Proteomic analysis of crocodile white blood cells reveals insights into the mechanism of the innate immune system.

Crocodiles have a particularly powerful innate immune system because their blood contains high levels of antimicrobial peptides. They can survive injuries that would be fatal to other animals, and they are rarely afflicted with diseases. To better understand the crocodile's innate immune response, proteomic analysis was performed on the white blood cells (WBC) of an Aeromonas hydrophila-infected crocodile. Levels of WBC and red blood cells (RBC) rapidly increased within 1 h. In WBC, there were 109 up-regulated differentially expressed proteins (DEP) that were up-regulated. Fifty-nine DEPs dramatically increased expression from 1 h after inoculation, whereas 50 up-regulated DEPs rose after 24 h. The most abundant DEPs mainly had two biological functions, 1) gene expression regulators, for example, zinc finger proteins and histone H1 family, and 2) cell mechanical forces such as actin cytoskeleton proteins and microtubule-binding proteins. This finding illustrates the characteristic effective innate immune response mechanism of crocodiles that might occur via boosted transcription machinery proteins to accelerate cytoskeletal protein production for induction of phagocytosis, along with the increment of trafficking proteins to transport essential molecules for combating pathogens. The findings of this study provide new insights into the mechanisms of the crocodile's innate immune system.

Assessment of safety and intranasal neutralizing antibodies of HPMC-based human anti-SARS-CoV-2 IgG1 nasal spray in healthy volunteers.

An HPMC-based nasal spray solution containing human IgG1 antibodies against SARS-CoV-2 (nasal antibody spray or NAS) was developed to strengthen COVID-19 management. NAS exhibited potent broadly neutralizing activities against SARS-CoV-2 with PVNT50 values ranging from 0.0035 to 3.1997 µg/ml for the following variants of concern (ranked from lowest to highest): Alpha, Beta, Gamma, ancestral, Delta, Omicron BA.1, BA.2, BA.4/5, and BA.2.75. Biocompatibility assessment showed no potential biological risks. Intranasal NAS administration in rats showed no circulatory presence of human IgG1 anti-SARS-CoV-2 antibodies within 120 h. A double-blind, randomized, placebo-controlled trial (NCT05358873) was conducted on 36 healthy volunteers who received either NAS or a normal saline nasal spray. Safety of the thrice-daily intranasal administration for 7 days was assessed using nasal sinuscopy, adverse event recording, and self-reporting questionnaires. NAS was well tolerated, with no significant adverse effects during the 14 days of the study. The SARS-CoV-2 neutralizing antibodies were detected based on the signal inhibition percent (SIP) in nasal fluids pre- and post-administration using a SARS-CoV-2 surrogate virus neutralization test. SIP values in nasal fluids collected immediately or 6 h after NAS application were significantly increased from baseline for all three variants tested, including ancestral, Delta, and Omicron BA.2. In conclusion, NAS was safe for intranasal use in humans to increase neutralizing antibodies in nasal fluids that lasted at least 6 h.

Efficiency Comparative Approach of Plant-Produced Monoclonal Antibodies against Rabies Virus Infection.

Rabies encephalitis is a fatal zoonotic viral disease caused by the neurotropic rabies virus. It remains a major public health concern as it causes almost 100% fatality and has no effective medication after the onset of the disease. However, this illness is preventable with the timely administration of effective post-exposure prophylaxis (PEP) consisting of the rabies vaccine and passive immune globulins (HRIG and ERIG). Recently, conventional PEP has been shown to have many limitations, resulting in little support for these expensive and heterologous globulins. Monoclonal antibody (mAb) production via recombinant technology in animal and human cell cultures, as well as a plant-based platform, was introduced to overcome the costly and high-tech constraints of former preparations. We used transient expression technology to produce two mAbs against the rabies virus in Nicotiana benthamiana and compared their viral neutralizing activity in vitro and in vivo. The expression levels of selective mAbs E559 and 62-71-3 in plants were estimated to be 17.3 mg/kg and 28.6 mg/kg in fresh weight, respectively. The plant-produced mAbs effectively neutralized the challenge virus CVS-11 strain in a cell-based RFFIT. In addition, the combination of these two mAbs in a cocktail protected hamsters from rabies virus infection more effectively than standard HRIG and ERIG. This study suggests that the plant-produced rabies antibody cocktail has promising potential as an alternative biological to polyclonal RIG in rabies PEP.

Rice ß-glucosidase Os12BGlu38 is required for synthesis of intine cell wall and pollen fertility.

