Accumulation Of Monomers Research Articles

Building a protective shield: The role of wound healing in reducing postharvest decay and preserving quality of citrus fruit1

Postharvest citrus fruit is susceptible to pathogenic infestation and quality reduction through wounds, leading to tremendous commercial losses. Herein, wound healing of citrus fruit was obviously at 25°C for five days to form a barrier effective against the development of infectious diseases and water dissipation. Combined with the results of transcriptional and metabolic levels, wound healing activated the expression of CsKCS4, CsKCS11, CsCYP704B1, CsFAH1, CsGPAT3 and CsGPAT9 genes in suberin biosynthesis pathway, and CsPMEI7, CsCesA-D3, CsXTH2, CsXTH6, CsXTH22, CsXTH23, CsXTH24, CsC4H and CsCAD genes in cell wall metabolism pathway, leading to the accumulation of suberin monomers and cell wall components. The results of microscopic observations proved wound healing promoted suberin deposition and cell wall strengthening. Meanwhile, wound healing required the provision of energy and precursor substances by carbohydrate metabolism and amino acid metabolism. We provide new insights into the regulatory mechanism of wound healing on improving disease resistance and maintaining the quality of citrus fruit.

Spatial accumulation of lignin monomers and cellulose underlying stalk strength in maize

Lodging largely affects yield, quality and mechanical harvesting of maize. Stalk strength is one of the major factors that affect maize lodging. Although plant cell wall components including lignin and cellulose were known to be associated with stalk strength and lodging resistance, spatial accumulation of specific lignin monomers and cellulose in different tissues and their association with stalk strength in maize was not clearly understood. In this study, we found that both G and S lignin monomers accumulate highest in root, stem rind and leaf vein. Consistently, most lignin biosynthetic genes were expressed higher in root and stem than in other tissues. However, cellulose appears to be lowest in root. There are only mild changes of G lignin and cellulose in different internodes. Instead, we noticed a dramatic decrease of S-lignin accumulation and lignin biosynthetic gene expression in 2nd to 4th internodes wherein stem breakage usually occurs, thereby revealing a few candidate lignin biosynthetic genes associated with stalk strength. Moreover, stalk strength is positively correlated with G, S lignin, and cellulose, but negatively correlated with S/G ratio based on data of maize lines with high or low stalk strength. Loss-of-function of a caffeic acid o-methyltransferase (COMT), which is involved in S lignin biosynthesis, in the maize bm3 mutant, leads to lower stalk strength. Our data collectively suggest that stalk strength is determined by tissue-specific accumulation of lignin monomers and cellulose, and manipulation of the cell wall components by genetic engineering is vital to improve maize stalk strength and lodging resistance.

Prolyl Endopeptidase Is Involved in Filaggrinolysis and Cornification

Filaggrin (FLG) is a well-known biomarker of atopic dermatitis and skin dryness. Its full proteolysis (or filaggrinolysis) produces the major constituents of the natural moisturizing factor. Some proteases/peptidases remain to be identified in this multistep process. Mining 16 omics analyses, we identified prolyl endopeptidase (PREP) as a candidate peptidase. Indirect immunofluorescence and confocal analysis demonstrated its localization in the granular and deep cornified layers, where it co-localized with FLG. Tandem mass spectroscopy and fluorescent quenching activity assays showed that PREP cleaved several synthetic peptides derived from the FLG sequence, at the carboxyl side of an internal proline. Deimination of these peptides increased PREP enzymatic efficiency. Specific inhibition of PREP in reconstructed human epidermis (RHEs) using benzyloxycarbonyl-Pro-Prolinal (ZPP) induced the accumulation of FLG monomers. Down-regulation of PREP expression in RHEs using RNA interference confirmed the impact of PREP on FLG metabolism, and highlighted a more general role of PREP in keratinocyte differentiation. Indeed, quantitative global proteomic, Western blotting and RT-qPCR analyses showed a strong reduction in the expression of bleomycin hydrolase, known to be involved in filaggrinolysis, and of several other actors of cornification like loricrin. Consequently, at the functional level, the trans-epidermal electric resistance was drastically reduced.

MYB24, MYB144, and MYB168 positively regulate suberin biosynthesis at potato tuber wounds during healing.

