Endogenous Production and Vibrational Analysis of Heavy-Isotope-Labeled Peptides from Cyanobacteria.

Stable isotope labeling is an extremely useful tool for characterizing the structure, tracing the metabolism, and imaging the distribution of natural products in living organisms using mass-sensitive measurement techniques. In this study, a cyanobacterium was cultured in 15 N/13 C-enriched media to endogenously produce labeled, bioactive oligopeptides. The extent of heavy isotope incorporation in these peptides was determined with LC-MS, while the overall extent of heavy isotope incorporation in whole cells was studied with nanoSIMS and AFM-IR. Up to 98 % heavy isotope incorporation was observed in labeled cells. Three of the most abundant peptides, microcystin-LR (MCLR), cyanopeptolin-A (CYPA), and aerucyclamide-A (ACAA), were isolated and further studied with Raman and FTIR spectroscopies and DFT calculations. This revealed several IR and Raman active vibrations associated with functional groups not common in ribosomal peptides, like diene, ester, thiazole, thiazoline, and oxazoline groups, which could be suitable for future vibrational imaging studies. More broadly, this study outlines a simple and relatively inexpensive method for producing heavy-labeled natural products. Manipulating the bacterial culture conditions by the addition of specific types and amounts of heavy-labeled nutrients provides an efficient means of producing heavy-labeled natural products for mass-sensitive imaging studies.

Creation of a Plant Metabolite Spectral Library for Untargeted and Targeted Metabolomics.

Large-scale high throughput metabolomic technologies are indispensable components of systems biology in terms of discovering and defining the metabolite parts of the system. However, the lack of a plant metabolite spectral library limits the metabolite identification of plant metabolomic studies. Here, we have created a plant metabolite spectral library using 544 authentic standards, which increased the efficiency of identification for untargeted metabolomic studies. The process of creating the spectral library was described, and the mzVault library was deposited in the public repository for free download. Furthermore, based on the spectral library, we describe a process of creating a pseudo-targeted method, which was applied to a proof-of-concept study of Arabidopsis leaf extracts. As authentic standards become available, more metabolite spectra can be easily incorporated into the spectral library to improve the mzVault package.

Mitogen-Activated Protein Kinase 4-Regulated Metabolic Networks.

Mitogen-activated protein kinase 4 (MPK4) was first identified as a negative regulator of systemic acquired resistance. It is also an important kinase involved in many other biological processes in plants, including cytokinesis, reproduction, and photosynthesis. Arabidopsis thaliana mpk4 mutant is dwarf and sterile. Previous omics studies including genomics, transcriptomics, and proteomics have revealed new functions of MPK4 in different biological processes. However, due to challenges in metabolomics, no study has touched upon the metabolomic profiles of the mpk4 mutant. What metabolites and metabolic pathways are potentially regulated by MPK4 are not known. Metabolites are crucial components of plants, and they play important roles in plant growth and development, signaling, and defense. Here we used targeted and untargeted metabolomics to profile metabolites in the wild type and the mpk4 mutant. We found that in addition to the jasmonic acid and salicylic acid pathways, MPK4 is involved in polyamine synthesis and photosynthesis. In addition, we also conducted label-free proteomics of the two genotypes. The integration of metabolomics and proteomics data allows for an insight into the metabolomic networks that are potentially regulated by MPK4.

Rapid Highly-Efficient Digestion and Peptide Mapping of Adeno-Associated Viruses.

Adeno-associated viruses (AAVs) comprise an area of rapidly growing interest due to their ability to act as a gene delivery vehicle in novel gene therapy strategies and vaccine development. Peptide mapping is a common technique in the biopharmaceutical industry to confirm the correct sequence, product purity, post-translational modifications (PTMs), and stability. However, conventional peptide mapping is time-consuming and has proven difficult to reproduce with viral capsids because of their high structural stability and the suboptimal localization of trypsin cleavage sites in the AAV protein sequences. In this study, we present an optimized peptide mapping-based workflow that provides thorough characterization within 1 day. This workflow is also highly reproducible due to its simplicity having very few steps and is easy to perform proteolytic digestion utilizing thermally stable pepsin, which is active at 70 °C in acidic conditions. The acidic conditions of the peptic digestions drive viral capsid denaturation and improve cleavage site accessibility. We characterized the efficiency and ease of digestion through peptide mapping of the AAV2 viral capsid protein. Using nanoflow liquid chromatography coupled with tandem mass spectrometry, we achieved 100% sequence coverage of the low-abundance VP1 capsid protein with a digestion process taking only 10 min to prepare and 45 min to complete the digestion.

