Continuous,high-resolution biospeckle imaging reveals a discrete zone of activity at the root apex that responds to contact with obstacles

Background and Aims

Shining a laser onto biological material produces light speckles termed biospeckles. Patterns of biospeckle activity reflect changes in cell biochemistry, developmental processes and responses to the environment. The aim of this work was to develop methods to investigate the biospeckle activity in roots and to characterize the distribution of its intensity and response to thigmostimuli.

Methods

Biospeckle activity in roots of Zea mays, and also Jatropha curcas and Citrus limonia, was imaged live and in situ using a portable laser and a digital microscope with a spatial resolution of 10 μm per pixel and the ability to capture images every 0·080 s. A procedure incorporating a Fujii algorithm, image restoration using median and Gaussian filters, image segmentation using maximum-entropy threshold methods and the extraction of features using a tracing algorithm followed by spline fitting were developed to obtain quantitative information from images of biospeckle activity. A wavelet transform algorithm was used for spectral decomposition of biospeckle activity and generalized additive models were used to attribute statistical significance to changes in patterns of biospeckle activity.

Key Results

The intensity of biospeckle activity was greatest close to the root apex. Higher frequencies (3–6 Hz) contributed most to the total intensity of biospeckle activity. When a root encountered an obstacle, the intensity of biospeckle activity decreased abruptly throughout the root system. The response became attenuated with repeated thigmostimuli.

Conclusions

The data suggest that at least one component of root biospeckle activity resulted from a biological process, which is located in the zone of cell division and responds to thigmostimuli. However, neither individual cell division events nor root elongation is likely to be responsible for the patterns of biospeckle activity.     

Live biospeckle laser imaging of root tissues

Live imaging is now a central component for the study of plant developmental processes. Currently, most techniques are extremely constraining: they rely on the marking of specific cellular structures which generally apply to model species because they require genetic transformations. The biospeckle laser (BSL) system was evaluated as an instrument to measure biological activity in plant tissues. The system allows collecting biospeckle patterns from roots which are grown in gels. Laser illumination has been optimized to obtain the images without undesirable specular reflections from the glass tube. Data on two different plant species were obtained and the ability of three different methods to analyze the biospeckle patterns are presented. The results showed that the biospeckle could provide quantitative indicators of the molecular activity from roots which are grown in gel substrate in tissue culture. We also presented a particular experimental configuration and the optimal approach to analyze the images. This may serve as a basis to further works on live BSL in order to study root development.     

Biospeckle‐characterization of hairy root cultures using laser speckle photometry

Monitoring is indispensable for the optimization and simulation of biotechnological processes. Hairy roots (hr, plant tissue cultures) are producers of valuable relevant secondary metabolites. The genetically stable cultures are characterized by a rapid filamentous growth, making monitoring difficult with standard methods. This article focuses on the application of laser speckle photometry (LSP) as an innovative, non‐invasive method to characterize Beta vulgaris (hr). LSP is based on the analysis of time‐resolved interference patterns. Speckle interference patterns of a biological object, known as biospeckles, are characterized by a dynamic behavior that is induced by physical and biological phenomena related to the object. Speckle contrast, a means of measuring the dynamic behavior of biospeckles, was used to assess the biospeckle activity. The biospeckle activity corresponds to processes modifying the object and correlates with the biomass growth. Furthermore, the stage of the cultures’ physiological development was assessed by speckle contrast due to the differentiation between active and low active behavior. This method is a new means of monitoring and evaluating the biomass growth of filamentous cultures in real time. As a potential tool to characterize hairy roots, LSP is non‐invasive, time‐saving, can be used online and stands out for its simple, low‐cost setup.     

The effects of cytohalasin B and dimethyl sulfoxide on the movement of mouse kidney cells. I. Effect on the outgrowth of cells

The following experiments were undertaken to determine the effect of cytochalasin B (CB) on the movement of mouse kidney epitheliocytes in primary culture. Fragments of mouse kidney were cultured in Sykes-Moore chambers with control medium (McCoy's 5a, modified), CB in dimethyl sulfoxide (DMSO), and DMSO. CB, 10 μg/ml, in 1% DMSO completely inhibited the outgrowth of cells from the kidney fragments. These effects were reversible. Unexpectedly, 1% DMSO suppressed the outgrowth of the kidney cells. A myriad of phenomenological effects of CB on mammalian cells have been reported in the recent literature. Most investigators report no effect of DMSO on the cells. These results suggest that DMSO is affecting the movement of mouse kidney cells.     

