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Background: Systemic abnormalities in the immune system may contribute to rosacea pathogenesis. Several studies have found a higher prevalence of abnormal bacterial growth, such as Helicobacter pylori ( H. pylori ) and small intestinal bacterial overgrowth (SIBO) in rosacea subjects. However, discrepancies remain in the literature, likely perpetuated by inconsistent testing methods and incomplete controlling for potential confounders., Objective: We aimed to evaluate the prevalence of H. pylori and SIBO in rosacea subjects after controlling for several potential confounders., Methods: This cross-sectional study evaluated subjects with papulopustular or erythematotelangiectatic rosacea. Subjects with previous or existing gastrointestinal (GI) disease, GI surgery, autoimmune disorders, immunosuppression, or significant comorbidities were excluded. Certain medication use (antibiotics, steroids, GI-modulating medications, anti-inflammatories) required an appropriate washout period. Rosacea history and severity were assessed. Subjects answered questions regarding their rosacea and GI health. H. pylori andSIBO were evaluated by 13 C-urea breath test and glucose-breath test methods, respectively., Results: Of 27 subjects, 14.8% (N=4) tested positive for H. pylori and 33.3% (N=9) tested positive for SIBO. Compared to the general population prevalence, the proportion of H. pylori in the rosacea cohort was significantly less (p=0.02). Though the estimated population prevalence of SIBO had a wider range, compared to midrange, the prevalence of SIBO in the rosacea cohort was greater (p<0.001). There were no significant associations between demographics, rosacea characteristics, or GI symptoms and H. pylori or SIBO positivity.  Conclusion: When eliminating several potential confounders, SIBO is more prevalent in subjects with rosacea compared to the general population. Thus, SIBO may be associated with rosacea, though it remains incompletely understood whether SIBO itself contributes to rosacea pathophysiology or rather SIBO prevalence and rosacea are both downstream effects of abnormalities in systemic immunity. Future studies are warranted to elucidate this relationship further, though this observed association may be promising for novel therapeutic targets in rosacea treatment., Competing Interests: Human subjects: Consent for treatment and open access publication was obtained or waived by all participants in this study. University of Michigan Medical School Institutional Review Board (IRBMED) issued approval HUM00113147. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: This work was supported by a clinical research grant to JMR (American Acne and Rosacea Society) and an NCATS grant to JPT (UM1TR004404). The financial sponsors had no involvement in the study. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Nelson et al.)

We report three new cases of a germline heterozygous gain-of-function missense (p.(Met1141Lys)) mutation in the C2 domain of phospholipase C gamma 2 (PLCG2) associated with symptoms consistent with previously described auto-inflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) syndrome and pediatric common variable immunodeficiency (CVID). Functional evaluation showed platelet hyper-reactivity, increased B cell receptor-triggered calcium influx and ERK phosphorylation. Expression of the altered p.(Met1141Lys) variant in a PLCγ2-knockout DT40 cell line showed clearly enhanced BCR-triggered influx of external calcium when compared to control-transfected cells. Our results further expand the molecular basis of pediatric CVID and phenotypic spectrum of PLCγ2-related defects.

During the development of a therapeutic monoclonal antibody (mAb-1), the charge variant profile obtained by pH-gradient cation exchange chromatography (CEX) contained two main peaks, each of which exhibited a unique intrinsic fluorescence profile and demonstrated inter-convertibility upon reinjection of isolated peak fractions. Domain analysis of mAb-1 by CEX and liquid chromatography-mass spectrometry indicated that the antigen-binding fragment chromatographed as two separate peaks that had identical mass. Surface plasmon resonance binding analysis to antigen demonstrated comparable kinetics/affinity between these fractionated peaks and unfractionated starting material. Subsequent molecular modeling studies revealed that the relatively long and flexible complementarity-determining region 3 (CDR3) loop on the heavy chain could adopt two discrete pH-dependent conformations: an "open" conformation at neutral pH where the HC-CDR3 is largely solvent exposed, and a "closed" conformation at lower pH where the solvent exposure of a neighboring tryptophan in the light chain is reduced and two aspartic acid residues near the ends of the HC-CDR3 loop have atypical pKa values. The pH-dependent equilibrium between "open" and "closed" conformations of the HC-CDR3, and its proposed role in the anomalous charge variant profile of mAb-1, were supported by further CEX and hydrophobic interaction chromatography studies. This work is an example of how pH-dependent conformational changes and conformation-dependent changes to net charge can unexpectedly contribute to perceived instability and require thorough analytical, biophysical, and functional characterization during biopharmaceutical drug product development.