The essence of wound healing is the accumulation of suberin at wounds, which is formed by suberin polyphenolic (SPP) and suberin polyaliphatic (SPA). The biosynthesis of SPP and SPA monomers is catalyzed by several enzyme classes related to phenylpropanoid metabolism and fatty acid metabolism, respectively. However, how suberin biosynthesis is regulated at the transcriptional level during potato (Solanum tuberosum) tuber wound healing remains largely unknown. Here, 6 target genes and 15 transcription factors related to suberin biosynthesis in tuber wound healing were identified by RNA-seq technology and qRT-PCR. Dual luciferase and yeast one-hybrid assays showed that StMYB168 activated the target genes StPAL, StOMT, and St4CL in phenylpropanoid metabolism. Meanwhile, StMYB24 and StMYB144 activated the target genes StLTP, StLACS, and StCYP in fatty acid metabolism, and StFHT involved in the assembly of SPP and SPA domains in both native and wound periderms. More importantly, virus-induced gene silencing in S. tuberosum and transient overexpression in Nicotiana benthamiana assays confirmed that StMYB168 regulates the biosynthesis of free phenolic acids, such as ferulic acid. Furthermore, StMYB24/144 regulated the accumulation of suberin monomers, such as ferulates, α, ω-diacids, and ω-hydroxy acids. In conclusion, StMYB24, StMYB144, and StMYB168 have an elaborate division of labor in regulating the synthesis of suberin during tuber wound healing.

The Arabidopsis cytochrome P450 enzyme CYP96A4 is involved in the wound-induced biosynthesis of cuticular wax and cutin monomers.

The plant cuticle is composed of cuticular wax and cutin polymers and plays an essential role in plant tolerance to diverse abiotic and biotic stresses. Several stresses, including water deficit and salinity, regulate the synthesis of cuticular wax and cutin monomers. However, the effect of wounding on wax and cutin monomer production and the associated molecular mechanisms remain unclear. In this study, we determined that the accumulation of wax and cutin monomers in Arabidopsis leaves is positively regulated by wounding primarily through the jasmonic acid (JA) signaling pathway. Moreover, we observed that a wound- and JA-responsive gene (CYP96A4) encoding an ER-localized cytochrome P450 enzyme was highly expressed in leaves. Further analyses indicated that wound-induced wax and cutin monomer production was severely inhibited in the cyp96a4 mutant. Furthermore, CYP96A4 interacted with CER1 and CER3, the core enzymes in the alkane-forming pathway associated with wax biosynthesis, and modulated CER3 activity to influence aldehyde production in wax synthesis. In addition, transcripts of MYC2 and JAZ1, key genes in JA signaling pathway, were significantly reduced in cyp96a4 mutant. Collectively, these findings demonstrate that CYP96A4 functions as a cofactor of the alkane synthesis complex or participates in JA signaling pathway that contributes to cuticular wax biosynthesis and cutin monomer formation in response to wounding.

Methyl jasmonate promotes suberin biosynthesis by stimulating transcriptional activation of AchMYC2 on AchFHT in wound healing of kiwifruit

Suberin consisting of the polyaliphatic and polyphenolic domain confers the hydrophobic barrier of wound fruit and vegetables, providing essential protection from excessive water loss, ion outflow and pathogen invasion. The ω-hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (FHT) is required for cross-linking of the polyaliphatic and polyphenolic domain in suberin biosynthesis. The transcription factor AchMYC2 acts as a positive activator in suberin biosynthesis but it is not known how it interacts with AchFHT in kiwifruit. In this study, overexpression of AchFHT in kiwifruit increased accumulation of suberin monomers, such as ferulate and ω-hydroxyacids. Yeast one-hybrid (Y1H) and dual-luciferase reporter (DLR) assays showed that AchMYC2 activated AchFHT transcript via interacting with the gene promoter. Overexpression of AchMYC2 in kiwifruit resulted in the upregulation of AchFHT, the accumulation of FHT protein and suberin monomers. Further, using yeast two-hybrid (Y2H) assay screened a jasmonate ZIM-domain protein (AchJAZ1, a key repressor of JA signaling pathway) which interacted with AchMYC2. Methyl jasmonate (MeJA) treatment increased expression levels of AchMYC2 and AchFHT, and the content of FHT protein, accompanying by the rapid wound healing process, as indicated by rapid suberin deposition, the reduced weight loss and relative electrolyte leakage. These results indicate that AchMYC2 activates AchFHT transcript to involve in MeJA-mediated suberin biosynthesis in wound healing of kiwifruit.