Glycoside hydrolase family1 ß-glucosidases play a variety of roles in plants, but their in planta functions are largely unknown in rice (Oryza sativa). In this study, the biological function of Os12BGlu38, a rice ß-glucosidase, expressed in bicellular to mature pollen, was examined. Genotype analysis of progeny of the self-fertilized heterozygous Os12BGlu38 T-DNA mutant, os12bglu38-1, found no homozygotes and a 1:1 ratio of wild type to heterozygotes. Reciprocal cross analysis demonstrated that Os12BGlu38 deficiency cannot be inherited through the male gamete. In cytological analysis, the mature mutant pollen appeared shrunken and empty. Histochemical staining and TEM showed that mutant pollen lacked intine cell wall, which was rescued by introduction of wild-type Os12BGlu38 genomic DNA. Metabolite profiling analysis revealed that cutin monomers and waxes, the components of the pollen exine layer, were increased in anthers carrying pollen of os12bglu38-1 compared with wild type and complemented lines. Os12BGlu38 fused with green fluorescent protein was localized to the plasma membrane in rice and tobacco. Recombinant Os12BGlu38 exhibited ß-glucosidase activity on the universal substrate p-nitrophenyl ß-d-glucoside and some oligosaccharides and glycosides. These findings provide evidence that function of a plasma membrane-associated ß-glucosidase is necessary for proper intine development.

Action of Multiple Rice ß-Glucosidases on Abscisic Acid Glucose Ester.

Conjugation of phytohormones with glucose is a means of modulating their activities, which can be rapidly reversed by the action of ß-glucosidases. Evaluation of previously characterized recombinant rice ß-glucosidases found that nearly all could hydrolyze abscisic acid glucose ester (ABA-GE). Os4BGlu12 and Os4BGlu13, which are known to act on other phytohormones, had the highest activity. We expressed Os4BGlu12, Os4BGlu13 and other members of a highly similar rice chromosome 4 gene cluster (Os4BGlu9, Os4BGlu10 and Os4BGlu11) in transgenic Arabidopsis. Extracts of transgenic lines expressing each of the five genes had higher ß-glucosidase activities on ABA-GE and gibberellin A4 glucose ester (GA4-GE). The ß-glucosidase expression lines exhibited longer root and shoot lengths than control plants in response to salt and drought stress. Fusions of each of these proteins with green fluorescent protein localized near the plasma membrane and in the apoplast in tobacco leaf epithelial cells. The action of these extracellular ß-glucosidases on multiple phytohormones suggests they may modulate the interactions between these phytohormones.

Physicochemical properties and oxygen affinity of glutaraldehyde polymerized crocodile hemoglobin: the new alternative hemoglobin source for hemoglobin-based oxygen carriers.

Hemoglobin-based oxygen carriers (HBOCs) are modified stroma-free hemoglobin molecules used in developing a blood substitute for therapeutic usage. In order to prevent hemoglobin dissociation, glutaraldehyde (GTA) was used to generate high-molecular weight heterogeneous crocodile hemoglobin (Poly-cHb). This work, Poly-cHb was created using various GTA concentrations, ranging from 0.025-0.150% (v/v). Physicochemical properties were investigated that were comparable GTA polymerized human hemoglobin (Poly-hHb). This study has revealed that GTA polymerization increases the molecular size of Native-cHbs from 14.10 nm over a range from 16.31 to 54.27 nm. Moreover, this polymerization alters the secondary structure and heme environment by decreasing the helicity ratio from 1.00 to 0.95 at the highest condition and exhibits hypochromic shift of the Soret band to be 0.88 times lower than the native. However, all Poly-cHbs still possessed higher oxygen affinity than that of Poly-hHbs with average P50 values of 13 and 21 mmHg, respectively. Although, polymerization affected the overall Poly-cHb structure slightly, but compensated by decreasing the denaturation level to lower than 10%. Thus, it is interesting to note that Poly-cHb may advantageously provide effective oxygen carriage and ability for pasteurization, which may benefit the search for new alternative hemoglobin sources for HBOC development.

Demonstration of monolignol ß-glucosidase activity of rice Os4BGlu14, Os4BGlu16 and Os4BGlu18 in Arabidopsis thaliana bglu45 mutant.

