Tear Proteins Altered in Patients with Persistent Eye Pain after Refractive Surgery: Biomarker Candidate Discovery.

Some patients develop persistent eye pain after refractive surgery, but factors that cause or sustain pain are unknown. We tested whether tear proteins of patients with pain 3 months after surgery differ from those of patients without pain. Patients undergoing refractive surgery (laser in situ keratomileusis or photorefractive keratectomy ) were recruited from 2 clinics, and tears were collected 3 months after surgery. Participants rated their eye pain using a numerical rating scale (NRS, 0-10; no pain-worst pain) at baseline, 1 day, and 3 months after surgery. Using tandem mass tag proteomic analysis, we examined tears from patients with pain [NRS ≥ 3 at 3 months (n = 16)] and patients with no pain [NRS ≤ 1 at 3 months (n = 32)] after surgery. A subset of proteins (83 of 2748 detected, 3.0%) were associated with pain 3 months after surgery. High-dimensional statistical models showed that the magnitude of differential expression was not the only important factor in classifying tear samples from pain patients. Models utilizing 3 or 4 proteins had better classification performance than single proteins and represented differences in both directions (higher or lower in pain). Thus, patterns of protein differences may serve as biomarkers of postsurgical eye pain as well as potential therapeutic targets.

Aging-related Alterations in mTOR Signaling Promote Platelet Hyperreactivity and Thrombosis.

BACKGROUND AND OBJECTIVES: Aging is an independent risk factor for the development of cardiovascular, thrombotic and other chronic diseases. However, mechanisms of platelet hyperactivation in aging remain poorly understood. Here, we examine whether and how aging alters intracellular signaling in platelets to support platelet hyperactivity and thrombosis. METHODS: Quantitative mass spectrometry with tandem mass tag (TMT) labeling systematically measured protein phosphorylation in platelets from healthy aged (>65 years) and young human (<45 years) subjects. The role of platelet mTOR in aging-induced platelet hyperreactivity was assessed using pharmacological mTOR inhibition and a platelet-specific mTOR-deficient mouse model (mTORplt-/-). RESULTS: Quantitative phosphoproteomics uncovered differential site-specific protein phosphorylation within mTOR, Rho GTPase and MAPK pathways in platelets from aged donors. Western blot confirmed constitutive activation of the mTOR pathway in platelets from both aged humans and mice, which was associated with increased aggregation compared to young controls. Inhibition of mTOR either with Torin 1 in aged humans, or genetic deletion in aged mice, reversed platelet hyperreactivity. In a collagen-epinephrine pulmonary thrombosis model, aged wild-type (mTORplt+/+) mice succumbed significantly faster compared to young controls, while time to death of aged mTORplt-/- mice was similar to young mTORplt+/+ mice. Mechanistically, we noted increased Rac1 activation and levels of mitochondrial reactive oxygen species in resting platelets from aged mice, as well as increased p38 phosphorylation upstream of thromboxane generation following agonist stimulation. CONCLUSION: Aging-related changes in mTOR phosphorylation enhance Rac1 and p38 activation, to enhance thromboxane generation, platelet hyperactivity and thrombosis.

Ocimum kilimandscharicum 4CL11 negatively regulates adventitious root development via accumulation of flavonoid glycosides.