The polyphenolic suberized peridermal skin of the Sikkim cucumber acts as an effective chemical barrier against Botrytis cinerea infection

Certain species of fleshy fruit that undergo skin cracking are capable of forming a specialized polyphenolic periderm to seal the wounded skin. Some of the genetic and metabolic components pertaining periderm formation have been identified, however it remains unknown if, and to what extent, this specialized tissue can act as an efficient barrier against the invasion of pathogenic fungi. We monitored the infection process of Botrytis cinerea on the cuticle-coated skin of the common cucumber (Cucumis sativus) or on the polyphenolic suberized peridermal skin of the Sikkim cucumber (C. sativus var. sikkimensis). In vivo inoculation assays inferred that while B. cinerea can successfully penetrate and form necrotic lesions on sativus fruit, it is incapable of penetrating the peridermal skin of sikkimensis fruit. Gas chromatography-mass spectrometry (GC-MS) profiling of fruit skin extracts, in vitro conidial germination and germ tube elongation assays, and qRT-PCR cutinase-encoding gene expression analyses, inferred that the accumulation of suberin monomers in the peridermal skin of sikkimensis fruit stimulate increased elongation of B. cinerea germ tubes and significantly alters the differential expression of cutinase-encoding genes. Among them, Bcin0607010, Bcin08g01580 and Bcin07g06480, three yet unidentified cutinases, whose expression is specifically increased in the presence of sikkimensis fruit skin extracts. Our data demonstrate that a polyphenolic suberized peridermal skin in cucumber fruit can shape the interactions with B. cinerea and implies that suberization of fleshy fruit may provide a means for reducing damage imposed by this and other pathogens.

Tubulin isotype regulation maintains asymmetric requirement for α-tubulin over β-tubulin.

How cells regulate α- and β-tubulin to meet the demand for αβ-heterodimers and avoid consequences of monomer imbalance is not understood. We investigate the role of gene copy number and how shifting expression of α- or β-tubulin genes impacts tubulin proteostasis and microtubule function in Saccharomyces cerevisiae. We find that α-tubulin gene copy number is important for maintaining excess α-tubulin protein compared to β-tubulin protein. Excess α-tubulin prevents accumulation of super-stoichiometric β-tubulin, which leads to loss of microtubules, formation of non-microtubule assemblies of tubulin, and disrupts cell proliferation. In contrast, sub-stoichiometric β-tubulin or overexpression of α-tubulin has minor effects. We provide evidence that yeast cells equilibrate α-tubulin protein concentration when α-tubulin isotype expression is increased. We propose an asymmetric relationship between α- and β-tubulins, in which α-tubulins are maintained in excess to supply αβ-heterodimers and limit the accumulation of β-tubulin monomers.

Overexpression of β-Ketoacyl CoA Synthase 2B.1 from Chenopodium quinoa Promotes Suberin Monomers' Production and Salt Tolerance in Arabidopsis thaliana.

Very-long-chain fatty acids (VLCFAs) are precursors for the synthesis of various lipids, such as triacylglycerols, sphingolipids, cuticular waxes, and suberin monomers, which play important roles in plant growth and stress responses. However, the underlying molecular mechanism regulating VLCFAs' biosynthesis in quinoa (Chenopodium quinoa Willd.) remains unclear. In this study, we identified and functionally characterized putative 3-ketoacyl-CoA synthases (KCSs) from quinoa. Among these KCS genes, CqKCS2B.1 showed high transcript levels in the root tissues and these were rapidly induced by salt stress. CqKCS2B.1 was localized to the endoplasmic reticulum. Overexpression of CqKCS2B.1 in Arabidopsis resulted in significantly longer primary roots and more lateral roots. Ectopic expression of CqKCS2B.1 in Arabidopsis promoted the accumulation of suberin monomers. The occurrence of VLCFAs with C22-C24 chain lengths in the overexpression lines suggested that CqKCS2B.1 plays an important role in the elongation of VLCFAs from C20 to C24. The transgenic lines of overexpressed CqKCS2B.1 showed increased salt tolerance, as indicated by an increased germination rate and improved plant growth and survival under salt stress. These findings highlight the significant role of CqKCS2B.1 in VLCFAs' production, thereby regulating suberin biosynthesis and responses to salt stress. CqKCS2B.1 could be utilized as a candidate gene locus to breed superior, stress-tolerant quinoa cultivars.