Electrical potential oscillations – movement relations in circumnutating sunflower stem and effect of ion channel and proton pump inhibitors on circumnutation

The physiological control and molecular mechanism of circumnutation (CN) has not yet been fully understood. To gain information on the CN mechanism, the relationship between the changes of electrical potential and movement in the circumnutating sunflower stem and effect of ion channels and proton pump inhibitors on CN parameters were evaluated. Long‐term electrophysiological measurements and injection of solutions of ion channel inhibitors (ICI) into sunflower stem with the simultaneous time‐lapse recording of the movement were made. The oscillations of electrical potential (OEP) – movement relations – consist of cells depolarization on the deflected side of the stem and, at this same time, cells hyperpolarization on the opposite side of the stem. The delay of organ movement in relation to electrical changes of approximately 28 min (22% of the period) may indicate that the ionic fluxes causing the OEP are the primary phenomenon. The biggest decrease of CN period was observed after injection of proton pump (approximately 26%) and cation channel (approximately 25%) inhibitors, while length and amplitude were reduced mainly by calcium channel inhibitors (approximately 67%). Existence of OEP only in circumnutating part of sunflower stem and reduction of CN parameters and OEP amplitude after application of ICI prove that the CN cellular mechanism is associated with transmembrane ion transport.     

Inhibition of anodic galvanotaxis of green paramecia by T-type calcium channel inhibitors

Calcium ion (Ca2+) is one of the key regulatory elements for ciliary movements in the Paramecium species. It has long been known that members of Paramecium species including green paramecia (Paramecium bursaria) exhibit galvanotaxis which is the directed movement of cells toward the anode by swimming induced in response to an applied voltage. However, our knowledge on the mode of Ca2+ action during green paramecia anodic galvanotactic response is still largely limited. In the present study, quantification of anodic galvanotaxis was carried out in the presence and absence of various inhibitors of calcium signaling and calcium channels. Interestingly, galvanotactic movement of the cells was completely inhibited by a variety of Ca2+-related inhibitors. Such inhibitors include a Ca2+ chelator (EGTA), general calcium channel blockers (such as lanthanides), inhibitors of intracellular Ca2+ release (such as ruthenium red and neomycin), and inhibitors of T-type calcium channels (such as NNC 55-0396, 1-octanol and Ni2+). However, L-type calcium channel inhibitors such as nimodipine, nifedipine, verapamil, diltiazem and Cd2+ showed no inhibitory action. This may be the first implication for the involvement of T-type calcium channels in protozoan cellular movements.     

Growth inhibition of murine melanoma by butyric acid and dimethylsulfoxide

Treatment of B16-F10 melanoma cells with dimethylsulfoxide (DMSO) or butyric acid (BA) inhibits cell growth and delays tumor appearance in syngeneic mice. Both agents induce morphological changes in these cells. Treatment of melanoma cells with DMSO results in a marked increase in tyrosinase activity and melanin content. BA, on the other hand, does not increase melanin content and decreases tyrosinase activity. The data show that there are marked differences in the effect of DMSO and BA on melanin biosynthesis, whereas both agents inhibit cell growth and cause a delay in tumor appearance. These findings indicate that decreased proliferation of melanoma cells and induction of melanin biosynthesis are not necessarily associated phenomena.     

Inhibitors such as staurosporine,H-7 or polymyxin B cannot be used in skeletal muscle to prove the role of protein kinase C on insulin action

The precise role of protein kinase C in insulin action in skeletal muscle is not well defined. Based on the fact that inhibitors of protein kinase C block some insulin effects, it has been concluded that some of the biological actions of insulin are mediated via protein kinase C. In this study, we present evidence that inhibitors of protein kinase C such as staurosporine, H-7 or polymyxin B cannot be used to ascertain the role of protein kinase C in skeletal muscle. This is based on the following experimental evidences: a) staurosporine, H-7 and polymyxin B markedly block in muscle the effect of insulin on System A transport activity; however, this effect of insulin is not mimicked in muscle by TPA-induced stimulation of protein kinase C, b) H-7 and polymyxin B block insulin action on System A transport activity in an additive manner to the inhibitory effect of phorbol esters, c) staurosporine, H-7 and polymyxin B block the effect of insulin on lactate production, a process that is activated by insulin and TPA in an additive fashion, and d) staurosporine completely blocks the tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle.Abbreviations MeAIB a-(methyl)aminoisobutyric acid - TPA 12-O-tetradecanoylphorbol-13-acetate - H-7 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine     

