1- Introduction

2- Material and methods

3- Results

4- Discussion

References

Abstract

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites.

Introduction

Mites and ticks are members of the chelicerates, the largest group of terrestrial animals after insects. The two-spotted spider mite, T. urticae, and other spider mites are important crop pests worldwide. This herbivore species is at the extreme end of the generalist-to specialist spectrum and can feed on a staggering 1,100 plant species. Not surprisingly, it is currently reported as the ‘most resistant’ pest worldwide, as it developed resistance to more than 90 acaricides (Mota-Sanchez and Wise, 2019; Van Leeuwen and Dermauw, 2016; Van Leeuwen et al., 2015). In 2011, a 90 Mb high-quality Sanger-sequenced genome became available for this species (Grbic et al., 2011). This allowed to disentangle some of the molecular mechanisms underlying resistance, whether to man-made pesticides or plant secondary compounds. The extreme adaptation potential of T. urticae was associated with specific gene expansions in known detoxification enzyme families, such as cytochrome P450 monooxygenases, glutathione-S-transferases, carboxyl-choline esterases, an unexpected repertoire of ABC and MFS transporters, and a proliferation of cysteine peptidases (Dermauw et al., 2013a; Dermauw et al., 2013b; Grbic et al., 2011; Santamaría et al., 2012).

Introduction

Materials and Methods

Results

Discussion

References

Abstract

Introduction: Elevated serum apolipoprotein B and the apolipoprotein B/A1 ratio have been associated with ischemic stroke and intracranial atherosclerotic disease. We sought to assess the relationship between serum levels of apolipoprotein B, apolipoprotein A1, and the apolipoprotein B/A1 ratio with ischemic stroke subtypes and large artery atherosclerosis location. Materials and Methods: We evaluated serum apolipoprotein B and apolipoprotein A1 levels in consecutive, statin-naïve, adult ischemic stroke patients admitted to an academic medical center in southern India. We evaluated for differences in the mean serum levels of apolipoprotein B, apolipoprotein A1, and the apolipoprotein B/A1 ratio between patients with ischemic stroke attributed to intracranial atherosclerotic disease, extracranial atherosclerotic disease, small vessel disease, and cardioembolism. In secondary analysis, we assessed for differences in these serum apolipoproteins between patients with moderate-severe intracranial atherosclerotic disease and extracranial atherosclerotic disease, irrespective of ischemic stroke subtype. Results: Among the 156 ischemic stroke patients enrolled in this study, there were no significant differences in serum levels of apolipoprotein B, apolipoprotein A1, and the apolipoprotein B/A1 ratio between patients with distinct ischemic stroke subtypes. No significant differences were found in serum levels of apolipoprotein B, A1 and the apolipoprotein B/A1 ratio between patients with moderate-severe intracranial atherosclerotic disease and moderate-severe extracranial atherosclerotic disease. Discussion: Serum levels of apolipoprotein B and A1 did not differ between ischemic stroke subtypes. Additional studies are needed to validate our findings and to better understand the relationship between serum apolipoproteins and stroke.

Introduction

Apolipoproteins (Apo) form the protein component of circulating lipoproteins and function in lipid transport and metabolism. Apo B is a major apolipoprotein component of the pro-atherogenic very low-, intermediate-, and low-density lipoproteins (LDL) whereas Apo A1 is the major apolipoprotein of high-density lipoprotein (HDL). Apo B has been suggested to be a better treatment target for statin therapy compared to LDL and a superior marker of cardiovascular disease risk.1 INTERSTROKE, an international case-control study, demonstrated that an elevated serum apolipoprotein (Apo) B/A1 ratio was associated with a higher odds of ischemic stroke. Elevated serum levels of Apo B and the Apo B/A1 ratio have been associated with ischemic stroke in case-control and population-based cohort studies, and have been demonstrated to be predictive of ischemic stroke in transient ischemic attack (TIA) patients. Elevated Apo B/A1 ratio has also been associated with carotid intima-media thickness among patients with cerebral infarction. Furthermore, higher serum Apo B and an elevated Apo B/A1 ratio have been shown to be independent predictors of intracranial atherosclerotic disease (ICAD) and have been associated with asymptomatic deep subcortical ischemia in patients with ICAD.