Impact of water deficiency on leaf cuticle lipids and gene expression networks in cotton (Gossypium hirsutum L.)

BackgroundWater deficit (WD) has serious effect on the productivity of crops. Formation of cuticular layer with increased content of wax and cutin on leaf surfaces is closely related to drought tolerance. Identification of drought tolerance associated wax components and cutin monomers and the genes responsible for their biosynthesis is essential for understanding the physiological and genetic mechanisms underlying drought tolerance and improving crop drought resistance.ResultIn this study, we conducted comparative phenotypic and transcriptomic analyses of two Gossypium hirsutum varieties that are tolerant (XL22) or sensitive (XL17) to drought stress. XL17 consumed more water than XL22, particularly under the WD conditions. WD significantly induced accumulation of most major wax components (C29 and C31 alkanes) and cutin monomers (palmitic acid and stearic acid) in leaves of both XL22 and XL17, although accumulation of the major cutin monomers, i.e., polyunsaturated linolenic acid (C18:3n-3) and linoleic acid (C18:2n-6), were significantly repressed by WD in both XL22 and XL17. According to the results of transcriptome analysis, although many genes and their related pathways were commonly induced or repressed by WD in both XL22 and XL17, WD-induced differentially expressed genes specific to XL22 or XL17 were also evident. Among the genes that were commonly induced by WD were the GhCER1 genes involved in biosynthesis of alkanes, consistent with the observation of enhanced accumulation of alkanes in cotton leaves under the WD conditions. Interestingly, under the WD conditions, several GhCYP86 genes, which encode enzymes catalyzing the omega-hydroxylation of fatty acids and were identified to be the hub genes of one of the co-expression gene modules, showed a different expression pattern between XL22 and XL17 that was in agreement with the WD-induced changes of the content of hydroxyacids or fatty alcohols in these two varieties.ConclusionThe results contribute to our comprehending the physiological and genetic mechanisms underlying drought tolerance and provide possible solutions for the difference of drought resistance of different cotton varieties.

Fluorescence imaging analysis of distribution of indocyanine green in molecular and nanoform in tumor model

BackgroundThe efficient intraoperative identification of tumors requires the development of highly specific near-infrared (NIR) probes as contrast agents. One of the most effective dyes existing in clinic oncology is Indocyanine Green (ICG). However, ICG has a rapid excretion, thus ruling out its extended accumulation in pathological tissues therefore limiting its clinical applications. ICG colloid solution (ICG NPs) consists predominantly of J-aggregates and to a lesser extent of H-aggregates and monomers. In the present study we assessed the spectral properties of ICG nanoforms in preclinical models. MethodsWe used optical spectroscopy and video fluorescence navigation to monitor accumulation and distribution of ICG monomers and ICG NPs in various tissues in mice with xenografted laryngopharyngeal carcinoma after intravenous drugs injection. ResultsAfter i.v. injection, the molecular form of ICG was not retained in the tumor and its circulation cycle averaged 5 min. Alternatively, the nanoform of the drug had a different pharmacokinetics, reaching maximum accumulation 24 h after intravenous injection. Moreover, once in the circulation, we observed a progressive accumulation in the tumor of both ICG H-aggregates and ICG monomers, but not J-aggregates. ConclusionSpectral characteristics of ICG NPs indicated the presence of several fractions, namely, J- and H-aggregates along with molecular forms. These fractions had different fluorescence spectra, allowing us to track the transformation of the drug in vivo conditions. After ICG NPs administration, J-aggregates induce accumulation of monomeric forms in the tumor, enabling extended intraoperative diagnostic, and as such further studies of J-aggregates for theranostic applications in oncological surgery are of great interest.

Curcumin Complex Analogues as Near-Infrared Fluorescent Probes for Monitoring all Aβ Species in the Early Alzheimer's Disease Model.