4-Coumarate-CoA Ligase (4CL) is an important enzyme in the phenylpropanoid biosynthesis pathway. Multiple 4CLs are identified in Ocimum species; however, their in planta functions remain enigmatic. In this study, we independently overexpressed three Ok4CL isoforms from Ocimum kilimandscharicum (Ok4CL7, -11, and -15) in Nicotiana benthamiana. Interestingly, Ok4CL11 overexpression (OE) caused a rootless or reduced root growth phenotype, whereas overexpression of Ok4CL15 produced normal adventitious root (AR) growth. Ok4CL11 overexpression in N. benthamiana resulted in upregulation of genes involved in flavonoid biosynthesis and associated glycosyltransferases accompanied by accumulation of specific flavonoid-glycosides (kaempferol-3-rhamnoside, kaempferol-3,7-O-bis-alpha-l-rhamnoside [K3,7R], and quercetin-3-O-rutinoside) that possibly reduced auxin levels in plants, and such effects were not seen for Ok4CL7 and -15. Docking analysis suggested that auxin transporters (PINs/LAXs) have higher binding affinity to these specific flavonoid-glycosides, and thus could disrupt auxin transport/signaling, which cumulatively resulted in a rootless phenotype. Reduced auxin levels, increased K3,7R in the middle and basal stem sections, and grafting experiments (intra and inter-species) indicated a disruption of auxin transport by K3,7R and its negative effect on AR development. Supplementation of flavonoids and the specific glycosides accumulated by Ok4CL11-OE to the wild-type N. benthamiana explants delayed the AR emergence and also inhibited AR growth. While overexpression of all three Ok4CLs increased lignin accumulation, flavonoids, and their specific glycosides were accumulated only in Ok4CL11-OE lines. In summary, our study reveals unique indirect function of Ok4CL11 to increase specific flavonoids and their glycosides, which are negative regulators of root growth, likely involved in inhibition of auxin transport and signaling.

Chitinase inhibition induces transcriptional dysregulation altering ecdysteroid-mediated control of Spodoptera frugiperda development.

Chitinases and ecdysteroid hormones are vital for insect development. Crosstalk between chitin and ecdysteroid metabolism regulation is enigmatic. Here, we examined chitinase inhibition effect on Spodoptera frugiperda ecdysteroid metabolism. In vitro studies suggested that berberine inhibits S. frugiperda chitinase 5 (SfCht5). The Berberine feeding resulted in defective S. frugiperda development. Berberine-fed insects showed higher SfCht5 and Chitinase 7 expression and cumulative chitinase activity. Chitinase inhibition led to overexpression of chitinases, ecdysteroid biosynthesis, and responsive genes. SfCht5 silencing and overexpression resulted in ecdysone receptor deregulation. Transcription factors, like Broad Complex Z4, regulate the ecdysteroid metabolism and showed high expression upon berberine ingestion. Broad Complex Z4 binding in 5' UTR of Ecdysone receptor, SfCht5, Chitinase 7, Phantom, Neverland, and other ecdysteroid biosynthesis genes might lead to their upregulation in berberine-fed insects. As a result, berberine-fed insects showed ecdysone overaccumulation. These findings underscore chitinase activity's impact on ecdysone biosynthesis and its transcriptional crosstalk.

Aminoacylase efficiently hydrolyses fatty acid amino acid conjugates of Helicoverpa armigera potentially to increase the pool of glutamine.

One of the most prevalent bioactive molecules present in the oral secretion (OS) of lepidopteran insects is fatty acid amino acid conjugates (FACs). Insect dietary components have influence on the synthesis and retaining the pool of FACs in the OS. We noted differential and diet-specific accumulation of FACs in the OS of Helicoverpa armigera by using Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry. Interestingly, we identified FACs hydrolyzing enzyme aminoacylase (HaACY) in the OS of H. armigera through proteomic analysis. Next, we have cloned, expressed, and purified active recombinant HaACY in the bacterial system. Recombinant HaACY hydrolyzes all the six identified FACs in the OS of H. armigera larvae fed on host and non-host plants and releases respective fatty acid and glutamine. In these six FACs, fatty acid moieties vary while amino acid glutamine was common. Glutamine obtained upon hydrolysis of FACs by HaACY might serve as an amino acid pool for insect growth and development. To understand the substrate choices of HaACY, we chemically synthesized, purified, and characterized all the six FACs. Interestingly, rHaACY also shows hydrolysis of synthetic FACs into respective fatty acid and glutamine. Our results underline the importance of diet on accumulation of FACs and role of aminoacylase(s) in regulating the level of FACs and glutamine.

The amniotic fluid proteome changes across gestation in humans and rhesus macaques.