TRPP2 ion channels: Critical regulators of organ morphogenesis in health and disease

Ion channels control the membrane potential and mediate transport of ions across membranes. Archetypical physiological functions of ion channels include processes such as regulation of neuronal excitability, muscle contraction, or transepithelial ion transport. In that regard, transient receptor potential ion channel polycystin 2 (TRPP2) is remarkable, because it controls complex morphogenetic processes such as the establishment of properly shaped epithelial tubules and left-right-asymmetry of organs. The fascinating question of how an ion channel regulates morphogenesis has since captivated the attention of scientists in different disciplines. Four loosely connected key insights on different levels of biological complexity ranging from protein to whole organism have framed our understanding of TRPP2 physiology: 1) TRPP2 is a non-selective cation channel; 2) TRPP2 is part of a receptor-ion channel complex; 3) TRPP2 localizes to primary cilia; and 4) TRPP2 is required for organ morphogenesis. In this review, we will discuss the current knowledge in these key areas and highlight some of the challenges ahead.     

The use of dimethylsulfoxide as a solvent in enzyme inhibition studies: the case of aldose reductase

Aldose reductase (AR) is an enzyme devoted to cell detoxification and at the same time is strongly involved in the aetiology of secondary diabetic complications and the amplification of inflammatory phenomena. AR is subjected to intense inhibition studies and dimethyl sulfoxide (DMSO) is often present in the assay mixture to keep the inhibitors in solution. DMSO was revealed to act as a weak but well detectable AR differential inhibitor, acting as a competitive inhibitor of the L-idose reduction, as a mixed type of non-competitive inhibitor of HNE reduction and being inactive towards 3-glutathionyl-4-hydroxynonanal transformation. A kinetic model of DMSO action with respect to differently acting inhibitors was analysed. Three AR inhibitors, namely the flavonoids neohesperidin dihydrochalcone, rutin and phloretin, were used to evaluate the effects of DMSO on the inhibition studies on the reduction of L-idose and HNE.     

Crystal structure of Natratoxin, a novel snake secreted phospholipaseA2 neurotoxin from Naja atra venom inhibiting A-type K+ currents

Snake secreted phospholipasesA2 (sPLA2s) are widely used as pharmacological tools to investigate their role in diverse pathophysiological processes. Some members of snake venom sPLA2s have been found to block voltage-activated K(+) channels (K(v) channels). However, most studies involved in their effects on ion channels were indirectly performed on motor nerve terminals while few studies were directly done on native neurons. Here, a novel snake sPLA2 peptide neurotoxin, Natratoxin, composed of 119 amino acid residues and purified from Naja atra venom was reported. It was characterized using whole-cell patch-clamp in acutely dissociated rat dorsal root ganglion (DRG) neurons. It was found to effectively inhibit A-type K(+) currents and cause alterations of channel gating characters, such as the shifts of steady-state activation and inactivation curves to hyperpolarization direction and changes of V(1/2) and slope factor. Therefore, Natratoxin was suggested to be a gating modifier of K(v) channel. In addition, this inhibitory effect was found to be independent of its enzymatic activity. These results suggested that the toxin enacted its inhibitory effect by binding to K(v) channel. To further elucidate the structural basis for this electrophysiological phenomenon, we determined the crystal structure of Natratoxin at 2.2 A resolution by molecular replacement method and refined to an R-factor of 0.190. The observed overall fold has a different structural organization from other K(+) channel inhibitors in animal toxins. Compared with other K(v) channel inhibitors, a similar putative functional surface in its C-terminal was revealed to contribute to protein-protein interaction in such a blocking effect. Our results demonstrated that the spatial distribution of key amino acid residues matters most in the recognition of this toxin towards its channel target rather than its type of fold.     

Interleukin-4 activates ion channels in B lymphocytes.

The lymphokine interleukin-4 (IL-4) has been shown to induce dramatic changes in the physiology of resting B cells. We have applied the patch clamp technique in the cell attached and inside/out configurations to resting and IL-4-treated B cells to determine whether specific ion conductances result as a consequence of IL-4 action. We report here that two distinct ion channel events occur in B lymphocytes after treatment with IL-4, (i) induction of an inward rectifying K+ channel that is not observed in untreated cells, and (ii) activation of a large conductance anion channel that is normally silent in non-treated cells in the cell attached patch configuration. These data present the first evidence of a direct effect by IL-4 on ion channels and we suggest roles for these two ionic conductances in IL-4-induced B cell activation.     