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer 1,2 . Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies 3 . However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.

Background: Oral vismodegib therapy and photodynamic therapy (PDT) are non-invasive treatments for basal cell carcinoma (BCC) with overlapping utility in widespread BCCs and patients who are poor surgical candidates. There is no published study to date investigating the combination use of PDT with vismodegib to optimize individual response rates., Objective: To evaluate the combination of red light PDT and vismodegib therapy in patients with multiple nodular BCCs. The primary objective was to determine the safety of this combination therapy. Secondary outcomes included evaluation of the overall response rate, treatment-related pain, and cosmesis., Methods: An open label pilot study of immunocompetent patients with multiple BCCs treated with 3 months of continuous vismodegib therapy (150 mg daily) and 3 consecutive ALA PDT sessions. Outcomes were assessed following each PDT session and 30 days post-treatment., Results: Four patients with multiple nodular BCC (median=5) were enrolled in the trial between January and August of 2016. Three patients completed the full intervention phase trial and a total of 19 lesions were treated. One patient completed 2 months of vismodegib and 2 PDT sessions. One PDT session was sufficient for small lesions, whereas larger lesions required all 3 sessions. The fifteen evaluable lesions at the end of the 3 PDT sessions showed complete responses. At 30-day follow-up, one of the treated lesions was noted to have clinical evidence of disease. Overall response rate showed 90% complete response and 10% partial response for the study. Combination therapy was well tolerated and yielded a similar or superior side effect profile to that of individual therapies with excellent cosmesis., Conclusion: Combination PDT-vismodegib is a potential safe & effective therapy for the treatment of multiple BCCs that may enhance efficacy of individual therapies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Atopic dermatitis (AD) is the most common inflammatory skin disease with no well-delineated cause or effective cure. Here we show that the p53 family member p63, specifically the ΔNp63, isoform has a key role in driving keratinocyte activation in AD. We find that overexpression of ΔNp63 in transgenic mouse epidermis results in a severe skin phenotype that shares many of the key clinical, histological and molecular features associated with human AD. This includes pruritus, epidermal hyperplasia, aberrant keratinocyte differentiation, enhanced expression of selected cytokines and chemokines and the infiltration of large numbers of inflammatory cells including type 2  T-helper cells - features that are highly representative of AD dermatopathology. We further demonstrate several of these mediators to be direct transcriptional targets of ΔNp63 in keratinocytes. Of particular significance are two p63 target genes, IL-31 and IL-33, both of which are key players in the signaling pathways implicated in AD. Importantly, we find these observations to be in good agreement with elevated levels of ΔNp63 in skin lesions of human patients with AD. Our studies reveal an important role for ΔNp63 in the pathogenesis of AD and offer new insights into its etiology and possible therapeutic targets.

In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

As a label-free technology, mass spectrometry (MS) enables assays to be generated that monitor the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods. Although traditional liquid chromatography (LC)-MS methods are relatively slow for a high-throughput screening (HTS) paradigm, with cycle times typically ≥ 60 s per sample, the Agilent RapidFire High-Throughput Mass Spectrometry (HTMS) System, with a cycle time of 5-7 s per sample, enables rapid analysis of compound numbers compatible with HTS. By monitoring changes in mass directly, HTMS assays can be used as a triaging tool by eliminating large numbers of false positives resulting from fluorescent compound interference or from compounds interacting with hydrophobic fluorescent dyes appended to substrates. Herein, HTMS assays were developed for multiple protease programs, including cysteine, serine, and aspartyl proteases, and applied as a confirmatory assay. The confirmation rate for each protease assay averaged <30%, independent of the primary assay technology used (i.e., luminescent, fluorescent, and time-resolved fluorescent technologies). Importantly, >99% of compounds designed to inhibit the enzymes were confirmed by the corresponding HTMS assay. Hence, HTMS is an effective tool for removing detection-based false positives from ultrahigh-throughput screening, resulting in hit lists enriched in true actives for downstream dose response titrations and hit-to-lead efforts., (© 2014 Society for Laboratory Automation and Screening.)