Stress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules form in part through intermolecular RNA-RNA interactions, and to better understand how RNA-based condensation occurs, we demonstrate that RNA is effectively recruited to the surfaces of RNA or RNP condensates in vitro. We demonstrate that, through ATP-dependent RNA binding, the DEADbox protein eIF4A reduces RNA condensation in vitro and limits stress granule formation in cells. This defines a function for eIF4A to limit intermolecular RNA-RNA interactions in cells. These results establish an important role for eIF4A, and potentially other DEAD-box proteins, as ATP-dependent RNA chaperones that limit the condensation of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates.

Introduction

Eukaryotic cells contain ribonucleoprotein (RNP) granules in the nucleus and cytosol, including P-bodies (PBs) and stress granules (SGs) (Anderson and Kedersha, 2006; Banani et al., 2017). SGs are cytosolic condensates composed of non-translating RNPs that are involved in the stress response, neurodegeneration, and viral infection (Protter and Parker, 2016; Ivanov et al., 2019). SGs typically form in response to translation shutoff induced by noxious stimuli such as arsenite, heat shock, and endogenous inflammatory molecules like prostaglandins, which all lead to phosphorylation of eIF2a and the activation of the integrated stress response (Aulas et al., 2017; Tauber and Parker, 2019). SGs can also form independently of eIF2a phosphorylation in response to inhibition of the eIF4F complex or osmotic stress (Aulas et al., 2017). SGs and other RNP condensates are thought to form in part through multimeric RNA binding proteins crosslinking RNPs into larger networks (Banani et al., 2017; Shin and Brangwynne, 2017). Recent evidence suggests that intermolecular RNA-RNA interactions are involved in SG formation. SGs require a substantial pool of non-translating RNA to form (Protter and Parker, 2016). Thus, elevating the non-translating RNA concentration by injecting exogenous RNA induces SGs (Mahadevan et al., 2013). Furthermore, certain RNAs can seed foci in human cell lysates that recruit many SG proteins (Fay et al., 2017). Strikingly, modest concentrations of yeast total RNA readily condense in physiological salt and polyamine conditions, recapitulating the SG transcriptome in a protein-free context (Khong et al., 2017; Van Treeck et al., 2018), arguing that trans RNA-RNA interactions contribute to SG formation. Messenger RNPs (mRNPs) are recruited to SGs in a biphasic manner, first engaging in transient docking interactions with the SG surface, which then transition into stable locking interactions that leave the RNA immobile within the granule (Moon et al., 2019), implying that surface recruitment of RNAs to SGs is a precursor to a more stable RNP assembly.

Translation of a genetic codon without a cognate tRNA gene is affected by both the cognate tRNA availability and the interaction with non-cognate isoacceptor tRNAs. Moreover, two consecutive slow codons (slow di-codons) lead to a much slower translation rate. Calculating the composition of host specific slow codons and slow di-codons in the viral protein coding sequences can predict the order of viral protein synthesis rates between different virus strains. Comparison of human-specific slow codon and slow di-codon compositions in the genomes of 590 coronaviruses infect humans revealed that the protein synthetic rates of 2019 novel coronavirus (2019-nCoV) and severe acute respiratory syndrome-related coronavirus (SARS-CoV) may be much faster than other coronaviruses infect humans. Analysis of host-specific slow codon and di-codon compositions provides links between viral genomic sequences and capability of virus replication in host cells that may be useful for surveillance of the transmission potential of novel viruses.

Introduction

No organism has a full set of tRNA species for all 61 amino acid-encoded codons.1 Codons without cognate tRNA gene can be referred to as slow codons of the organism. These codons are organism specific codons because different organisms have different tRNA gene compositions. For example, 13 amino acid-encoding codons have no cognate tRNA genes in the human genome. Studies have revealed by ribosomal profiling experiments that translation of codons by rare tRNAs and non-cognate isoacceptor tRNAs (by wobble base pairing of codons and tRNAs) reduces translation efficiency2,3, . 4 Moreover, it has been reported that the efficiency of translating a particular codon is affected by the nature of the immediately adjacent codons.5,6 Two consecutive slow codons (slow di-codons) lead to a much slower translation rate. Conversely, coding sequences (CDSs) with low composition of slow codons and slow dicodons may possess fast translation rates. Translation of viral proteins is completely dependent on the translation machinery of host cells. Therefore, the proportions of host-specific slow codons and slow di-codons in the viral CDSs can be used to predict the order of viral protein synthesis rates between different viruses of different genera, serotypes, and strains.

