Large-scale genetic perturbations reveal regulatory networks and an abundance of gene-specific repressors.

To understand regulatory systems, it would be useful to uniformly determine how different components contribute to the expression of all other genes. We therefore monitored mRNA expression genome-wide, for individual deletions of one-quarter of yeast genes, focusing on (putative) regulators. The resulting genetic perturbation signatures reflect many different properties. These include the architecture of protein complexes and pathways, identification of expression changes compatible with viability, and the varying responsiveness to genetic perturbation. The data are assembled into a genetic perturbation network that shows different connectivities for different classes of regulators. Four feed-forward loop (FFL) types are overrepresented, including incoherent type 2 FFLs that likely represent feedback. Systematic transcription factor classification shows a surprisingly high abundance of gene-specific repressors, suggesting that yeast chromatin is not as generally restrictive to transcription as is often assumed. The data set is useful for studying individual genes and for discovering properties of an entire regulatory system.

Functional overlap and regulatory links shape genetic interactions between signaling pathways.

To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy, and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multiprocess control.

Improving the Accuracy, Robustness, and Dynamic Range of Digital Bead Assays.

We report methods that improve the quantification of digital bead assays (DBA)─such as the digital enzyme-linked immunosorbent assay (ELISA)─that have found widespread use for high sensitivity measurement of proteins in clinical research and diagnostics. In digital ELISA, proteins are captured on beads, labeled with enzymes, individual beads are interrogated for activity from one or more enzymes, and the average number of enzymes per bead (AEB) is determined based on Poisson statistics. The widespread use of digital ELISA has revealed limitations to the original approaches to quantification that can lead to inaccurate AEB. Here, we have addressed the inaccuracy in AEB due to deviations from Poisson distribution in a digital ELISA for Aß-40 by changing the AEB calculation from a fixed threshold between digital counting and average normalized intensity to a smooth, continuous combination of digital counting and intensity. We addressed issues with determining the average product fluorescence intensity from single enzymes on beads by allowing outlier, high intensity arrays to be removed from average intensities, and by permitting the use of a wider range of arrays. These approaches improved the accuracy of a digital ELISA for tau protein that was affected by aggregated detection antibodies. We increased the dynamic range of a digital ELISA for IL-17A from AEB ∼25 to ∼130 by combining long and short exposure images at the product emission wavelength to create virtual images. The methods reported will significantly improve the accuracy and robustness of DBA based on imaging─such as single molecule arrays (Simoa)─and flow detection.

The specificity and topology of chromatin interaction pathways in yeast.

Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. The data are assembled into a network of chromatin interaction pathways. The network is function based, has a branched, interconnected topology, and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are important for which genes and predicts additional interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin.

Well-defined palladium nanoparticles supported on siliceous mesocellular foam as heterogeneous catalysts for the oxidation of water.

Herein, we describe the use of Pd nanoparticles immobilized on an amino-functionalized siliceous mesocellular foam for the catalytic oxidation of H2O. The Pd nanocatalyst proved to be capable of mediating the four-electron oxidation of H2O to O2, both chemically and photochemically. The Pd nanocatalyst is easy to prepare and shows high chemical stability, low leaching, and recyclability. Together with its promising catalytic activity, these features make the Pd nanocatalyst of potential interest for future sustainable solar-fuel production.

Tight cooperation between Mot1p and NC2ß in regulating genome-wide transcription, repression of transcription following heat shock induction and genetic interaction with SAGA.

TATA-binding protein (TBP) is central to the regulation of eukaryotic transcription initiation. Recruitment of TBP to target genes can be positively regulated by one of two basal transcription factor complexes: SAGA or TFIID. Negative regulation of TBP promoter association can be performed by Mot1p or the NC2 complex. Recent evidence suggests that Mot1p, NC2 and TBP form a DNA-dependent protein complex. Here, we compare the functions of Mot1p and NC2ßduring basal and activated transcription using the anchor-away technique for conditional nuclear depletion. Genome-wide expression analysis indicates that both proteins regulate a highly similar set of genes. Upregulated genes were enriched for SAGA occupancy, while downregulated genes preferred TFIID binding. Mot1p and NC2ß depletion during heat shock resulted in failure to downregulate gene expression after initial activation, which was accompanied by increased TBP and RNA pol II promoter occupancies. Depletion of Mot1p or NC2ß displayed preferential synthetic lethality with the TBP-interaction module of SAGA. Our results support the model that Mot1p and NC2ß directly cooperate in vivo to regulate TBP function, and that they are involved in maintaining basal expression levels as well as in resetting gene expression after induction by stress.