Recent cumulative data show that various transcription factors are recruited to the chromatin in an iron-responsive manner to affect diverse cellular functions in the pathogenic fungus Candida albicans. Here we identified groups of iron-responsive genes in C. albicans by chromatin remodelling analysis at gene promoters, using micrococcal nuclease (MNase) digestion followed by deep sequencing. Chromatin in the promoter regions of iron uptake and utilization genes showed repressed and active configuration, respectively, under iron-replete conditions. GO Term enrichment analysis of genes with differentially remodelled chromatin, in respective promoter locales, suggested that many genes involved in adhesion are also iron-responsive. C. albicans was observed to be more self-adherent (twofold increase) and formed higher biofilm mass (77% increase) in the presence of iron. Furthermore, we identified various known and novel adhesion-related genes with iron-dependent active chromatin profiles that are indicative of potential upregulation under iron-replete conditions. Transcription factor Cph1 that is activated upon Cek1 phosphorylation also showed an active chromatin profile under iron-replete conditions and cells showed iron-responsive Cek1 MAPK phosphorylation in the presence of iron. Thus, iron affects diverse biological functions by modulating chromatin profiles of large gene sets and by signalling through Cek1 MAPK in C. albicans., (© 2014 John Wiley & Sons Ltd.)

Eukaryotic DNA is wrapped around histone octamers, known as nucleosomes, in an orderly fashion that provides the primary structure of chromatin organization. The compaction of DNA into nucleosomal repeats not only allows the tight packaging of the large eukaryotic genomes into the nucleus, it also dictates the accessibility of genetic information. Thus, in order to understand how nucleosomes can affect the dynamics of DNA-protein interactions, such as those associated with transcriptional regulatory mechanisms, it is important to define nucleosomal positioning and occupancy along genomic DNA. Here we describe a method that relies on the enzymatic activity of micrococcal nuclease (MNase) to determine nucleosomal footprints and boundaries. By pairing this technique with next generation sequencing techniques (i.e., MNase-seq), it is possible to generate a genome-wide detailed map of chromatin architecture.

We have used micrococcal nuclease (MNase) digestion followed by deep sequencing in order to obtain a higher resolution map than previously available of nucleosome positions in the fission yeast, Schizosaccharomyces pombe. Our data confirm an unusually short average nucleosome repeat length, ∼152 bp, in fission yeast and that transcriptional start sites (TSSs) are associated with nucleosome-depleted regions (NDRs), ordered nucleosome arrays downstream and less regularly spaced upstream nucleosomes. In addition, we found enrichments for associated function in four of eight groups of genes clustered according to chromatin configurations near TSSs. At replication origins, our data revealed asymmetric localization of pre-replication complex (pre-RC) proteins within large NDRs-a feature that is conserved in fission and budding yeast and is therefore likely to be conserved in other eukaryotic organisms.

Demand for fast, inexpensive, and accurate DNA sequencing data has led to the birth and dominance of a new generation of sequencing technologies. So-called "next-generation" sequencing technologies enable rapid generation of data by sequencing massive amounts of DNA in parallel using diverse methodologies which overcome the limitations of Sanger sequencing methods used to sequence the first human genome. Despite opening new frontiers of genomics research, the fundamental shift away from the Sanger sequencing that next-generation technologies has created has also left many unaware of the capabilities and applications of these new technologies, especially those in the clinical realm. Moreover, the brisk evolution of sequencing technologies has flooded the market with commercially available sequencing platforms, whose unique chemistries and diverse applications stand as another obstacle restricting the potential of next-generation sequencing. This review serves to provide a primer on next-generation sequencing technologies for clinical researchers and physician scientists. We provide an overview of the capabilities and clinical applications of DNA sequencing technologies to raise awareness among researchers about the power of these novel genomic tools. In addition, we discuss that key sequencing principles provide a comparison between existing and near-term technologies and outline key advantages and disadvantages between different sequencing platforms to help researchers choose an appropriate platform for their research interests., (©2012 AACR.)