1- Introduction

2- Materials and methods

3- Results and discussion

4- Conclusion

References

Abstract

The daily output of orange residue from the orange juice production enterprise in the Three Gorges Reservoir Area (TGRA) of China is approximately 100 t/d, which seriously pollutes the environment of the TGRA. The key challenge of handling this waste is maximizing profitability. In this study, high-protein feed with low levels of crude fiber and pectin was produced by solid fermentation of orange waste. The synergistic and antagonistic effects of microorganisms on one another significantly influenced the quality of single cell protein (SCP) feed. The added true protein (ATP) content increased gradually as pectin degradation (PD) and crude fiber degradation (CFD) were enhanced because of the synergistic effects. However, ATP decreased because of antagonistic effects as PD and CFD were increased beyond certain values. Aspergillus oryzae (A. oryzae) and Trichoderma koningii (T. koningii) mutually promoted each other, but the growth of Candida tropicalis was inhibited by A. oryzae and T. koningii as polygalacturonase and carboxymethyl cellulase accumulated. Synergistic and antagonistic effects existed simultaneously during microorganic fermentation of orange wastes. In large-scale fermentation, ATP, PD, and CFD were increased by 14.20%, 15.80%, and 9.15%, respectively, in comparison with the flask test. The profit achieved by reusing orange waste in the Chongshou Agricultural Park as SCP feed was calculated to be 48500 USD per year, whereas the cost of disposing of the orange waste was 7560 USD. This study provides insight into how microorganic synergistic and antagonistic effects influence the quality of SCP feed and provides a potential route for recycling agricultural waste into valuable materials.

Introduction

China is the foremost orange producer in the world. In 2014, the orange planting area of China was 2.211
1010 m2 , and the annual output was 2.75
1010 kg (Shan, 2014). Oranges are consumed worldwide in the forms of pulp, peel and juice. Approximately 8
109 -2
1010 kg of solid and liquid residue is produced as waste during orange processing every year (Tripodo et al., 2004; Rezzadori et al., 2012). In most instances, this enormous amount of waste is scattered on soil in areas adjacent to the production facility, incinerated, placed in a land fill, or used as raw material for animal feed production (Martín et al., 2010; Patsalou et al., 2017). However, incineration of orange waste was found to lead to highly polluted surface and ground water with altered chemical oxygen demand and biological oxygen demand, as well as polluted soil (Rezzadori et al., 2012; Braddock, 1995). The term SCP refers to dead, dry cells of microorganisms such as yeast, bacteria, fungi and algae, which are utilized as a protein supplement in foods for humans or animal feed (Ofodile et al., 2011; Mahmood, 2012). SCP is made up of carbohydrates, fats, vitamins, minerals, and other cellular constituents (Mondal et al., 2012), and it can be produced from relatively inexpensive material or waste material (Santamaría-Fernandez et al., 2017).

1- Introduction

2- Theory

3- Material and methods

4- Results and discussion

5- Conclusions

References

Abstarct

During gastric digestion, hydrolysis of proteins by pepsin contributes largely to the breakdown of protein-rich food. We hypothesized that the effect of pepsin is limited by its diffusivity, which is co-determined by the food structure and the local pH in the food during digestion. To investigate the principle mechanism of enzyme diffusion in food matrices, we used enhanced green fluorescent protein (EGFP) as probe to study the diffusivity of proteins in whey protein isolate gels, using fluorescence correlation spectroscopy (FCS). Gels made with different ionic strength showed distinctive elastic moduli but did not show differences in diffusivity of EGFP. Some models for diffusion in hydrogels yield good description of the obtained data, and can approximate the enzyme diffusion in diverse food matrices. However, the enzyme pepsin is more complicated than the probe EGFP, to yield more accurate predictions, electrostatic and enzyme-substrate interaction also need to be considered.