The glycoside hydrolase family 1 members Os4BGlu14, Os4BGlu16, and Os4BGlu18 were proposed to be rice monolignol ß-glucosidases. In vitro studies demonstrated that the Os4BGlu16 and Os4BGlu18 hydrolyze the monolignol glucosides coniferin and syringin with high efficiency compared to other substrates. The replacement of the conserved catalytic acid/base glutamate residue by a nonionizable glutamine residue in Os4BGlu14 suggested that it may be inactive as a ß-glucosidase. Here, we investigated the activities of Os4BGlu14, Os4BGlu16, and Os4BGlu18 in planta by recombinant expression of their genes in the Arabidopsis bglu45-2 (monolignol ß-glucosidase) mutant and analysis of monolignol glucosides by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MSMS). The bglu45-2 line exhibits elevated monolignol glucoside levels, but lower amounts of coniferin, syringin, and p-coumaryl alcohol glucoside were seen in Arabidopsis bglu45-2 rescued lines complemented by the Os4BGlu14, Os4BGlu16, and Os4BGlu18 genes. These data suggest that the bglu45-2 mutant has a broader effect on monolignols than previously reported and that the Os4BGlu14, Os4BGlu16 and Os4BGlu18 proteins act as monolignol ß-glucosidases to complement the defect. An OsBGlu16-GFP fusion protein localized to the cell wall. This apoplastic localization and the effect of these enzymes on monolignol glucoside levels suggest monolignol glucosides from the vacuole may meet the monolignol ß-glucosidases, despite their different localization.

Comparative proteomic analysis of Chlamydomonas reinhardtii control and a salinity-tolerant strain revealed a differential protein expression pattern.

MAIN CONCLUSION: Proteins involved in membrane transport and trafficking, stress and defense, iron uptake and metabolism, as well as proteolytic enzymes, were remarkably up-regulated in the salinity-tolerant strain of Chlamydomonas reinhardtii. Excessive concentration of NaCl in the environment can cause adverse effects on plants and microalgae. Successful adaptation of plants to long-term salinity stress requires complex cellular adjustments at different levels from molecular, biochemical and physiological processes. In this study, we developed a salinity-tolerant strain (ST) of the model unicellular green alga, Chlamydomonas reinhardtii, capable of growing in medium containing 300 mM NaCl. Comparative proteomic analyses were performed to assess differential protein expression pattern between the ST and the control progenitor cells. Proteins involved in membrane transport and trafficking, stress and defense, iron uptake and metabolism, as well as protein degradation, were remarkably up-regulated in the ST cells, suggesting the importance of these processes in acclimation mechanisms to salinity stress. Moreover, 2-DE-based proteomic also revealed putative salinity-specific post-translational modifications (PTMs) on several important housekeeping proteins. Discussions were made regarding the roles of these differentially expressed proteins and the putative PTMs in cellular adaptation to long-term salinity stress.

ß-Glucosidases: Multitasking, moonlighting or simply misunderstood?

ß-Glucosidases have a wide range of functions in plants, including roles in recycling of cell-wall oligosaccharides, defense, phytohormone signaling, secondary metabolism, and scent release, among others. It is not always clear which one is responsible for a specific function, as plants contain a large set of ß-glucosidases. However, progress has been made in recent years in elucidating these functions. To help understand what is known and what remains ambiguous, we review the general approaches to investigating plant ß-glucosidase functions. We consider information that has been gained regarding glycoside hydrolase family 1 enzyme functions utilizing these approaches in the past decade. In several cases, one enzyme has been assigned different biological functions by different research groups. We suggest that, at least in some cases, the ambiguity of an enzyme's function may come from having multiple functions that may help coordinate the response to injury or other stresses.

Identification of Fatty Acid Glucose Esters as Os9BGlu31 Transglucosidase Substrates in Rice Flag Leaves.

Rice Os9BGlu31 transglucosidase transfers glucosyl moieties between various carboxylic acids and alcohols, including phenolic acids and flavonoids, in vitro. The role of Os9BGlu31 transglucosidase in rice plant metabolism has only been suggested to date. Methanolic extracts of rice bran and leaves were found to contain oleic acid and linoleic acid to which Os9BGlu31 could transfer glucose from the 4-nitrophenyl ß-D-glucoside (4NPGlc) donor to form 1-O-acyl glucose esters. Os9BGlu31 showed higher activity with oleic acid (18:1) and linoleic acid (18:2) than with stearic acid (18:0) and had both a higher kcat and a higher Km for linoleic than oleic acid in the presence of 8 mM 4NPGlc donor. Os9BGlu31 knockout mutant rice lines were found to have significantly larger amounts of fatty acid glucose esters than wild-type control lines. Because the transglucosylation reaction is reversible, these data suggest that fatty acid glucose esters act as glucosyl donor substrates for Os9BGlu31 transglucosidase in rice.