Background: The organization of eukaryotic DNA into chromatin has a strong influence on the accessibility and regulation of genetic information. The locations and occupancies of a principle component of chromatin, nucleosomes, are typically assayed through use of enzymatic digestion with micrococcal nuclease (MNase). MNase is an endo-exo nuclease that preferentially digests naked DNA and the DNA in linkers between nucleosomes, thus enriching for nucleosome-associated DNA. To determine nucleosome organization genome-wide, DNA remaining from MNase digestion is sequenced using high-throughput sequencing technologies (MNase-seq). Unfortunately, the results of MNase-seq can vary dramatically due to technical differences and this confounds comparisons between MNase-seq experiments, such as examining condition-dependent chromatin organizations., Results: In this study we use MNase digestion simulations to demonstrate how MNase-seq signals can vary for different nucleosome configuration when experiments are performed with different extents of MNase digestion. Signal variation in these simulations reveals an important DNA sampling bias that results from a neighborhood effect of MNase digestion techniques. The presence of this neighborhood effect ultimately confounds comparisons between different MNase-seq experiments. To address this issue we present a standardized chromatin preparation which controls for technical variance between MNase-based chromatin preparations and enables the collection of similarly sampled (matched) chromatin populations. Standardized preparation of chromatin includes a normalization step for DNA input into MNase digestions and close matching of the extent of digestion between each chromatin preparation using gel densitometry analysis. The protocol also includes directions for successful pairing with multiplex sequencing reactions., Conclusions: We validated our method by comparing the experiment-to-experiment variation between biological replicates of chromatin preparations from S. cerevisiae. Results from our matched preparation consistently produced MNase-seq datasets that were more closely correlated than other unstandardized approaches. Additionally, we validated the ability of our approach at enabling accurate downstream comparisons of chromatin structures, by comparing the specificity of detecting Tup1-dependent chromatin remodeling events in comparisons between matched and un-matched wild-type and tup1Δ MNase-seq datasets. Our matched MNase-seq datasets demonstrated a significant reduction in non-specific (technical) differences between experiments and were able to maximize the detection of biologically-relevant (Tup1-dependent) changes in chromatin structure.

Hyperosmotic stress activates an array of cellular detoxification mechanisms, including the high-osmolarity glycerol (HOG) pathway. We report here that vacuolar H(+)-ATPase (V-ATPase) activity helps provide osmotic tolerance in Saccharomyces cerevisiae. V-ATPase subunit genes exhibit complex haploinsufficiency interactions with HOG pathway components. vma mutants lacking V-ATPase function are sensitive to high concentrations of salt and exhibit Hog1p activation even at low salt concentrations, as demonstrated by phosphorylation of Hog1p, a shift in Hog1-green fluorescent protein localization, transcriptional activation of a subset of HOG pathway effectors, and transcriptional inhibition of parallel mitogen-activated protein kinase pathway targets. vma2Δ hog1Δ and vma3Δ pbs2Δ double mutants have a synthetic growth phenotype, poor salt tolerance, and an aberrant, hyper-elongated morphology on solid media, accompanied by activation of a filamentous response element-LacZ construct, indicating cross talk into the filamentous growth pathway. Vacuoles isolated from wild-type cells briefly exposed to salt show higher levels of V-ATPase activity, and Na(+)/H(+) exchange in isolated vacuolar vesicles suggests a biochemical basis for the genetic interactions observed. V-ATPase activity is upregulated during salt stress by increasing assembly of the catalytic V(1) sector with the membrane-bound V(o) sector. Together, these data suggest that the V-ATPase acts in parallel with the HOG pathway in order to mediate salt detoxification.