Successful Intravascular Treatment of an Intraosseous Arteriovenous Fistula in Fibrous Dysplasia.

Fibrous dysplasia (FD) is a benign bone disease characterized by expansile lesions that typically stabilize with age. Rarely, FD can undergo malignant transformation, presenting with atypical, rapid growth and destruction of adjacent bone. Other potential causes of rapid FD expansion include secondary lesions, such as aneurysmal bone cysts. We describe a case of an aggressive occipital lesion that presented with pain associated with diplopia and tinnitus, raising concern for malignant transformation. A massive intraosseous arteriovenous fistula was identified giving rise to an anomalous vein coursing to the cavernous sinus with compression of the abducens nerve. The vascular anomaly was mapped and after embolization symptoms resolved; a biopsy with extensive genetic analyses excluded malignancy. The differential diagnosis for expanding FD lesions includes aggressive FD, malignant transformation, and secondary vascular anomalies. In cases when traditional radiographic and histologic assessments are nondescript, use of additional radiographic modalities and genetic analyses are required to make an accurate diagnosis and guide treatment. When vascular anomalies are suspected, detailed angiography with embolization is necessary to define and treat the lesion. However, to rule out malignant transformation, genetic screening is recommended.

Free Dermal Fat Autografting for Complex Craniofacial Wounds.

Complex craniofacial wounds (CCW) are those refractory to initial treatment and may involve chronic infection, exposed hardware, irradiated tissue, and soft tissue volume loss. Typical reconstruction with microvascular flaps involves considerable morbidity. While free dermal fat autografting (DFA) is used extensively in many applications, its use treating CCW remains an unexplored but attractive possibility. Data from a retrospective cohort of 34 consecutive patients (13 male; 21 female and aged 2-79-years), who underwent free DFA between 1985 and 2018 for CCW by a single plastic surgeon, were analyzed. Post-operative follow-up was 1-24 years (M = 6.53, SD = 7.91). Many patients had several concomitant wound complications. Primary pre-operative wound complications were dominated by infection (N = 20), of which over 75% (N = 15) were associated with non-autogenic material. Eighteen had resolution of their pre-operative infection. Of the total (N = 34), 79.41% had stable grafts at follow-up [X(3) = 54, P < 0.001], with only 3 experiencing observable atrophy and 1 graft necrosis. Most of the cohort was complication free [X(1) = 7.53, P = 0.006], with 73.53% experiencing no problems involving the graft. Twenty-nine (85.29%) of 34 patients had therapeutic success with free DFA [X(1) = 28.65, P < 0.001]. Pre-operative wound status (ß = 1.13, P < 0.001) predicted therapeutic success [R = 0.87, F(7,9) = 8.94, P = 0.002]. While 5 (14.71%) did not have therapeutic success, no additional problems arose related to grafts. Free DFA appears to be beneficial and show low morbidity. Future studies must evaluate these findings. In this context, their use should be considered in recalcitrant craniofacial wounds.

Identification and Recent Approaches for Evaluation and Management of Dentofacial and Otolaryngologic Concerns for Patients With Freeman-Burian Syndrome: Principles for Global Treatment.

Freeman-Burian syndrome (FBS), formerly Freeman-Sheldon syndrome, is a complex myopathic craniofacial syndrome. Functional craniofacial deformities resulting in respiratory, eating, auditory, or speech impairments often are present to varying degrees in this unique population. There are few references in the literature addressing diagnosis, evaluation, operative counseling, and craniofacial management of FBS, and guidance was absent. As part of a clinical practice guideline development process for FBS, the authors have reviewed dental and oral health concerns, hearing loss, paranasal sinusitis, dysphagia, and dysphasia management for patients with FBS. Searching PubMed and Google Scholar has yielded 14 results describing dentofacial and otorhinolaryngologic concerns in FBS. There is a significant paucity of scholarship on FBS, presenting considerable knowledge gaps. Craniofacial muscles may be preferentially impacted by fibrous tissue replacement. The lack of available objective data should not reduce clinical vigilance to the possibility that fibrous tissue replacement may influence almost any aspect of the patient's presentation, thus necessitating nonstandard treatment deviations. Based on the decades of experience with this challenging patient population, the authors feel much can be done to afford patients with FBS a good and productive quality of life through exquisite medical surveillance, rapid intervention in acute upper respiratory disturbances, conservative operative intervention, and longitudinal lifestyle structuring by the patients.

