscles via shear wave elastography serves as an assessment of biological changes in individuals with lower back pain in response to interventions.

WASH phosphorylation balances endosomal versus cortical actin network integrities during epithelial morphogenesis.

Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation.

Change in the circulation and activity of the lower erector spinae muscles after repeated trunk extension movement.

BACKGROUND: The optimum repetition number of standing back-extension exercise (SBEE) effective for the prevention and improvement of low back pain (LBP) is unknown. OBJECTIVE: To determine the effect of physiotherapy on LBP by investigating the optimum repetition number of SBEE via optical analysis and electromyographic (EMG) examination of the multifidus muscles. METHODS: Hemodynamics and multifidus muscle activity were examined in 16 healthy adult men using near-infrared spectroscopy and surface EMG after performing repetitive SBEE. RESULTS: Oxidized hemoglobin (Oxy-Hb) levels significantly increased in the second extension phase but decreased in the third and subsequent extension phases; deoxidized hemoglobin (deOxy-Hb) levels increased in the third and subsequent extension phases. In the standing phase, no significant difference was observed; in the third and subsequent phases, Oxy-Hb levels decreased and deOxy-Hb levels increased. Muscular activity significantly decreased in the second standing phase but increased in the third and subsequent phases. No significant difference was observed in the extension phase with respect to the number of SBEE repetitions performed. In healthy individuals, hemodynamics improved up to second repetition of SBEE; subsequent repetitions may decrease hemodynamics because of increased activity of the multifidus muscles. CONCLUSIONS: In healthy individuals, hemodynamics improved up to second repetition of SBEE.

Gas Exchange Rates Decrease and Leaf Temperature Increases in Nicotiana benthamiana Leaves Transiently Overexpressing Hemagglutinin in an Agrobacterium-Assisted Viral Vector System.

In this study, gas exchange characteristics and temperature of Nicotiana benthamiana leaves transiently overexpressing hemagglutinin (HA), an influenza vaccine antigen, with an Agrobacterium tumefaciens-assisted viral vector were investigated. Inoculation of leaves with an empty viral vector not containing the HA gene decreased the net photosynthetic rate (Pn) and transpiration rate (T) from 2 to 3 days post-infiltration (DPI) in the A. tumefaciens suspension. Expression of HA with the vector decreased Pn and T to much lower levels until 4 DPI. Such significant decreases were not observed in leaves infiltrated with suspension of A. tumefaciens not carrying the viral vector or in uninfiltrated leaves. Thus, viral vector inoculation itself decreased Pn and T to a certain extent and the HA expression further decreased them. The decreases in Pn and T in empty vector-inoculated and HA expression vector-inoculated leaves were associated with decreases in stomatal conductance, suggesting that the reduction of gas exchange rates was caused at least in part by stomatal closure. More detailed gas exchange and chlorophyll fluorescence analyses revealed that in HA vector-inoculated leaves, the capacity of ribulose-1,5-bisphosphate carboxylase/oxygenase to assimilate CO2 and the capacity of photosynthetic electron transport in planta were downregulated, which contributed also to the decrease in Pn. Leaf temperature (LT) increased in viral vector-inoculated leaves, which was associated with the decrease in T. When HA vector-inoculated leaves were grown at air temperatures (ATs) of 21, 23, and 26°C post-infiltration, HA accumulated earlier in leaves and the days required for HA content to attain its peak became shorter, as AT was higher. The highest LT was found 1-2 days earlier than the highest leaf HA content under all post-infiltration AT conditions. This phenomenon could be applicable in a non-destructive technique to detect the optimum harvesting date for individual plants to determine the day when leaf HA content reaches its maximum level, irrespective of spatiotemporal variation of AT, in a plant growth facility.

A Mathematical Model of Photosynthetic Electron Transport in Response to the Light Spectrum Based on Excitation Energy Distributed to Photosystems.