Introduction

The digestion kinetics of food are dependent on the structure of the food that is digested. Food structure influences the oral processing, the gastric disintegration rate, and the consequent gastric emptying towards the duodenum (Singh, Ye, & Ferrua, 2015). The gastric disintegration of food invovles physical and chemical processes, including the peristalsis of the stomach, acid hydrolysis and enzymatic reactions (Bornhorst & Singh, 2014). Among these processes, hydrolysis of proteins by pepsin contributes largely to the breakdown of protein-rich food in the stomach. This hydrolysis can limited by the diffusion of pepsin and the local pH in the solid food matrix during digestion. The hydrolysis kinetics of egg white protein gels and whey protein gels differed strongly from that of the same proteins in solution, which is likely due to the diffusion limitation in gels for both the pepsin and the hydrolysates (Luo, Boom, & Janssen, 2015). Compared to acid-induced dairy gels, a rennet-induced casein gel consists of compact protein aggregates in the acidic gastric environment, and the rennet gel had much slower proteolysis kinetics than that of acid-induced gels (Floury et al., 2018). Thus, a quantitative investigation of pepsin diffusion in food structures may contribute to the understanding of food breakdown and digestion kinetics. We previously measured the diffusivity of pepsin in whey protein isolate (WPI) gels by fluorescence correlation spectroscopy (FCS). We found that the pepsin does not penetrate deep into the gel but remains in a thin layer below the surface of the gel. A second finding was that the diffusivity of pepsin depends strongly on the concentration of the protein gels (Luo, Borst, Westphal, Boom, & Janssen, 2017). Fluorescence Correlation Spectroscopy (FCS) was used for its non-invasiveness and suitability to be used within protein gels. In FCS, a confocal laser microscope is coupled with a photon detector to measure fluorescence intensity fluctuations in a small focal volume. If these fluctuations originate primarily from the diffusion of the fluorophores through the focal volume, autocorrelation analysis can quantify the diffusion rate of the fluorophore. Whey protein gels were used before as model for protein-based solid foods (Luo et al., 2017). Whey protein gelation is generally a two-step process. After heat denaturation, protein oligomers form primary aggregates with different shapes and sizes depending on the pH and the salt concentration. These primary aggregates then form large self-similar aggregates that precipitate or gel above a critical concentration (Aymard et al., 1996; Nicolai, Britten, & Schmitt, 2011).

Recycling ethanol fermentation stillage from food waste could help to resolve the pollution problems, but it would lead to the accumulation of lactic acid (LA), which had a negative effect on ethanol fermentation. In this study, bipolar membrane electrodialysis was used to recover LA as well as other fatty acids in the stillage, and the corresponding parameters that affected this process were investigated. Pretreatment via centrifugation and ultrafiltration reduced the turbidity successfully. Experiments on simulated and real stillage showed that organic acids such as formic acid and acetic acid had a slight effect on the separation of LA, but the existence of chloride ions could form strong anion competition with lactate ions. Simulation experiments with sodium chloride reduced the recovery of LA from 98% to 92%. The recovery rate of LA from real stillage could reach 71.2%. After electrodialysis, the stillage can be reused in ethanol fermentation, the fermentation time was shortened to 24 h, and the ethanol yield increased.

Introduction

For the sustainable development of society, the recycling of waste has become more and more important. Food waste (FW) accounts for a large proportion of municipal solid waste (15%–63%) [1]. It has high organic, volatile solid (85%–95%), and moisture content (75%–85%) and is easily degraded and deteriorated. There are many ways of reusing the waste, such as composting and production of pig feed, biomethane, biodiesel, ethanol, and lactic acid (LA) [2]. The use of FW to produce ethanol can take full advantage of the rich nutrients in FW and obtain fuel. However, a large quantity of stillage will be produced; one way for dealing it is stillage recycle fermentation [3]. This method can reduce pollution and utilize proteins, amino acids, and various metal ions in stillage [4]. In our previous study, LA was determined to be the major accumulated by-product along with stillage recyling. A significant inhibition phenomenon was observed after 5 times recycling. Although the addition of calcium carbonate could alleviate the influence of LA on ethanol fermentation, if we can separate the accumulated LA in the stillage, it will provide better economic benefits. LA is an important natural organic acid. It has received increasing attention as one of the most important building blocks for the production of polylactic acid [5,6]. At present, the main methods of LA production are microbial fermentation and chemical synthesis. Biological fermentation is inexpensive, utilizes a wide range of raw materials, and produces optically pure L- or D-lactic acid, which make it superior to chemical synthesis. More attention has also been paid to the utilization of renewable carbohydrates as a carbon source for LA fermentation [7,8]. However, the major cost of LA production is due to its downstream separation and purification steps [9]. As LA exists in the form of lactate salt in the fermentation broth, acidification and removal of impurities are required for future separation [10]. The extraction methods of LA include precipitation, solution extraction, adsorption, distillation, and nanofiltration [11]. Electrodialysis has the advantages of diversity, sophisticated functions, and technological compatibility [12,13]. Danner et al. used monopolar electrodialysis to extract LA from grass silages and obtained press extraction yields of 31–96 g of lactate per kilogram of silage dry matter (g LA/kg DM) [14]. Chen used conventional electrodialysis (CED) to remove 80% lactate from acid whey [15]. Lopez-Garzon used bipolar membrane electrodialysis (BMED) to recycle LA and other carboxylic acids in the fermentation broth [16].