Findings, Phenotypes, Diagnostic Accuracy, and Treatment in Freeman-Burian Syndrome.

Freeman-Burian syndrome (FBS) is a rare congenital myopathic craniofacial syndrome. Since publication of the genotype-correlated clinical diagnostic criteria, no complete survey of the literature has been accomplished. As part of the clinical practice guideline development, we evaluate diagnostic accuracy for FBS from 1938 to 2019 and range of findings, complications, treatments, and outcomes. Published manuscripts in PubMed, Google Scholar, and OMIM describing cases with a reported diagnosis of FBS, Sheldon-Hall syndrome, and distal arthrogryposes type 1 and 3 are initially included. Articles with sufficient case-level data for diagnosis verification are analyzed further. Of 724 unique papers considered, 188 papers describing 304 unique patients are included; 101 papers and 119 patients reflect an FBS diagnosis, with 80 patients meeting the full diagnostic criteria. Most cases are re-screened as distal arthrogryposis type 1. Among all cases re-screened as FBS, the presence of FBS pathognomonic craniofacial findings is not correlated with other physical findings. There are no significant differences between patients meeting the full diagnostic criteria and those not, but both are distinct from other diagnoses. Plastic surgery demonstrates the highest cumulative diagnostic accuracy for FBS overall (86.66%), while orthopedic surgery shows the lowest (44.83%). No statistically usable treatment-related or psychosocial data are available. Quality of case reports and patient data vary widely, reducing the statistical strength and significance. Major knowledge gaps exist in treatment, psychosocial, and longitudinal outcomes. At this point, it is impossible to derive clinical practice guidelines exclusively from the literature.

S-Nitroso-Proteome Revealed in Stomatal Guard Cell Response to Flg22.

Nitric oxide (NO) plays an important role in stomata closure induced by environmental stimuli including pathogens. During pathogen challenge, nitric oxide (NO) acts as a second messenger in guard cell signaling networks to activate downstream responses leading to stomata closure. One means by which NO's action is achieved is through the posttranslational modification of cysteine residue(s) of target proteins. Although the roles of NO have been well studied in plant tissues and seedlings, far less is known about NO signaling and, more specifically, protein S-nitrosylation (SNO) in stomatal guard cells. In this study, using iodoTMTRAQ quantitative proteomics technology, we analyzed changes in protein SNO modification in guard cells of reference plant Arabidopsis thaliana in response to flg22, an elicitor-active peptide derived from bacterial flagellin. A total of 41 SNO-modified peptides corresponding to 35 proteins were identified. The proteins cover a wide range of functions, including energy metabolism, transport, stress response, photosynthesis, and cell-cell communication. This study creates the first inventory of previously unknown NO responsive proteins in guard cell immune responses and establishes a foundation for future research toward understanding the molecular mechanisms and regulatory roles of SNO in stomata immunity against bacterial pathogens.

MPK4 Phosphorylation Dynamics and Interacting Proteins in Plant Immunity.

Arabidopsis MAP kinase 4 (MPK4) has been proposed to be a negative player in plant immunity, and it is also activated by pathogen-associated molecular patterns (PAMPs), such as flg22. The molecular mechanisms by which MPK4 is activated and regulates plant defense remain elusive. In this study, we investigated Arabidopsis defense against a bacterial pathogen Pseudomonas syringae pv tomato ( Pst) DC3000 when Brassica napus MPK4 ( BnMPK4) is overexpressed. We showed an increase in pathogen resistance and suppression of jasmonic acid (JA) signaling in the BnMPK4 overexpressing (OE) plants. We also showed that the OE plants have increased sensitivity to flg22-triggered reactive oxygen species (ROS) burst in guard cells, which resulted in enhanced stomatal closure compared to wild-type (WT). During flg22 activation, dynamic phosphorylation events within and outside of the conserved TEY activation loop were observed. To elucidate how BnMPK4 functions during the defense response, we used immunoprecipitation coupled with mass spectrometry (IP-MS) to identify BnMPK4 interacting proteins in the absence and presence of flg22. Quantitative proteomic analysis revealed a shift in the MPK4-associated protein network, providing insight into the molecular functions of MPK4 at the systems level.