Recombinant expression and characterization of the cytoplasmic rice ß-glucosidase Os1BGlu4.

The Os1BGlu4 ß-glucosidase is the only glycoside hydrolase family 1 member in rice that is predicted to be localized in the cytoplasm. To characterize the biochemical function of rice Os1BGlu4, the Os1bglu4 cDNA was cloned and used to express a thioredoxin fusion protein in Escherichia coli. After removal of the tag, the purified recombinant Os1BGlu4 (rOs1BGlu4) exhibited an optimum pH of 6.5, which is consistent with Os1BGlu4's cytoplasmic localization. Fluorescence microscopy of maize protoplasts and tobacco leaf cells expressing green fluorescent protein-tagged Os1BGlu4 confirmed the cytoplasmic localization. Purified rOs1BGlu4 can hydrolyze p-nitrophenyl (pNP)-ß-D-glucoside (pNPGlc) efficiently (kcat/Km  =  17.9 mM(-1) · s(-1)), and hydrolyzes pNP-ß-D-fucopyranoside with about 50% the efficiency of the pNPGlc. Among natural substrates tested, rOs1BGlu4 efficiently hydrolyzed ß-(1,3)-linked oligosaccharides of degree of polymerization (DP) 2-3, and ß-(1,4)-linked oligosaccharide of DP 3-4, and hydrolysis of salicin, esculin and p-coumaryl alcohol was also detected. Analysis of the hydrolysis of pNP-ß-cellobioside showed that the initial hydrolysis was between the two glucose molecules, and suggested rOs1BGlu4 transglucosylates this substrate. At 10 mM pNPGlc concentration, rOs1BGlu4 can transfer the glucosyl group of pNPGlc to ethanol and pNPGlc. This transglycosylation activity suggests the potential use of Os1BGlu4 for pNP-oligosaccharide and alkyl glycosides synthesis.

Rice Os9BGlu31 is a transglucosidase with the capacity to equilibrate phenylpropanoid, flavonoid, and phytohormone glycoconjugates.

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu(169)), catalytic nucleophile residues (Glu(387)), and His(386), the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 ß-glucosidases that utilize a double displacement retaining mechanism.

Proteomic analysis of salinity-stressed Chlamydomonas reinhardtii revealed differential suppression and induction of a large number of important housekeeping proteins.

Salinity stress is one of the most common abiotic stresses that hamper plant productivity worldwide. Successful plant adaptations to salt stress require substantial changes in cellular protein expression. In this work, we present a 2-DE-based proteomic analysis of a model unicellular green alga, Chlamydomonas reinhardtii, subjected to 300 mM NaCl for 2 h. Results showed that, in addition to the protein spots that showed partial up- or down-regulation patterns, a number of proteins were exclusively present in the proteome of the control cells, but were absent from the salinity-stressed samples. Conversely, a large number of proteins exclusively appeared in the proteome of the salinity-stressed samples. Of those exclusive proteins, we could successfully identify, via LC-MS/MS, 18 spots uniquely present in the control cells and 99 spots specific to NaCl-treated cells. Interestingly, among the salt-exclusive protein spots, we identified several important housekeeping proteins like molecular chaperones and proteins of the translation machinery, suggesting that they may originate from post-translational modifications rather than from de novo biosynthesis. The possible role and the salt-specific modification of these proteins by salinity stress are discussed.

Proteomic analysis of a model unicellular green alga, Chlamydomonas reinhardtii, during short-term exposure to irradiance stress reveals significant down regulation of several heat-shock proteins.

Oxygenic photosynthetic organisms often suffer from excessive irradiance, which cause harmful effects to the chloroplast proteins and lipids. Photoprotection and the photosystem II repair processes are the mechanisms that plants deploy to counteract the drastic effects from irradiance stress. Although the protective and repair mechanisms seemed to be similar in most plants, many species do confer different level of tolerance toward high light. Such diversity may originate from differences at the molecular level, i.e., perception of the light stress, signal transduction and expression of stress responsive genes. Comprehensive analysis of overall changes in the total pool of proteins in an organism can be performed using a proteomic approach. In this study, we employed 2-DE/LC-MS/MS-based comparative proteomic approach to analyze total proteins of the light sensitive model unicellular green alga Chlamydomonas reinhardtii in response to excessive irradiance. Results showed that among all the differentially expressed proteins, several heat-shock proteins and molecular chaperones were surprisingly down-regulated after 3-6 h of high light exposure. Discussions were made on the possible involvement of such down regulation and the light sensitive nature of this model alga.