The effects of boric acid on sister chromatid exchanges and chromosome aberrations in cultured human lymphocytes

The aim of this study was to determine the possible genotoxic effects of boric acid (BA) (E284), which is used as an antimicrobial agent in food, by using sister chromatid exchange (SCEs) and chromosome aberration (CAs) tests in human peripheral lymphocytes. The human lymphocytes were treated with 400, 600, 800, and 1000 μg/mL concentrations of BA dissolved in dimethyl sulfoxide (DMSO), for 24 h and 48 h treatment periods. BA did not increase the SCEs for all the concentrations and treatment periods when compared to control and solvent control (DMSO). BA induced structural and total CAs at all the tested concentrations for 24 and 48 h treatment periods. The induction of the total CAs was dose dependent for the 24 h treatment period. However, BA did not cause numerical CAs. BA showed a cytotoxic effect by decreasing the replication index (RI) and mitotic index (MI). BA decreased the MI in a dose-dependent manner for the 24 h treatment period.     

Biospeckle activity in coffee seeds is associated non‐destructively with seedling quality

Seeds age during storage, resulting in a decline in germination and seedling quality. Seed quality tests are important to monitor this decline. However, such tests are usually destructive and require large seed numbers and long time. For coffee seeds the standard germination test and assessment of seedling quality takes 30 days. Biospeckle has been used previously as a non‐destructive optical tool to monitor biological activity in a range of tissues. Biospeckle was applied 3–6 days after imbibition (DAI) to investigate an association with coffee seedling quality after 30 days. Two distinct areas of biospeckle activity were demonstrated, concurring with the locations of the embryonic axis and the cotyledons in the apical and central seed parts, respectively. Moisture content analysis revealed that embryos of imbibed seeds contained more water than endosperm. Different areas within the endosperm did not differ in moisture content, while the moisture content of the axis was higher than that of the cotyledons, and this did not change from 4 DAI. Therefore, it was concluded that high biospeckle activity was not the result of increased water content in any seed part, but more likely of growth and metabolism in the axis and cotyledons, which had been described previously. A threshold biospeckle ratio apical : central of 1.02 after 6 days distinguished between seeds that produced dead and viable seedlings after 30 days and provided similar results as a tetrazolium test, a widely acknowledged but destructive test for seed quality. Thus, biospeckle data provided a non‐destructive early parameter for seedling quality, based on embryo growth during germination.     

PDZ domains and their binding partners: structure, specificity, and modification

PDZ domains are abundant protein interaction modules that often recognize short amino acid motifs at the C-termini of target proteins. They regulate multiple biological processes such as transport, ion channel signaling, and other signal transduction systems. This review discusses the structural characterization of PDZ domains and the use of recently emerging technologies such as proteomic arrays and peptide libraries to study the binding properties of PDZ-mediated interactions. Regulatory mechanisms responsible for PDZ-mediated interactions, such as phosphorylation in the PDZ ligands or PDZ domains, are also discussed. A better understanding of PDZ protein-protein interaction networks and regulatory mechanisms will improve our knowledge of many cellular and biological processes.     

Identification of the pore-lining residues of the BM2 ion channel protein of influenza B virus

The influenza B virus BM2 proton-selective ion channel is essential for virus uncoating, a process that occurs in the acidic environment of the endosome. The BM2 channel causes acidification of the interior of the virus particle, which results in dissociation of the viral membrane protein from the ribonucleo-protein core. The BM2 protein is similar to the A/M2 protein ion channel of influenza A virus (A/M2) in that it contains an HXXXW motif. Unlike the A/M2 protein, the BM2 protein is not inhibited by the antiviral drug amantadine. We used mutagenesis to ascertain the pore-lining residues of the BM2 ion channel. The specific activity (relative to wild type), reversal voltage, and susceptibility to modification by (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide of cysteine mutant proteins were measured in oocytes. It was found that mutation of transmembrane domain residues Ser(9), Ser(12), Phe(13), Ser(16), His(19), and Trp(23) to cysteine were most disruptive for ion channel function. These cysteine mutants were also most susceptible to (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide modification. Furthermore, considerable amounts of dimer were formed in the absence of oxidative reagents when cysteine was introduced at positions Ser(9), Ser(12), Ser(16), or Trp(23). Based on these experimental data, a BM2 transmembrane domain model is proposed. The presence of polar residues in the pore is a probable explanation for the amantadine insensitivity of the BM2 protein and suggests that related but more polar compounds might serve as useful inhibitors of the protein.     