Amniotic fluid is a complex biological medium that offers protection to the fetus and plays a key role in normal fetal nutrition, organogenesis, and potentially fetal programming. Amniotic fluid is also critically involved in longitudinally shaping the in utero milieu during pregnancy. Yet, the molecular mechanism(s) of action by which amniotic fluid regulates fetal development is ill-defined partly due to an incomplete understanding of the evolving composition of the amniotic fluid proteome. Prior research consisting of cross-sectional studies suggests that the amniotic fluid proteome changes as pregnancy advances, yet longitudinal alterations have not been confirmed because repeated sampling is prohibitive in humans. We therefore performed serial amniocenteses at early, mid, and late gestational time-points within the same pregnancies in a rhesus macaque model. Longitudinally-collected rhesus amniotic fluid samples were paired with gestational-age matched cross-sectional human samples. Utilizing LC-MS/MS isobaric labeling quantitative proteomics, we demonstrate considerable cross-species similarity between the amniotic fluid proteomes and large scale gestational-age associated changes in protein content throughout pregnancy. This is the first study to compare human and rhesus amniotic fluid proteomic profiles across gestation and establishes a reference amniotic fluid proteome. The non-human primate model holds promise as a translational platform for amniotic fluid studies.

Unraveling the role of TGFß signaling in thoracic aortic aneurysm and dissection using Fbn1 mutant mouse models.

Although abnormal TGFß signaling is observed in several heritable forms of thoracic aortic aneurysms and dissections including Marfan syndrome, its precise role in aortic disease progression is still disputed. Using a mouse genetic approach and quantitative isobaric labeling proteomics, we sought to elucidate the role of TGFß signaling in three Fbn1 mutant mouse models representing a range of aortic disease from microdissection (without aneurysm) to aneurysm (without rupture) to aneurysm and rupture. Results indicated that reduced TGFß signaling and increased mast cell proteases were associated with microdissection. In contrast, increased abundance of extracellular matrix proteins, which could be reporters for positive TGFß signaling, were associated with aneurysm. Marked reductions in collagens and fibrillins, and increased TGFß signaling, were associated with aortic rupture. Our data indicate that TGFß signaling performs context-dependent roles in the pathogenesis of thoracic aortic disease.

MicroRNA137-loaded lipid nanoparticles regulate synaptic proteins in the prefrontal cortex.

Genome-wide association studies indicate that allele variants in MIR137, the host gene of microRNA137 (miR137), confer an increased risk of schizophrenia (SCZ). Aberrant expression of miR137 and its targets, many of which regulate synaptic functioning, are also associated with an increased risk of SCZ. Thus, miR137 represents an attractive target aimed at correcting the molecular basis for synaptic dysfunction in individuals with high genetic risk for SCZ. Advancements in nanotechnology utilize lipid nanoparticles (LNPs) to transport and deliver therapeutic RNA. However, there remains a gap in using LNPs to regulate gene and protein expression in the brain. To study the delivery of nucleic acids by LNPs to the brain, we found that LNPs released miR137 cargo and inhibited target transcripts of interest in neuroblastoma cells. Biodistribution of LNPs loaded with firefly luciferase mRNA remained localized to the mouse prefrontal cortex (PFC) injection site without circulating to off-target organs. LNPs encapsulating Cre mRNA preferentially co-expressed in neuronal over microglial or astrocytic cells. Using quantitative proteomics, we found miR137 modulated glutamatergic synaptic protein networks that are commonly dysregulated in SCZ. These studies support engineering the next generation of brain-specific LNPs to deliver RNA therapeutics and improve symptoms of central nervous system disorders.

Domain Shuffling and Site-Saturation Mutagenesis for the Enhanced Inhibitory Potential of Amaranthaceae α-Amylase Inhibitors.

Amaranthaceae α-amylase inhibitors (AAIs) are knottin-type proteins with selective inhibitory potential against coleopteran α-amylases. Their small size and remarkable stability make them exciting molecules for protein engineering to achieve superior selectivity and efficacy. In this report, we have designed a set of AAI pro- and mature peptides chimeras. Based on in silico analysis, stable AAI chimeras having a stronger affinity with target amylases were selected for characterization. In vitro studies validated that chimera of the propeptide from Chenopodium quinoa α-AI and mature peptide from Beta vulgaris α-AI possess 3, 7.6, and 4.26 fold higher inhibition potential than parental counterparts. Importantly, recombinant AAI chimera retained specificity towards target coleopteran α-amylases. In addition, to improve the inhibitory potential of AAI, we performed in silico site-saturation mutagenesis. Computational analysis followed by experimental data showed that substituting Asparagine at the 6th position with Methionine had a remarkable increase in the specific inhibition potential of Amaranthus hypochondriacus α-AI. These results provide structural-functional insights into the vitality of AAI propeptide and a potential hotspot for mutagenesis to enhance the AAI activity. Our investigation will be a toolkit for AAI's optimization and functional differentiation for future biotechnological applications.