Aggregation of amyloid β-peptide (Aβ) is closely related to the pathology of Alzheimer's disease (AD). In this pathology, the beginning stage is characterized by excessive accumulation of Aβ monomers due to imbalanced Aβ in the process of clearance. The Aβ peptide exists in many forms such as soluble and insoluble Aβ species, both of which coexist during the progression of AD and contribute to AD pathology. Thus, probes capable of monitoring all Aβ species are highly desirable. While there are several fluorescent probes for detecting insoluble Aβ, it is still challenging to monitor all Aβ forms by using probes. Here, we describe a near-infrared fluorescent chemical probe, termed AD-1, developed through complexation of curcumin analogues with a stabilizer, which has good photophysical properties and shows high binding to all Aβ species in solution tests. Furthermore, AD-1 exhibited good blood-brain barrier penetrating ability and low cytotoxicity. More importantly, it was successfully applied to 4-month-young APP/PS1 mice imaging noninvasively.

Thiopurines activate an antiviral unfolded protein response that blocks influenza A virus glycoprotein accumulation.

Influenza A viruses (IAVs) utilize host shutoff mechanisms to limit antiviral gene expression and redirect translation machinery to the synthesis of viral proteins. Previously, we showed that IAV replication is sensitive to protein synthesis inhibitors that block translation initiation and induce formation of cytoplasmic condensates of untranslated messenger ribonucleoprotein complexes called stress granules (SGs). In this study, using an image-based high-content screen, we identified two thiopurines, 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo), that triggered SG formation in IAV-infected cells and blocked IAV replication in a dose-dependent manner without eliciting SG formation in uninfected cells. 6-TG and 6-TGo selectively disrupted the synthesis and maturation of IAV glycoproteins hemagglutinin (HA) and neuraminidase (NA) without affecting the levels of the viral RNAs that encode them. By contrast, these thiopurines had minimal effect on other IAV proteins or the global host protein synthesis. Disruption of IAV glycoprotein accumulation by 6-TG and 6-TGo correlated with activation of unfolded protein response (UPR) sensors activating transcription factor-6 (ATF6), inositol requiring enzyme-1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), leading to downstream UPR gene expression. Treatment of infected cells with the chemical chaperone 4-phenylbutyric acid diminished thiopurine-induced UPR activation and partially restored the processing and accumulation of HA and NA. By contrast, chemical inhibition of the integrated stress response downstream of PERK restored accumulation of NA monomers but did not restore processing of viral glycoproteins. Genetic deletion of PERK enhanced the antiviral effect of 6-TG without causing overt cytotoxicity, suggesting that while UPR activation correlates with diminished viral glycoprotein accumulation, PERK could limit the antiviral effects of drug-induced ER stress. Taken together, these data indicate that 6-TG and 6-TGo are effective host-targeted antivirals that trigger the UPR and selectively disrupt accumulation of viral glycoproteins.IMPORTANCESecreted and transmembrane proteins are synthesized in the endoplasmic reticulum (ER), where they are folded and modified prior to transport. Many viruses rely on the ER for the synthesis and processing of viral glycoproteins that will ultimately be incorporated into viral envelopes. Viral burden on the ER can trigger the unfolded protein response (UPR). Much remains to be learned about how viruses co-opt the UPR to ensure efficient synthesis of viral glycoproteins. Here, we show that two FDA-approved thiopurine drugs, 6-TG and 6-TGo, induce the UPR, which represents a previously unrecognized effect of these drugs on cell physiology. This thiopurine-mediated UPR activation blocks influenza virus replication by impeding viral glycoprotein accumulation. Our findings suggest that 6-TG and 6-TGo may have broad antiviral effect against enveloped viruses that require precise tuning of the UPR to support viral glycoprotein synthesis.

Methyl jasmonate promotes wound healing by activation of phenylpropanoid metabolism in harvested kiwifruit

A rapid wound healing in vegetables and fruit is instrumental in maintaining fruit quality and extension of shelf life. In this study, wounded kiwifruit were separately treated with methyl jasmonate (MeJA) and diethyldithiocarbamic acid (DIECA, an inhibitor of jasmonate biosynthesis) to evaluate the effect of MeJA on wound healing of kiwifruit. The results showed that MeJA treatment leads to increases of endogenous JA biosynthesis, lipoxygenase (LOX) and 12-oxo-phytodienoic acid reductase (OPR) activities, and JA synthesis genes expression, while DIECA represses these processes. Meanwhile, MeJA promoted the autofluorescence of suberin polyphenolic (SPP) and the accumulation of SPP monomers. MeJA-treated group demonstrated significantly enhanced total phenols, flavonoids, and lignin contents. Besides, MeJA promoted the activities of phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (C4H), 4-COumaric-COA ligase (4CL), peroxidase (POD), polyphenol oxidase (PPO), and the respective gene expression levels. Results suggest that wound healing in kiwifruit could be promoted by MeJA via activation of phenylpropanoid metabolism.