Identification and Recent Approaches for Evaluation, Operative Counseling, and Management in Patients With Freeman-Burian Syndrome: Principles for Global Treatment.

For many, the experience of a complex craniofacial malformation condition, such as Freeman-Burian syndrome (FBS), formerly Freeman-Sheldon syndrome, is deeply distressing. There are few references in the literature addressing initial evaluation and operative counseling for FBS, and guidance is absent. Two major outcomes of FBS are explored, namely diagnostic accuracy and therapeutic result, to identify factors influencing optimal clinical care in (1) diagnosis, (2) evaluation, (3) general and craniofacial operative counseling, and (4) craniofacial management.PubMed searches have yielded 15 results describing craniofacial surgery in FBS and 29 manuscripts describing psychosocial aspects of surgery and patient and family counseling and education in other non-intellectually impairing craniofacial malformation conditions. Research in this area of scholarship is plagued by problems, especially considerable knowledge gaps and an absence of study data for operative outcomes. As a result, the literature remains unsettled, though our experience presents a much more clear picture of the clinical reality for this challenging patient population. While many challenges and limitations to treatment are present, much can be done to afford these patients a good and productive quality of life through operative intervention and longitudinal psychosocial support.

The Human Eye Proteome Project: Updates on an Emerging Proteome.

The human eye is a complex organ consisting of multiple compartments with unique and specialized properties that reflect their varied functions. Although there have been advancements in ocular imaging and therapeutics over the past decade, the pathogenesis of many common eye diseases remains poorly understood. Proteomics is an invaluable tool to gain insight into pathogenesis, diagnosis, and treatment of eye diseases. By 2013, when the Human Eye Proteome Project (also known as the EyeOme) was founded, there were 4842 nonredundant proteins identified in the human eye. Twenty-three recent papers on the human eye proteome were identified in PubMed searches. These papers were used to compile an updated resource of 9782 nonredundant proteins in the human eye. This updated catalogue sheds light on the molecular makeup of previously undescribed proteomes within the human eye, including optic nerve, sclera, iris, and ciliary body, while adding additional proteins to previously characterized proteomes such as aqueous humor, lens, vitreous, retina, and retinal pigment epithelium/choroid. Although considerable advances have been made to characterize the complete proteome of the human eye, additional high-quality data are needed to confirm and quantify previously discovered eye proteins in both health and disease.

Revisiting the Many Names of Freeman-Sheldon Syndrome.

While officially designated as distal arthrogryposis type 2A, the condition commonly referred to as Freeman-Sheldon syndrome (FSS) also historically has been termed craniocarpotarsal dystrophy, whistling face syndrome, and craniocarpotarsal dysplasia and classified at different times as a skeletal dysplasia, nonprogressive myopathy, craniofacial syndrome, and distal arthrogryposis. Having previously provided evidence for FSS being a complex myopathic craniofacial syndrome with extra-craniofacial features in most patients, the rationale for revising the FSS eponym and supplanting the current official designation with a new one was based on considerations for educational usefulness, historical accuracy, communication fluency, and nosologic clarity underpinned by genetic, pathologic, and operative experience and outcomes.

Head First, Not Feet First: Freeman-Sheldon Syndrome as Primarily a Craniofacial Condition.

The historical and clinical basis for classification of Freeman-Sheldon syndrome as a craniofacial syndrome and explanation of the rationale underlying this decision is provided. Correctly classifying the condition will avoid confusion and may help to clarify the vernacular employed and eventually aid in improving diagnosis.

A proteomic approach to understanding the pathogenesis of idiopathic macular hole formation.