Introduction

Evaluation of hypothesis

Prolactin-inducible protein (PIP)

Current challenges and consequences of the hypothesis

Conclusion

References

Abstract

Primary acquired nasolacrimal duct obstruction (PANDO) is a syndrome of unknown etiology, predominantly affecting post-menopausal females, characterized by progressive inflammation, fibrosis and subsequent obstruction of the nasolacrimal duct. Numerous factors have been proposed as possible etiologic factors and include anatomical configuration, ocular and nasal infections, peri-lacrimal vascular disorders, hormonal influence, lacrimal drainage lymphoid tissue, gastroesophageal reflux disease, topical medications, swimming pool exposure, smoking, genetic factors, autonomic and lysosomal dysregulation. The authors hypothesize Prolactin (PRL) and Prolactin-inducible protein (PIP) play a role in the etiopathogenesis of primary acquired nasolacrimal duct obstruction.

Introduction

Primary acquired nasolacrimal duct obstruction (PANDO) is a syndrome of unknown etiology, predominantly affecting post-menopausal females, characterized by progressive inflammation, fibrosis and subsequent obstruction of the nasolacrimal duct [1–3]. The resultant clinical manifestations include epiphora and discharge, occasional development of lacrimal sac mucocele or acute dacryocystitis [2]. Numerous factors have been proposed as possible etiologic factors and include anatomical configuration, ocular and nasal infections, peri-lacrimal vascular disorders, hormonal influence, lacrimal drainage lymphoid tissue, gastroesophageal reflux disease, topical medications, swimming pool exposure, smoking, genetic factors, autonomic and lysosomal dysregulation [1–7]. Prolactin receptors have been identified in the epithelia and submucosal glands of the lacrimal sac and nasolacrimal ducts [6]. They have been proposed as possible extra-pituitary sites of prolactin synthesis. Significant proportions of Prolactin-inducible protein (PIP) has been detected in lacrimal sac extracts [7].

In Bioinformatics Protein Fold Recognition (PFR) and Structural Class Prediction (SCP) is a significant problem in predicting protein with a three dimensional structure. Extraction of valuable features of protein that consists of 20 amino acids to acquire more desirable classifiers is fundamental to this PFR and SCP. Feature extraction technique predominantly exploits Forward Consecutive Search Scheme (FCS) that supplements syntacticalbased, evolutionary-based and physicochemical-based information. In this research work, a classifier known as Enhanced Artificial Neural Network (ANN) is employed as it is more efficient than Forward Consecutive Search scheme in order to improve the performance of PFR and SCP. The Enhanced ANN algorithm is an improved version of Artificial Neural Network when compared with various existing algorithms such as Support Vector Machine (SVM), ANN, K-Nearest Neighbor (KNN) and the Bayesian. The experiments are conducted on four datasets namely DD, EDD, TG and RDD. Ultimately, the statistical imputation of Enhanced ANN algorithm hypothesizes gives better results than other algorithms to improve the performance of PFR and SCP.