A BAHD-type acyltransferase concludes the biosynthetic pathway of non-bitter glycoalkaloids in ripe tomato fruit.

Tomato is the highest value fruit and vegetable crop worldwide, yet produces α-tomatine, a renowned toxic and bitter-tasting anti-nutritional steroidal glycoalkaloid (SGA) involved in plant defense. A suite of modifications during tomato fruit maturation and ripening converts α-tomatine to the non-bitter and less toxic Esculeoside A. This important metabolic shift prevents bitterness and toxicity in ripe tomato fruit. While the enzymes catalyzing glycosylation and hydroxylation reactions in the Esculeoside A pathway have been resolved, the proposed acetylating step remains, to date, elusive. Here, we discovered that GAME36 (GLYCOALKALOID METABOLISM36), a BAHD-type acyltransferase catalyzes SGA-acetylation in cultivated and wild tomatoes. This finding completes the elucidation of the core Esculeoside A biosynthetic pathway in ripe tomato, allowing reconstitution of Esculeoside A production in heterologous microbial and plant hosts. The involvement of GAME36 in bitter SGA detoxification pathway points to a key role in the evolution of sweet-tasting tomato as well as in the domestication and breeding of modern cultivated tomato fruit.

Proteomic profiling of retina and retinal pigment epithelium combined embryonic tissue to facilitate ocular disease gene discovery.

To expedite gene discovery in eye development and its associated defects, we previously developed a bioinformatics resource-tool iSyTE (integrated Systems Tool for Eye gene discovery). However, iSyTE is presently limited to lens tissue and is predominantly based on transcriptomics datasets. Therefore, to extend iSyTE to other eye tissues on the proteome level, we performed high-throughput tandem mass spectrometry (MS/MS) on mouse embryonic day (E)14.5 retina and retinal pigment epithelium combined tissue and identified an average of 3300 proteins per sample (n = 5). High-throughput expression profiling-based gene discovery approaches-involving either transcriptomics or proteomics-pose a key challenge of prioritizing candidates from thousands of RNA/proteins expressed. To address this, we used MS/MS proteome data from mouse whole embryonic body (WB) as a reference dataset and performed comparative analysis-termed "in silico WB-subtraction"-with the retina proteome dataset. In silico WB-subtraction identified 90 high-priority proteins with retina-enriched expression at stringency criteria of ≥ 2.5 average spectral counts, ≥ 2.0 fold-enrichment, false discovery rate < 0.01. These top candidates represent a pool of retina-enriched proteins, several of which are associated with retinal biology and/or defects (e.g., Aldh1a1, Ank2, Ank3, Dcn, Dync2h1, Egfr, Ephb2, Fbln5, Fbn2, Hras, Igf2bp1, Msi1, Rbp1, Rlbp1, Tenm3, Yap1, etc.), indicating the effectiveness of this approach. Importantly, in silico WB-subtraction also identified several new high-priority candidates with potential regulatory function in retina development. Finally, proteins exhibiting expression or enriched-expression in the retina are made accessible in a user-friendly manner at iSyTE ( https://research.bioinformatics.udel.edu/iSyTE/ ), to allow effective visualization of this information and facilitate eye gene discovery.

Proteomic profiling of retina and retinal pigment epithelium combined embryonic tissue to facilitate ocular disease gene discovery.