The Mutagenic Plasticity of the Cholera Toxin B-Subunit Surface Residues: Stability and Affinity.

Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools.

Malignancies in Immunoglobulin G4-related ophthalmic disease.

PURPOSE: Clinical phenotypes in Immunoglobulin G4-related disease (IgG4-RD) according to the patterns of affecting organs have different risks of malignancies. We attempt to determine the association of malignancies with IgG4-related ophthalmic disease (IgG4-ROD). DESIGN: Retrospective cohort study. METHODS: Review of medical records, orbital images and histopathology reports in a territory-wide cohort of biopsy proven IgG4-ROD patients from 2005-2019. FINDINGS: Among 122 patients who had biopsies taken from adnexal lesions including lacrimal glands (n = 108), orbital mass (n = 30), infiltrated orbital fat (n = 10), conjunctiva (n = 2) or extraocular muscles (n = 3), 13% (16/122) developed malignancies over 73 ± 48months' follow-up. There were 9 cases of ocular adnexal lymphoma (OAL) and 7 extra-orbital malignancies. Compared with the general population, the incidence of OAL was significantly higher (standardized incidence ratios, SIRs = 10.0, 95%CI = 4.5-17.6) while that of extra-orbital malignancies was similar. The SIRs was highest within the first year (SIR = 46.7, 95%CI = 18.5-87.6) when 7 OAL were concomitantly diagnosed. Patients who developed OAL or extra-orbital malignancies were older than other patients at IgG4-ROD diagnosis (64.9 ± 7.1, 68.3 ± 8.5 versus 55.2 ± 15.0 years, P < 0.05). Asymmetric lacrimal gland enlargement (78% versus 13%), lack of frontal (0% versus 12%) or infraorbital nerve enlargement (0% versus 36%) were associated with OAL (all P < 0.05). Pre-treatment serum IgG4 level or extra-orbital IgG4-RD involvement was similar among patients with or without malignancies. CONCLUSION: In this biopsy-proven IgG4-ROD cohort, 7% developed OAL which was 10 times higher than the general population. Patients with asymmetric lacrimal gland enlargement or without trigeminal nerves involvement radiologically were associated with OAL.

Oxidative addition or Werner coordination complex? Reactivity of ß-diketiminate supported main group and first-row transition metal complexes towards ammonia.

A series of neutral LM (L = [HC{(H3C)C(Dipp)N}2], Dipp = 2,6-iPr2C6H3, M = group 13: B-In, TM: Fe, Co, Ni, Cu) and L'M (L' = [HC{(CCH2)(CCH3)(DippN)2}], M = group 14: C-Pb) compounds including a main group 13/14 and first-row transition metal complexes were studied computationally by density functional theory (DFT). The optimised complexes were assessed in terms of structural parameters and electronic structures to find trends and characteristics that could be used to predict their reactivity towards ammonia. In addition, the differences in oxidative addition and Werner coordination complex formation depending on the identity of the central element were investigated and the Werner complexes were evaluated by QTAIM and EDA-NOCV approaches. The computational results complement the earlier experimental studies and shed light on the feasibility of isolating novel main group Werner complexes or transition metal oxidative addition products.

Ultrasensitive multiplexed chemiluminescent enzyme-linked immunosorbent assays in 384-well plates.