Mutation analysis of candidate genes SCN1B, KCND3 and ANK2 in patients with clinical diagnosis of long QT syndrome

The long QT syndrome (LQTS) is a monogenic disorder characterized by prolongation of the QT interval on electrocardiogram and syncope or sudden death caused by polymorphic ventricular tachycardia (torsades de pointes). In general, mutations in cardiac ion channel genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2) have been identified as a cause for LQTS. About 50-60 % of LQTS patients have an identifiable LQTS causing mutation in one of mentioned genes. In a group of 12 LQTS patients with no identified mutations in these genes we have tested a hypothesis that other candidate genes could be involved in LQTS pathophysiology. SCN1B and KCND3 genes encode ion channel proteins, ANK2 gene encodes cytoskeletal protein interacting with ion channels. To screen coding regions of genes SCN1B, KCND3, and 10 exons of ANK2 following methods were used: PCR, SSCP, and DNA sequencing. Five polymorphisms were found in screened candidate genes, 2 polymorphisms in KCND3 and 3 in SCN1B. None of found polymorphisms has coding effect nor is located close to splice sites or has any similarity to known splicing enhancer motifs. Polymorphism G246T in SCN1B is a novel one. No mutation directly causing LQTS was found. Molecular mechanism of LQTS genesis in these patients remains unclear.     

新型冠状病毒抗体和小分子抑制剂的研究现状

世界卫生组织已宣布新型冠状病毒感染（coronavirus disease 2019,COVID-19）的爆发为全球大流行。中和抗体和小分子抑制剂在预防及治疗COVID-19中发挥重要作用。尽管已开发出了多种中和抗体以及疫苗,但是随着病原体严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)的不断变异,现有的抗体及疫苗面临巨大的挑战。小分子抑制剂主要通过干扰病毒与宿主的结合以及病毒自身的复制达到消灭病毒以及抑制病毒感染的作用,并且对SARS-CoV-2突变株具有广谱抑制作用,是当前研究的热点。近年来国内外学者对SARS-CoV-2的小分子抑制剂做了大量的研究工作,本文根据中和抗体识别的抗原表位以及小分子抑制剂的作用机制分别对用于预防及治疗COVID-19的中和抗体和小分子抑制剂进行综述,讨论其研究现状,并展望小分子抑制剂的应用前景,以期为该领域的进一步研究提供参考。     

Differential effects of dimethyl sulfoxide and sodium butyrate on alpha-fetoprotein, albumin, and transferrin production by rat hepatomas in culture

Radioimmunoassay was used to determine alpha-fetoprotein (AFP), albumin, and transferrin production (ng/10(5) cells/24 h) by two cell lines (7777 and 8994) derived from chemically induced rat hepatomas. alpha-Fetoprotein production was high (2000 to 4400) in 7777, but was very low (0.2 to 0.4) in 8994. Albumin production varied from 0.4-0.8 (7777) to 14-26 (8994). Both lines produced substantial amounts of transferrin (180 to 240 by 7777 and 29 to 42 by 8994). Addition of dimethyl sulfoxide (DMSO, 1 to 4%) or sodium butyrate (BA, 0.5 to 2.0 mM) to the medium inhibited growth in both lines, but 8994 was more sensitive to these agents than 7777. Dimethyl sulfoxide treatment (2 to 4%) resulted in a dose-related decrease (less than 10% of control at 4% DMSO) in AFP, albumin, and transferrin production by 7777, but in 8994, DMSO (1 to 2%) resulted in an increase (up to sixfold) in albumin and transferrin production, without affecting AFP production. By contrast, BA (2 to 4 mM) stimulated the production of all three proteins in both lines, most notably that of albumin (up to sixfold) by 7777 and that of AFP (up to 20-fold) by 8994. It is concluded that both DMSO and BA can enhance the expression of differentiated functions of the hepatoma cell, and that DMSO at the same time can suppress the expression of an oncofetal function. However, neither DMSO nor BA is selective in its effects on specific genes (i.e., normal, adult vs. oncofetal genes), and it appears that their effects may be the result of a more general phenomenon, the expression of which may be related to the stage of differentiation of the cell.