Introduction

Proteins are the components which play important roles in the activities of organisms. Protein’s function depends on the interactions with other proteins and its folding. Mismatch protein folding usually leads to changing in properties of the protein, which causes some diseases (Hashemi et al., 2009). To acquire knowledge about the protein function, interactions and regulations the prediction of protein structural classes is extremely useful (Jian-Yi Yang et al., 2010). To Increase the prediction accuracy of secondary structure and also to reduce the testimony of hunting scope in three dimensional structure predictions, the mastery of the structural class is helpful (Mohammad and AliYaghoubi, 2016). The SCP has become one of the most important features for characterizing the overall folding type of a protein in protein research. The first definition of protein structural class was introduced by Levitt and Chothia in 1976 and the globular proteins are normally classified into four structural classes such as (i) the all-α class consists of only little amount of strands, (ii) the all-β class consists of only little amount of helices, (iii) the α/β class consists of helices and almost all parallel strands, and α + β class consists of helices and almost all anti-parallel strands (Levitt and Chothia, 1976). Basically, the structural class of protein prediction from 20 amino acids is a significant task in the field of molecular biology. Proteins with unique length and similarities to be a part of the same fold having the identical significant protein secondary structure in the identical arrangement with the identical topology certainly they have a regular origin of evolutionary (Yang et al., 2011). PFR is used to model the proteins which have the similar fold as proteins of known structure, but do not have homologous proteins with known protein structure. PFR is the acquiring of three dimensional structure of the protein sequences independent from the sequence identities (Ding and Dubchak, 2001). PFR and SCP are prohibited as a transitional step for identifying the protein three dimensional structures. The PFR and SCP consist of two main concepts such as feature extraction techniques and classification techniques. The main goal of PFR and SCP is to allocate the novel protein sequence to a particular fold type and to a particular class type. Computational approaches considered more attention over the years due to the expense and the time involved in identifying the three dimensional structure of protein by using X-ray crystallography and Nuclear Magnetic Resonance (NMR) (Ibrahim and Abadeh, 2017).

Introduction

Coronary artery disease (CAD), a multifactorial heart disorder resulting from all kinds of predisposing environmental and genetic factors [1], is still one of the leading causes of disability and death around the world, accounting for 14.8% of global death [2]. Premature coronary artery disease (PCAD) is defined as CAD that occurs in males <55 years old or females <65 years old [3]. Even though PCAD constitutes only about 30% of all CAD subjects [4], [5], it is highly stressful for the patients’ families and generates a heavy social burden because of the longer period that the patient lives with the disease in comparison to older patients with CAD [6]. Large epidemiological studies have revealed that genetic factors have a strong influence on early onset CAD [7]. One known factor is the apolipoprotein E (APOE) gene [8].

Apolipoprotein E (apoE) is a multifunction glycoprotein containing 299 amino acids, which are encoded by the APOE gene [9]. It acts as cholesterol carrier and plays a major role in mediating the transportation and metabolism of lipids [10]. The APOE gene is situated at 19q13.2 of the human chromosome and has four exons and three introns [11]. As the genetic polymorphisms which are most widely studied in APOE, rs429358 and rs7412 together define the ε2, ε3, and ε4 alleles, which encode three major protein isoforms (E2, E3, E4) and generate six different genotypes (ε2/4, ε2/3, ε3/4, ε3/3, ε2/2, ε4/4) [12]. There is significant discrepancy in structure and function among the three protein isoforms [12]. ε3 is the wild type, and its amino acids at positions 112 and 158 are cysteine and arginine, respectively [10]. It has normal affinity for low-density lipoprotein receptors (LDLR) and modulates the clearance of lipoprotein remnants from the plasma [12]. When the 112th amino acid is replaced by arginine, ε3 mutates into ε4. Similarly, ε3 mutates into ε2 when the 158th amino acid is replaced by cysteine [13]. ε4 has similar high affinity for LDLR as ε3 and enhanced binding capacity for lipids, so it impairs the process of lipolysis and is associated with high levels of very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and total cholesterol [12]. As for ε2, it has defective affinity ability for LDLR, which would delay the clearance of VLDL. ε2 is associated with low levels of LDL and total cholesterol [12].

The plasma membrane (PM) H+ -ATPase is an important ion pump in the plant cell membrane. By extruding protons from the cell and generating a membrane potential, this pump energizes the PM, which is a prerequisite for growth. Modification of the autoinhibitory terminal domains activates PM H+ -ATPase activity, and on this basis it has been hypothesized that these regulatory termini are targets for physiological factors that activate or inhibit proton pumping. In this review, we focus on the posttranslational regulation of the PM H+ -ATPase and place regulation of the pump in an evolutionary and physiological context. The emerging picture is that multiple signals regulating plant growth interfere with the posttranslational regulation of the PM H+ -ATPase.