To enable us to analyze more systematically the effects of the spectral distribution of light (i.e. light quality) on photosynthetic electron transport, we propose a simple mathematical model which describes electron transport reactions under light-limited conditions based on the excitation energy distributed to the photosystems. The model assumes that the rate-limiting photosystem performs the photochemical reaction at its maximum yield, while the yield in the other photosystem is passively down-regulated to equalize the rates of linear electron transport through the photosystems. Using intact cucumber leaves, we tested the model by comparing actual and estimated photosynthetic parameters under several combinations of photon flux densities of red and far-red lights (R and FR, respectively). Simultaneously provided R and FR yielded greater gross photosynthetic rates than the sums of the rates under only R and only FR, which is known as the 'enhancement effect'. The present model reproduced these non-additive increases in the gross photosynthetic rates in response to supplemental FR to R and provided more accurate estimates than an existing method that did not take the enhancement effect into account (root mean square errors: 0.11 and 0.21 µmol m-2 s-1, respectively). Using the present model, the photon flux density of the supplemental FR which gives the changing point of rate-limiting photosystem and the photochemical yields of the non-rate-limiting photosystems were estimated reasonably well. The present study has therefore formulated a simplified quantitative electron transport model in response to the light spectrum based on generally accepted concepts and demonstrated its validity experimentally.

Effects of photosynthetic photon flux density, frequency, duty ratio, and their interactions on net photosynthetic rate of cos lettuce leaves under pulsed light: explanation based on photosynthetic-intermediate pool dynamics.

Square-wave pulsed light is characterized by three parameters, namely average photosynthetic photon flux density (PPFD), pulsed-light frequency, and duty ratio (the ratio of light-period duration to that of the light-dark cycle). In addition, the light-period PPFD is determined by the averaged PPFD and duty ratio. We investigated the effects of these parameters and their interactions on net photosynthetic rate (P n) of cos lettuce leaves for every combination of parameters. Averaged PPFD values were 0-500 µmol m-2 s-1. Frequency values were 0.1-1000 Hz. White LED arrays were used as the light source. Every parameter affected P n and interactions between parameters were observed for all combinations. The P n under pulsed light was lower than that measured under continuous light of the same averaged PPFD, and this difference was enhanced with decreasing frequency and increasing light-period PPFD. A mechanistic model was constructed to estimate the amount of stored photosynthetic intermediates over time under pulsed light. The results indicated that all effects of parameters and their interactions on P n were explainable by consideration of the dynamics of accumulation and consumption of photosynthetic intermediates.

Quantification of excitation energy distribution between photosystems based on a mechanistic model of photosynthetic electron transport.

Absorbed light energy is converted into excitation energy. The excitation energy is distributed to photosystems depending on the wavelength and drives photochemical reactions. A non-destructive, mechanistic and quantitative method for estimating the fraction of the excitation energy distributed to photosystem II (f) was developed. For the f values for two simultaneously provided actinic lights (ALs) with different spectral distributions to be estimated, photochemical yields of the photosystems were measured under the ALs and were then fitted to an electron transport model assuming the balance between the electron transport rates through the photosystems. For the method to be tested using leaves with different properties in terms of the long-term and short-term acclimation (adjustment of photosystem stoichiometry and state transition, respectively), the f values for red and far-red light (R and FR) were estimated in leaves grown (~1 week) under white light without and with supplemental FR and adapted (~10 min) to R without and with supplemental FR. The f values for R were clearly greater than those for FR and those of leaves grown with and adapted to supplemental FR tended to be higher than the controls. These results are consistent with previous studies and therefore support the validity of the proposed method.

Genome-wide identification of Grainy head targets in Drosophila reveals regulatory interactions with the POU domain transcription factor Vvl.

Grainy head (Grh) is a conserved transcription factor (TF) controlling epithelial differentiation and regeneration. To elucidate Grh functions we identified embryonic Grh targets by ChIP-seq and gene expression analysis. We show that Grh controls hundreds of target genes. Repression or activation correlates with the distance of Grh-binding sites to the transcription start sites of its targets. Analysis of 54 Grh-responsive enhancers during development and upon wounding suggests cooperation with distinct TFs in different contexts. In the airways, Grh-repressed genes encode key TFs involved in branching and cell differentiation. Reduction of the POU domain TF Ventral veins lacking (Vvl) largely ameliorates the airway morphogenesis defects of grh mutants. Vvl and Grh proteins additionally interact with each other and regulate a set of common enhancers during epithelial morphogenesis. We conclude that Grh and Vvl participate in a regulatory network controlling epithelial maturation.