Idiopathic macular holes (IMH) are full-thickness defects of retinal tissue that cause severe vision loss due to disruption of the anatomic fovea. Abnormal vitreous traction is involved in the formation of macular holes. Both glial cells and hyalocytes contribute to epiretinal membrane formation in IMH. In order to gain further insight into the pathophysiology of IMH, we conducted a discovery phase investigation of the vitreous proteome in four patients with macular holes and six controls using one-dimensional gel fractionation and liquid chromatography-tandem mass spectrometry analyses on an Orbitrap Elite mass spectrometer. Of a total of 5912 vitreous proteins, 32 proteins had increased and 39 proteins had decreased expression in IMH compared with controls, using a false discovery rate approach with p value < 0.001 and q value < 0.05. IMH was associated with increased expression of proteins in the complement pathway, α-2-macroglobulin, a major inducer of Müller glial cell migration, fibrinogen, and extracellular matrix proteins, and decreased expression of proteins involved in protein folding and actin filament binding. A proteomic approach revealed proteins and biological pathways that may be involved in the pathogenesis of IMH and could be targeted for future studies.

Anatomical differences of the protein profile in the rabbit sclera during growth.

We aimed to investigate the proteome changes in anatomical regions of sclera during growth and development of the rabbit. Sclera from New Zealand white rabbits of three ages (1 month, 2 months and 6 months) was dissected into three segments - anterior, equatorial, and posterior. A total of 36 samples were divided into groups by age and anatomical region. Tryptic digests of total proteins were analyzed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Label-free quantification based on spectral counts was used to determine the relative protein abundance and identify proteins with statistically significant differences between age groups or anatomical regions of the sclera. Western blotting was performed to validate some of the differentially expressed proteins. A total of 840 non-redundant proteins was identified in the sclera at different ages and regions with protein and peptide false discovery rate (FDR) at ≤1.0% and ≤0.1%, respectively. Differentially expressed proteins were identified by comparing age or anatomical region. Among these, periostin showed decreasing abundance with age, while myocilin, latent-transforming growth factor beta-binding protein 2, hyaluronan, proteoglycan link protein 1 and selenbp1 showed increasing abundance with age. In mature rabbits, alcohol dehydrogenase showed region-related differences in the sclera. Periostin showed an age-related decrease while selenbp1 showed an age-related increase in abundance in the anterior region. Vitronectin and extracellular superoxide dismutase had greater expression with age in the equatorial and posterior regions, respectively. The age related differential expression of periostin and selenbp1 was confirmed by western blotting. In conclusion, the protein profile of sclera showed age- and region-related differences. The differential protein profiles provide a baseline for understanding changes in the protein expression in the young and mature rabbit that appears to show regional changes. The changes observed in the present study add to the existing knowledge about regional alterations in biomechanical properties of sclera during growth.

The proteome of normal human retrobulbar optic nerve and sclera.

The optic nerve is a white matter tract that conveys visual information to the brain. The sclera comprises the white, protective outer layer of the eye. A characterization of the proteome of normal human retrobulbar optic nerve and sclera may facilitate studies of the eye. We conducted a proteomic analysis of optic nerve and sclera from five adults. Proteins were fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. We identified 2711 non-redundant proteins in retrobulbar optic nerve and 1945 non-redundant proteins in sclera. Optic nerve proteins included proteins expressed by oligodendrocytes (laminin, proteolipid protein, fibronectin), myelin proteins (myelin basic protein, myelin-associated glycoprotein), and paranodal structural proteins (ankyrin ß, spectrin). Sclera included 18 collagen protein chains, small leucine-rich proteoglycans (decorin, biglycan, lumican, keratocan, prolargin, fibromodulin, mimecan), non-collagenous glycoproteins (fibronectin, vitronectin, laminin), extracellular matrix proteins (thrombospondins 1-4, dystroglycan, transgelins 1-3), and integrins alpha-V, alpha-1 and 2, beta-1, -2, and -5. Twenty-one unambiguous alternative splicing protein isoforms were identified in optic nerve and ten unambiguous alternative splicing protein isoforms were identified in sclera. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001581.

Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid.

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false-positive rates of <0.1% and <1%, respectively. Forty-three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four "missing proteins" were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.