INTRODUCTION

The plasma membrane (PM) H+ -ATPase is an electrogenic pump that exports cellular protons. In addition to generating a transmembrane chemical gradient of H+ (DpH; acidic on the outside), it also establishes an electrical gradient (the membrane potential; negative on the inside). As almost all other transport proteins in the plant PM are energized by this combined electrochemical gradient of protons, it has been a long-standing hypothesis in plant physiology that the PM H+ – ATPase is a powerhouse for plant growth (reviewed in Palmgren, 2001; Arango et al., 2003; Sondergaard et al., 2004; Duby and Boutry, 2009). However, although the biochemistry of the pump is known in great detail and its structure has been determined (reviewed in Morth et al., 2011), strong evidence for its role in plant growth has remained sparse until recently, when strong links between posttranslational regulation of PM H+ -ATPase and essential physiological functions of plants have emerged. The aim of this review is to provide an overview of this exciting and rapidly developing field.

Abstract

Spectroscopic properties of tyrosine residues may be employed in structural studies of proteins. Here we discuss several different types of UV–Vis spectroscopy, like normal, difference and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, and corresponding optical properties of the tyrosine chromophore, phenol, which are used to study protein structure.

Introduction

Fluorescence and other spectral parameters of aromatic chromophores in proteins, tryptophan, tyrosine (Tyr) and phenylalanine, may be used as probes of protein structure. Using Tyr is an attractive choice because its chromophore, phenol, is expected to exhibit substantial responses to environmental changes (Fornander et al. 2014). We have found it interesting to review the development of structural studies of proteins based on detection of different spectroscopic features of tyrosines in the UV–Vis range, from the earliest investigations using UV absorbance measurements (Crammer and Neuberger 1943; Shugar 1952) to more recent resonance Raman (Larkin 2011; Reymer et al. 2014) or linear dichroism (Reymer et al. 2009) spectroscopy studies.

First, we recall the basic principles of different methods in UV–Vis spectrometry. We then describe properties of Tyr and related model compounds, i.e., phenol, para-cresol (p-cresol) and N-acetyl-l-tyrosine amide (N-Ac-Tyr-NH₂) as revealed by different UV–Vis spectroscopies. In Part 2, we present selected applications of UV–Vis spectrometry of tyrosines to probe protein structures.

Abstract

Plants are permanently situated in a fixed location and thus are well adapted to sense and respond to environmental stimuli and developmental cues. At the cellular level, several of these responses require delicate adjustments that affect the activity and steady-state levels of plasma membrane proteins. These adjustments involve both vesicular transport to the plasma membrane and protein internalization via endocytic sorting. A substantial part of our current knowledge of plant plasma membrane protein sorting is based on studies of PIN-FORMED (PIN) auxin transport proteins, which are found at distinct plasma membrane domains and have been implicated in directional efflux of the plant hormone auxin. Here, we discuss the mechanisms involved in establishing such polar protein distributions, focusing on PINs and other key plant plasma membrane proteins, and we highlight the pathways that allow for dynamic adjustments in protein distribution and turnover, which together constitute a versatile framework that underlies the remarkable capabilities of plants to adjust growth and development in their ever-changing environment.

Introduction

Plasma membrane proteins play key roles in mediating responses to the endogenous and environmental cues that regulate various developmental and metabolic events. Intrinsic membrane proteins have been implicated in various transport processes across membranes as well as in perception and further transmission of regulatory signals. Crosstalk at the plasma membrane interface is all the more important for sessile organisms such as higher plants, which are required to maintain a diverse spectrum of plasma membrane receptor and transport proteins in order to be able to properly sense and respond to fluctuations in their environment (Geldner and Robatzek, 2008; Otegui and Spitzer, 2008; Richter et al., 2009; Robinson et al., 2012; Korbei and Luschnig, 2013). In plants, as in any other eukaryote, this is achieved by a complex system of internal membranes that serves to transport proteins to their site of action and degradation. Exchange of material between these membranes is achieved by vesicles that bud off from one membrane, mature and fuse with another in a highly controlled fashion.