Hydrophilic meso-substituted cyanine dyes in solution and in complexes with serum albumins: spectral properties and molecular docking study

The absorption and fluorescence spectral properties of three meso-substituted hydrophilic thiacarbocyanine dyes were studied in solutions and in noncovalent complexes with human and bovine serum albumins (HSA and BSA, respectively). The presence of alkyl substituents at the meso-position of the polymethine chain of the dyes determines the occurrence of a cis–trans equilibrium. Dyes form aggregates in aqueous media; the effect of electrolyte (NaCl) on aggregation has been studied. The interaction of the dyes with albumins leads to the decomposition of the aggregates and is accompanied by a shift in the isomeric equilibrium. Complexation with HSA leads to accumulation of dye monomers in the trans-form. However, in the case of BSA the cis-to-trans isomeric shift is incomplete. Using the fluorescence data, the effective binding constants of the trans-isomers with albumins (Ka) and the detection limits of albumin molecules (LD and LQ) were determined. The data obtained are indicative of high selectivity of some dyes to HSA compared to BSA. The results of molecular docking experiments correspond to the data obtained from the spectra. The influence of the dyes on intrinsic fluorescence of HSA and BSA was also studied, and fluorescence quenching, static in nature, was detected.

Solid-state fermentation – assisted extraction of bioactive compounds from hass avocado seeds

The effect of solid-state fermentation (SSF) of avocado seed with A. niger GH1 on the release of bioactive compounds with antioxidant capacity was studied. The growing conditions of the fungus were optimized. Subsequently, The effect of sterile water and pontecorvo culture medium was evaluated in the SSF. The optimal conditions for SSF were particle size of 2.5 mm, 60 % of humidity, and 120 h. In the assessments of antioxidant capacity by the ABTS and DPPH methods, the increase with respect to the initial values was 33 and 77 % with water and 28 and 42 % with culture medium, respectively. The analysis HPLC showed degradation of catechin and epicatechin polymers, particularly procyanidin B2 dimers resulting in the accumulation of monomers. The ability of the fungus to degrade compounds present in the avocado seed was decisive to enhance the antioxidant capacity. This degradation was related to increased cellulase and xylanase activity.

Heterologous production and biochemical characterization of a new highly glucose tolerant GH1 β-glucosidase from Anoxybacillus thermarum

The enzymatic lignocellulosic biomass conversion into value-added products requires the use of enzyme-rich cocktails, including β-glucosidases that hydrolyze cellobiose and cellooligosaccharides to glucose. During hydrolysis occurs accumulation of monomers causing inhibition of some enzymes; thus, glucose/xylose tolerant β-glucosidases could overcome this drawback. The search of new tolerant enzymes showing additional properties, such as high activity, wide-pH range, and thermal stability is very relevant to improve the bioprocess. We describe a novel β-glucosidase GH1 from the thermophilic Anoxybacillus thermarum (BgAt), which stood out by the robustness combination of great glucose/xylose tolerance, thermal stability, and high Vmax. The recombinant his-tagged-BgAt was overexpressed in Escherichia coli, was purified in one step, showed a high glucose/xylose tolerance, and activity stimulation (presence of 0.4 M glucose/1.0 M xylose). The optimal activity was at 65 °C - pH 7.0. BgAt presented an extraordinary temperature stability (48 h – 50 °C), and pH stability (5.5–8.0). The novel enzyme showed outstanding Vmax values compared to other β-glucosidases. Using p-nitrophenyl-β-d-glucopyranoside as substrate the values were Vmax (7614 U/mg), and KM (0.360 mM). These values suffer a displacement in Vmax to 14,026 U/mg (glucose), 14,886 U/mg (xylose), and KM 0.877 mM (glucose), and 1.410 mM (xylose).