We have developed an ultrasensitive multiplexed immunoassay using 384-well microtiter plates capable of detecting proteins at subfemtomolar concentrations that requires as little as 2.5 µL of sample. Arrays of up to 4 capture antibodies were patterned on the bottom of the wells of a 384-well plate either by directly printing the capture antibodies or by printing anti-peptide tag anchor antibodies and incubating these arrays with capture antibodies conjugated to the corresponding peptide tags ("customized" assays). Samples were incubated with the antibody arrays and shaken orbitally at 2000 rpm to achieve the greatest sensitivity. Chemiluminescence (CL) from immunocomplexes labeled with horseradish peroxidase was imaged across the entire plate to quantify the amount of protein bound to each antibody spot of the arrays. The 384-well assay had a throughput 5-fold greater than 96-well plates that was achieved from simultaneous imaging of CL in all 384-wells and the use of automated pipettors to allow parallel processing of 384 assays. We developed 4 assays based on the 384-well CL ELISA: a direct print assay for IL-10 (limit of detection (LOD) = 0.075 fM); a customized assay for IL-6 (0.22 fM); a customized pharmacokinetic (PK) assay for measuring adalimumab (7.3 pg/mL); and a customized 4-plex assay for IL-5 (0.1 fM), IL-6 (0.52 fM), IL-10 (0.2 fM), and TNF-α (3.2 fM). The sensitivity and precision of the cytokine assays were comparable to current ultrasensitive protein detection methods in 96-well formats. The PK assay for adalimumab was 650 times more sensitive than a commercially available 96-well plate ELISA. We used the 384-well CL ELISAs to measure endogenous levels of the cytokines in the serum and plasma of healthy humans: the mean concentrations and precision were comparable to those from 96-well immunoassays. This 384-well format with subfemtomolar sensitivity will enable ultrasensitive multiplexed immunoassays to be performed with higher throughput and lower sample volumes than currently possible, a particularly important capability for clinical studies in drug development.

Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range.

We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ∼1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.

Thyroid Adenoma of Probable Ultimobranchial Body Origin: A Case Report.

Solid cell nests are generally believed to represent remnants of the ultimobranchial body, which can be found in the normal thyroid gland, occasionally associated with other branchial pouch remnants such as salivary gland, cartilage, and adipose tissue. We describe the case of a 44-year-old man incidentally found to have a large tumor in the left lobe of the thyroid. The tumor was a circumscribed growth consisting of distinctly lobulated proliferation of solid to cystic epidermoid cell nests and thyroid follicles in a fibromatous stroma, which merged into abundant adipose tissue and focally myxoid matrix. The solid epidermoid cell nests resembled solid cell nests and exhibited a p63+, GATA3+, galectin-3+, TTF1-, PAX8-, thyroglobulin- phenotypes, while the follicles were p63-, GATA3-, galectin-3-, TTF1+, PAX8+, and thyroglobulin+. RAS mutations were not found. This thyroid tumor may represent a hitherto undescribed "ultimobranchial body adenoma" in human.

Triterpenoidal alkaloids from Buxus natalensis and their acetylcholinesterase inhibitory activity.

Acetylcholinesterase (AChE) inhibition-directed phytochemical studies on the methanolic extract of Buxus natalensis, collected in South Africa, resulted in the isolation of 12 compounds: O(2)-natafuranamine (1), O(10)-natafuranamine (2), cyclonataminol (3), 31-demethylbuxaminol A (4), buxaminol A (5), buxafuranamide (6), buxalongifolamidine (7), buxamine A (8), cyclobuxophylline K (9), buxaminol C (10), methyl syringate (11), and p-coumaroylputrescine (12). Compounds 1-4 were new alkaloids, and compound 5 was isolated for the first time as a natural product. Their structures were elucidated with the aid of extensive NMR and mass spectroscopic studies. Compounds 1 and 2 are members of a rarely occurring class of Buxus alkaloids, having a tetrahydrofuran ring incorporated in their structures. Compounds 1-12 exhibited strong to moderate AChE inhibitory activity.

Composite Hemangioendothelioma With Neuroendocrine Marker Expression: Report of a "Paraganglioma-Like" Paravertebral Case.

Composite hemangioendothelioma is a rare vascular tumor morphologically comprising several distinct vascular components and exhibits a borderline malignant potential. We described the case of a 53-year-old female who presented with an infiltrative mass in the paravertebral soft tissue. The tumor showed discrete nests of synaptophysin-expressing epithelioid cells accompanied by rich vasculature, features highly reminiscent of sympathetic paraganglioma. Further analysis revealed areas resembling spindle cell hemangioma, retiform hemangioendothelioma, cavernous hemangioma/lymphangioma, and epithelioid hemangioendothelioma without the myxohyaline matrix in the tumor, and a final diagnosis of composite hemangioendothelioma with synaptophysin expression was made. Critical appraisal of this recently described entity and its possible pathogenic relationship with retiform hemangioendothelioma were discussed.