To expedite gene discovery in eye development and its associated defects, we previously developed a bioinformatics resource-tool iSyTE (integrated Systems Tool for Eye gene discovery). However, iSyTE is presently limited to lens tissue and is predominantly based on transcriptomics datasets. Therefore, to extend iSyTE to other eye tissues on the proteome level, we performed high-throughput tandem mass spectrometry (MS/MS) on mouse embryonic day (E)14.5 retina and retinal pigment epithelium combined tissue and identified an average of 3,300 proteins per sample (n=5). High-throughput expression profiling-based gene discovery approaches-involving either transcriptomics or proteomics-pose a key challenge of prioritizing candidates from thousands of RNA/proteins expressed. To address this, we used MS/MS proteome data from mouse whole embryonic body (WB) as a reference dataset and performed comparative analysis-termed "in silico WB-subtraction"-with the retina proteome dataset. In silico WB-subtraction identified 90 high-priority proteins with retina-enriched expression at stringency criteria of ³2.5 average spectral counts, ³2.0 fold-enrichment, False Discovery Rate <0.01. These top candidates represent a pool of retina-enriched proteins, several of which are associated with retinal biology and/or defects (e.g., Aldh1a1, Ank2, Ank3, Dcn, Dync2h1, Egfr, Ephb2, Fbln5, Fbn2, Hras, Igf2bp1, Msi1, Rbp1, Rlbp1, Tenm3, Yap1, etc.), indicating the effectiveness of this approach. Importantly, in silico WB-subtraction also identified several new high-priority candidates with potential regulatory function in retina development. Finally, proteins exhibiting expression or enriched-expression in the retina are made accessible in a user-friendly manner at iSyTE (https://research.bioinformatics.udel.edu/iSyTE/), to allow effective visualization of this information and facilitate eye gene discovery.

Assessment of the Risk of Venous Thromboembolism in Nonhospitalized Patients With COVID-19.

Importance: Patients hospitalized with COVID-19 have higher rates of venous thromboembolism (VTE), but the risk and predictors of VTE among individuals with less severe COVID-19 managed in outpatient settings are less well understood. Objectives: To assess the risk of VTE among outpatients with COVID-19 and identify independent predictors of VTE. Design, Setting, and Participants: A retrospective cohort study was conducted at 2 integrated health care delivery systems in Northern and Southern California. Data for this study were obtained from the Kaiser Permanente Virtual Data Warehouse and electronic health records. Participants included nonhospitalized adults aged 18 years or older with COVID-19 diagnosed between January 1, 2020, and January 31, 2021, with follow-up through February 28, 2021. Exposures: Patient demographic and clinical characteristics identified from integrated electronic health records. Main Outcomes and Measures: The primary outcome was the rate per 100 person-years of diagnosed VTE, which was identified using an algorithm based on encounter diagnosis codes and natural language processing. Multivariable regression using a Fine-Gray subdistribution hazard model was used to identify variables independently associated with VTE risk. Multiple imputation was used to address missing data. Results: A total of 398 530 outpatients with COVID-19 were identified. The mean (SD) age was 43.8 (15.8) years, 53.7% were women, and 54.3% were of self-reported Hispanic ethnicity. There were 292 (0.1%) VTE events identified over the follow-up period, for an overall rate of 0.26 (95% CI, 0.24-0.30) per 100 person-years. The sharpest increase in VTE risk was observed during the first 30 days after COVID-19 diagnosis (unadjusted rate, 0.58; 95% CI, 0.51-0.67 per 100 person-years vs 0.09; 95% CI, 0.08-0.11 per 100 person-years after 30 days). In multivariable models, the following variables were associated with a higher risk for VTE in the setting of nonhospitalized COVID-19: age 55 to 64 years (HR 1.85 [95% CI, 1.26-2.72]), 65 to 74 years (3.43 [95% CI, 2.18-5.39]), 75 to 84 years (5.46 [95% CI, 3.20-9.34]), greater than or equal to 85 years (6.51 [95% CI, 3.05-13.86]), male gender (1.49 [95% CI, 1.15-1.96]), prior VTE (7.49 [95% CI, 4.29-13.07]), thrombophilia (2.52 [95% CI, 1.04-6.14]), inflammatory bowel disease (2.43 [95% CI, 1.02-5.80]), body mass index 30.0-39.9 (1.57 [95% CI, 1.06-2.34]), and body mass index greater than or equal to 40.0 (3.07 [1.95-4.83]). Conclusions and Relevance: In this cohort study of outpatients with COVID-19, the absolute risk of VTE was low. Several patient-level factors were associated with higher VTE risk; these findings may help identify subsets of patients with COVID-19 who may benefit from more intensive surveillance or VTE preventive strategies.