Despite technical advances, the future of chromatin mapping studies requires an ability to draw accurate comparisons between different chromatin states to enhance our understanding of genome biology. In this study, we used matched chromatin preparations to enable specific and accurate comparisons of Saccharomyces cerevisiae chromatin structures in the presence and absence of the co-repressor protein Tup1. Analysis of wild-type and tup1 Δ chromatin data sets revealed unique organizational themes relating to the function of Tup1. Regulatory regions bound by Tup1 assumed a distinct chromatin architecture composed of a wide nucleosome-depleted region, low occupancy/poorly positioned promoter nucleosomes, a larger number and wider distribution of transcription factor-binding sites and downstream genes with enhanced transcription plasticity. Regions of Tup1-dependent chromatin structure were defined for the first time across the entire yeast genome and are shown to strongly overlap with activity of the chromatin remodeler Isw2. Additionally, Tup1-dependent chromatin structures are shown to relate to distinct biological processes and transcriptional states of regulated genes, including Tup1 stabilization of Minus 1 and Minus 2 promoter nucleosomes at actively repressed genes. Together these results help to enhance our mechanistic understanding of Tup1 regulation of chromatin structure and gene expression.

Maintaining the proper expression of the transcriptome during development or in response to a changing environment requires a delicate balance between transcriptional regulators with activating and repressing functions. The budding yeast transcriptional co-repressor Tup1-Ssn6 is a model for studying similar repressor complexes in multicellular eukaryotes. Tup1-Ssn6 does not bind DNA directly, but is directed to individual promoters by one or more DNA-binding proteins, referred to as Tup1 recruiters. This functional architecture allows the Tup1-Ssn6 to modulate the expression of genes required for the response to a variety of cellular stresses. To understand the targeting or the Tup1-Ssn6 complex, we determined the genomic distribution of Tup1 and Ssn6 by ChIP-chip. We found that most loci bound by Tup1-Ssn6 could not be explained by co-occupancy with a known recruiting cofactor and that deletion of individual known Tup1 recruiters did not significantly alter the Tup1 binding profile. These observations suggest that new Tup1 recruiting proteins remain to be discovered and that Tup1 recruitment typically depends on multiple recruiting cofactors. To identify new recruiting proteins, we computationally screened for factors with binding patterns similar to the observed Tup1-Ssn6 genomic distribution. Four top candidates, Cin5, Skn7, Phd1, and Yap6, all known to be associated with stress response gene regulation, were experimentally confirmed to physically interact with Tup1 and/or Ssn6. Incorporating these new recruitment cofactors with previously characterized cofactors now explains the majority of Tup1 targeting across the genome, and expands our understanding of the mechanism by which Tup1-Ssn6 is directed to its targets.

The V-ATPase H subunit (encoded by the VMA13 gene) activates ATP-driven proton pumping in intact V-ATPase complexes and inhibits MgATPase activity in cytosolic V1 sectors (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767). Yeast diploids heterozygous for a vma13Delta mutation show the pH- and calcium-dependent conditional lethality characteristic of mutants lacking V-ATPase activity, although they still contain one wild-type copy of VMA13. Vacuolar vesicles from this strain have approximately 50% of the ATPase activity of those from a wild-type diploid but do not support formation of a proton gradient. Compound heterozygotes with a second heterozygous deletion in another V1 subunit gene exhibit improved growth, vacuolar acidification, and ATP-driven proton transport in vacuolar vesicles. In contrast, compound heterozygotes with a second deletion in a Vo subunit grow even more poorly than the vma13Delta heterozygote, have very little vacuolar acidification, and have very low levels of V-ATPase subunits in isolated vacuoles. In addition, cytosolic V1 sectors from this strain and from the strain containing only the heterozygous vma13Delta mutation have elevated MgATPase activity. The results suggest that balancing levels of subunit H with those of other V-ATPase subunits is critical, both for allowing organelle acidification and for preventing unproductive hydrolysis of cytosolic ATP.

The purpose of this study was to investigate the comprehension abilities of children with impaired expressive language and to obtain information on the interrelationships among tests of comprehension. Forty preschool children, 20 with normal language and 20 with impaired expressive language, were given a set of auditory comprehension tests. As a group, the language-impaired children demonstrated deficits in comprehension when compared to the normal-language children. However, both groups scored near the ceiling on several tests, and on most tests that did differentiate the two groups, the mean scores of both groups when applied to performances of middle-class white children. An analysis of responses to selected groupings of analogous items revealed that a preschool child's correct response to a linguistic stimulus in one instance provides no assurance that the child will respond similarly to the stimulus in another linguistic environment with different task demands and different foil alternatives. In addition, the large majority of correlations among the test were nonsignificant, indicating that it is not clinically appropriate to regard these measures of language comprehension as equivalent.