Myxoid Spindle Cell Sarcoma With LMNA-NTRK Fusion: Expanding the Morphologic Spectrum of NTRK-Rearranged Tumors.

Neurotrophic tyrosine receptor kinase (NTRK)-rearranged spindle cell neoplasm is a recently described soft tissue tumor entity that occurs predominantly in children and young adults. The diagnosis of this tumor is difficult due to the nonspecific and highly variable morphology, yet it is of clinical relevance because of the availability of highly effective TRK inhibitors. In this article, we report the case of a 40-year-old female who presented with a mass in the left calf. Histologic examination revealed a low-grade sarcoma consisting of monomorphic spindle cells accompanied by abundant myxoid stroma, a feature that had not been emphasized in the reported cases of NTRK-rearranged tumors. The tumor cells expressed CD34 and S100 but not SOX10, and they showed positive staining for pan-TRK. Next-generation sequencing showed the presence of LMNA-NTRK1 fusion. The patient developed several episodes of lung metastases that eventually became unresectable. TRK inhibitor was given that led to near-complete resolution of the tumors.

A Novel Mouse Model of Autoimmune Thyroiditis Induced by Immunization with Adenovirus Containing Full-Length Thyroglobulin cDNA: Implications to Genetic Studies of Thyroid Autoimmunity.

Background: Thyroglobulin (TG) is a key autoantigen in autoimmune thyroid diseases (AITD). Several single nucleotide polymorphisms (SNPs) in the TG locus were shown to be strongly associated with disease susceptibility in both humans and mice, and autoimmune response to TG is the earliest event in the development of thyroid autoimmunity in mice. The classical model of experimental autoimmune thyroiditis (EAT) is induced by immunizing mice with TG protein together with an adjuvant to break down immune tolerance. The classical EAT model has limited utility in genetic studies of TG since it does not allow testing the effects of TG sequence variants on the development of autoimmune thyroiditis. In this study, we have immunized CBA-J mice, an EAT-susceptible strain, with an adenovirus vector encoding the full-length human TG (hTG) to generate a model of EAT in which the TG sequence can be manipulated to test AITD-associated TG SNPs. Methods: We immunized CBA-J mice with hTG-expressing adenovirus following the well-recognized experimental autoimmune Graves' disease protocol that also uses an adenovirus vector to deliver the immunogen. Results: After hTG adenovirus immunizations, mice developed higher T cell proliferative and cytokine responses to hTG and TG2098 (a major T cell epitope in AITD) and higher titers of TG and thyroperoxidase autoantibodies compared with mice immunized with control LacZ-expressing adenovirus. The mice, however, did not develop thyroidal lymphocytic infiltration and hypothyroidism. Conclusions: Our data describe a novel murine model of autoimmune thyroiditis that does not require the use of adjuvants to break down tolerance and that will allow investigators to test the effects of hTG variants in the pathoetiology of Hashimoto's thyroiditis.

Digital enzyme-linked immunosorbent assays with sub-attomolar detection limits based on low numbers of capture beads combined with high efficiency bead analysis.

We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule arrays (Simoa) with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (≤5000 vs.∼500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency) from ∼5% to ∼50%. Bead read efficiency was increased by: a) improving the loading of beads into arrays of microwells by combining capillary and magnetic forces in a method called magnetic-meniscus sweeping (MMS); b) using a centrifugal washer to minimize bead loss during the assay; and, c) improved optics and image analysis to enable the analysis of more microwells. Using this approach, we developed an assay for IL-17A with a limit of detection (LOD) of 0.7 aM, 437-fold more sensitive than standard digital ELISA. A digital ELISA with improved sensitivity was used to measure IL-17A in 100 serum and plasma samples with 100% detectability, compared to 51% for standard digital ELISA. Low numbers of capture beads yielded improved LODs for IL-12p70 (0.092 aM), p24 (9.1 aM), and interferon alpha (45.9 aM). IL-4 and PSA showed no improvements in sensitivity using fewer beads, primarily due to low antibody loading on beads and increased non-specific binding, respectively. The results were consistent with a kinetic model of binding that showed that combining capture antibodies with high on-rates with high antibodies per bead yields the greatest improvement in sensitivity.