Genomic Determinants of Entomopathogenic Fungi and Their Involvement in Pathogenesis.

Entomopathogenic fungi offer an effective and eco-friendly alternative to curb insect populations in biocontrol strategy. The evolutionary history of selected entomopathogenic fungi indicates their ancestral relationship with plant endophytes. During this host shifting, entomopathogenic fungi must have acquired multiple mechanisms, including a combination of various biomolecules that make them distinguishable from other fungi. In this review, we focus on understanding various biochemical and molecular mechanisms involved in entomopathogenesis. In particular, we attempt to explain the indispensable role of enlarged gene families of various virulent factors, viz. chitinases, proteases, lipases, specialized metabolites, and cytochrome P450, in entomopathogenesis. Our analysis suggests that entomopathogenic fungi recruit a different set of gene products during the progression of pathogenesis. Knowledge of these bio-molecular interactions between fungi and insect hosts will allow researchers to execute pointed efforts towards the development of improved entomopathogenic fungal strains.

Effect of antiplatelet agents and tyrosine kinase inhibitors on oxLDL-mediated procoagulant platelet activity.

Low-density lipoprotein (LDL) contributes to atherogenesis and cardiovascular disease through interactions with peripheral blood cells, especially platelets. However, mechanisms by which LDL affects platelet activation and atherothrombosis, and how to best therapeutically target and safely prevent such responses remain unclear. Here, we investigate how oxidized low-density lipoprotein (oxLDL) enhances glycoprotein VI (GPVI)-mediated platelet hemostatic and procoagulant responses, and how traditional and emerging antiplatelet therapies affect oxLDL-enhanced platelet procoagulant activity ex vivo. Human platelets were treated with oxLDL and the GPVI-specific agonist, crosslinked collagen-related peptide, and assayed for hemostatic and procoagulant responses in the presence of inhibitors of purinergic receptors (P2YR), cyclooxygenase (COX), and tyrosine kinases. Ex vivo, oxLDL enhanced GPVI-mediated platelet dense granule secretion, α-granule secretion, integrin activation, thromboxane generation and aggregation, as well as procoagulant phosphatidylserine exposure and fibrin generation. Studies of washed human platelets, as well as platelets from mouse and nonhuman primate models of hyperlipidemia, further determined that P2YR antagonists (eg, ticagrelor) and Bruton tyrosine kinase inhibitors (eg, ibrutinib) reduced oxLDL-mediated platelet responses and procoagulant activity, whereas COX inhibitors (eg, aspirin) had no significant effect. Together, our results demonstrate that oxLDL enhances GPVI-mediated platelet procoagulant activity in a manner that may be more effectively reduced by P2YR antagonists and tyrosine kinase inhibitors compared with COX inhibitors.

Protein kinase CK2 phosphorylates a conserved motif in the Notch effector E(spl)-Mγ.

Across metazoan animals, the effects of Notch signaling are mediated via the Enhancer of Split (E(spl)/HES) basic Helix-Loop-Helix-Orange (bHLH-O) repressors. Although these repressors are generally conserved, their sequence diversity is, in large part, restricted to the C-terminal domain (CtD), which separates the Orange (O) domain from the penultimate WRPW tetrapeptide motif that binds the obligate co-repressor Groucho. While the kinases CK2 and MAPK target the CtD and regulate Drosophila E(spl)-M8 and mammalian HES6, the generality of this regulation to other E(spl)/HES repressors has remained unknown. To determine the broader impact of phosphorylation on this large family of repressors, we conducted bioinformatics, evolutionary, and biochemical analyses. Our studies identify E(spl)-Mγ as a new target of native CK2 purified from Drosophila embryos, reveal that phosphorylation is specific to CK2 and independent of the regulatory CK2-ß subunit, and identify that the site of phosphorylation is juxtaposed to the WRPW motif, a feature unique to and conserved in the Mγ homologues over 50 × 106 years of Drosophila evolution. Thus, a preponderance of E(spl) homologues (four out of seven total) in Drosophila are targets for CK2, and the distinct positioning of the CK2 and MAPK sites raises the prospect that phosphorylation underlies functional diversity of bHLH-O proteins.