Lamin A-mediated nuclear lamina integrity is required for proper ciliogenesis.

The primary cilium is a sensory organelle that receives specific signals from the extracellular environment important for vertebrate development and tissue homeostasis. Lamins, the major components of the nuclear lamina, are required to maintain the nuclear structure and are involved in most nuclear activities. In this study, we show that deficiency in lamin A/C causes defective ciliogenesis, accompanied by increased cytoplasmic accumulation of actin monomers and increased formation of actin filaments. Disruption of actin filaments by cytochalasin D rescues the defective ciliogenesis in lamin A/C‐depleted cells. Moreover, lamin A/C‐deficient cells display lower levels of nesprin 2 and defects in recruiting Arp2, myosin Va, and tau tubulin kinase 2 to the basal body during ciliogenesis. Collectively, our results uncover a functional link between nuclear lamina integrity and ciliogenesis and implicate the malfunction of primary cilia in the pathogenesis of laminopathy.

In vitro stem cell modelling demonstrates a proof-of-concept for excess functional mutant TIMP3 as the cause of Sorsby fundus dystrophy.

Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disease of the macula that leads to bilateral loss of central vision and is caused by mutations in the TIMP3 gene. However, the mechanisms by which TIMP3 mutations cause SFD are poorly understood. Here, we generated human induced pluripotent stem cell-derived retinal pigmented epithelial (hiPSC-RPE) cells from three SFD patients carrying TIMP3 p.(Ser204Cys) and three non-affected controls to study disease-related structural and functional differences in the RPE. SFD-hiPSC-RPE exhibited characteristic RPE structure and physiology but showed significantly reduced transepithelial electrical resistance associated with enriched expression of cytoskeletal remodelling proteins. SFD-hiPSC-RPE exhibited basolateral accumulation of TIMP3 monomers, despite no change in TIMP3 gene expression. TIMP3 dimers were observed in both SFD and control hiPSC-RPE, suggesting that mutant TIMP3 dimerisation does not drive SFD pathology. Furthermore, mutant TIMP3 retained matrix metalloproteinase activity. Proteomic profiling showed increased expression of ECM proteins, endothelial cell interactions and angiogenesis-related pathways in SFD-hiPSC-RPE. By contrast, there were no changes in VEGF secretion. However, SFD-hiPSC-RPE secreted higher levels of monocyte chemoattractant protein 1, PDGF and angiogenin. Our findings provide a proof-of-concept that SFD patient-derived hiPSC-RPE mimic mature RPE cells and support the hypothesis that excess accumulation of mutant TIMP3, rather than an absence or deficiency of functional TIMP3, drives ECM and angiogenesis-related changes in SFD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

SUBERMAN regulates developmental suberization of the Arabidopsis root endodermis.

Root endodermis, the innermost cortical layer surrounding the root vasculature, serves as the foremost barrier to water, solutes, and nutrients taken up from soil. Endodermis barrier functionality is achieved via its hydrophobic coating of lignified Casparian strips and the suberin lamellae; nonetheless the regulatory mechanisms underlying endodermis suberization are still elusive. Here, we discovered that the Arabidopsis SUBERMAN (SUB) transcription factor controls the establishment of the root suberin lamellae. Transient expression of SUB in Nicotiana benthamiana leaves resulted in the induction of heterologous suberin genes, the accumulation of suberin-type monomers, and consequent deposition of suberin-like lamellae. We demonstrate that SUB exerts its regulatory roles by transactivating promoters of suberin genes. In Arabidopsis, SUB is expressed in patchy and continuous suberization root endodermal cells, and thus roots with higher or lower expression of SUB display altered suberin polymer deposition patterns and modified composition. While these changes did not interfere with Casparian strip formation they had a substantial effect on root uptake capacity, resulting in varied root and leaf ionomic phenotypes. Gene expression profiling revealed that SUB function impacts transcriptional networks associated with suberin, phenylpropanoids, lignin, and cuticular lipid biosynthesis, as well as root transport activities, hormone signalling, and cell wall modification. Our findings highlight SUB as a regulator of root endodermis suberization during normal development, and its characterization is thus a key step towards dissecting the molecular mechanisms partaking in root endodermal barrier functionalities.