Lymphadenopathy and lymph node rejection following facial vascularized composite allotransplantation.

BACKGROUND: Apart from the skin, little is known about the immunological processes in deeper tissues, which are typically not accessible to biopsy and inspection, of vascularized composite allografts (VCAs). Face transplant patients develop prominent adenopathy shortly after transplantation that resolves over time. The mechanisms underlying this process are not understood. MATERIALS AND METHODS: A retrospective cohort study was conducted on 9 patients who underwent 10 facial VCAs at the Brigham and Women's Hospital, Boston, MA, between April 2009 and July 2019. Clinical, radiological, and histological data related to lymphadenopathy of the head and neck were reviewed. RESULTS: Patients who received donor-derived lymph nodes (LNs) developed bilateral lymphadenopathy of the submental or submandibular superficial LNs. Median time of presentation was POD18 (range POD6-POM3). Notably, bilateral adenopathy of the neck was not observed in later stages of follow-up (mean follow-up, 115 months). Histology of 3 LNs showed increased histiocytes and apoptosis, with the features reminiscent of necrotizing histiocytic lymphadenitis, and B and T lymphocytes (mostly CD8 + T) admixed with CD163 + histiocytes and dendritic cells. Molecular chimerism analysis in one case showed the coexistence of donor (81%) and recipient (19%) derived lymphocytes. Granzyme B (GZMB) expression confirmed the presence of increased cytotoxic T cells in this LN sample. CONCLUSION: Our data suggested the involvement of an immunological process within the donor-derived LNs after facial allotransplantation between the recipient and donor cells. GZMB expression suggested LN rejection that can occurred independently of skin rejection. This finding supports the need to better define the role of donor-derived immune cells in the context of allograft rejection.

Accelerated Aging and Microsatellite Instability in Recessive Dystrophic Epidermolysis Bullosa-Associated Cutaneous Squamous Cell Carcinoma.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severely debilitating disorder caused by mutations in COL7A1 and is characterized by extreme skin fragility, chronic inflammation and fibrosis. A majority of RDEB patients develop squamous cell carcinoma (SCC), a highly aggressive skin cancer with limited treatment options currently available. In this study, we utilized an approach leveraging WGS and RNA-seq across three different tissues in a single RDEB patient to gain insight into possible mechanisms of RDEB-associated SCC progression and to identify potential therapeutic options. As a result, we identified PLK-1 as a possible candidate for targeted therapy and discovered microsatellite instability and accelerated aging as factors potentially contributing to the aggressive nature and early onset of RDEB SCC. By integrating multi-tissue genomic and transcriptomic analyses in a single patient, we demonstrate the promise of bridging the gap between genomic research and clinical applications for developing tailored therapies for patients with rare genetic disorders such as RDEB.

Development of a Patient-Derived 3D Immuno-Oncology Platform to Potentiate Immunotherapy Responses in Ascites-Derived Circulating Tumor Cells.

High-grade serous ovarian cancer (HGSOC) is responsible for the majority of gynecology cancer-related deaths. Patients in remission often relapse with more aggressive forms of disease within 2 years post-treatment. Alternative immuno-oncology (IO) strategies, such as immune checkpoint blockade (ICB) targeting the PD-(L)1 signaling axis, have proven inefficient so far. Our aim is to utilize epigenetic modulators to maximize the benefit of personalized IO combinations in ex vivo 3D patient-derived platforms and in vivo syngeneic models. Using patient-derived tumor ascites, we optimized an ex vivo 3D screening platform (PDOTS), which employs autologous immune cells and circulating ascites-derived tumor cells, to rapidly test personalized IO combinations. Most importantly, patient responses to platinum chemotherapy and poly-ADP ribose polymerase inhibitors in 3D platforms recapitulate clinical responses. Furthermore, similar to clinical trial results, responses to ICB in PDOTS tend to be low and positively correlated with the frequency of CD3+ immune cells and EPCAM+/PD-L1+ tumor cells. Thus, the greatest response observed with anti-PD-1/anti-PD-L1 immunotherapy alone is seen in patient-derived HGSOC ascites, which present with high levels of systemic CD3+ and PD-L1+ expression in immune and tumor cells, respectively. In addition, priming with epigenetic adjuvants greatly potentiates ICB in ex vivo 3D testing platforms and in vivo tumor models. We further find that epigenetic priming induces increased tumor secretion of several key cytokines known to augment T and NK cell activation and cytotoxicity, including IL-6, IP-10 (CXCL10), KC (CXCL1), and RANTES (CCL5). Moreover, epigenetic priming alone and in combination with ICB immunotherapy in patient-derived PDOTS induces rapid upregulation of CD69, a reliable early activation of immune markers in both CD4+ and CD8+ T cells. Consequently, this functional precision medicine approach could rapidly identify personalized therapeutic combinations able to potentiate ICB, which is a great advantage, especially given the current clinical difficulty of testing a high number of potential combinations in patients.

Single-cell profiling reveals unique features of diabetogenic T cells in anti-PD-1-induced type 1 diabetes mice.

Immune-related adverse events (irAEs) are a notable complication of PD-1 cancer immunotherapy. A better understanding of how these iatrogenic diseases compare with naturally arising autoimmune diseases is needed for treatment and monitoring of irAEs. We identified differences in anti-PD-1-induced type 1 diabetes (T1D) and spontaneous T1D in non-obese diabetic (NOD) mice by performing single-cell RNA-seq and TCR-seq on T cells from the pancreas, pancreas-draining lymph node (pLN), and blood of mice with PD-1-induced T1D or spontaneous T1D. In the pancreas, anti-PD-1 resulted in expansion of terminally exhausted/effector-like CD8+ T cells, an increase in T-bethi CD4+FoxP3- T cells, and a decrease in memory CD4+FoxP3- and CD8+ T cells in contrast to spontaneous T1D. Notably, anti-PD-1 caused increased TCR sharing between the pancreas and the periphery. Moreover, T cells in the blood of anti-PD-1-treated mice expressed markers that differed from spontaneous T1D, suggesting that the blood may provide a window to monitor irAEs rather than relying exclusively on the autoimmune target organ.

Cellular activation pathways and interaction networks in vascularized composite allotransplantation.

Vascularized composite allotransplantation (VCA) is an evolving field of reconstructive surgery that has revolutionized the treatment of patients with devastating injuries, including those with limb losses or facial disfigurement. The transplanted units are typically comprised of different tissue types, including skin, mucosa, blood and lymphatic vasculature, muscle, and bone. It is widely accepted that the antigenicity of some VCA components, such as skin, is particularly potent in eliciting a strong recipient rejection response following transplantation. The fine line between tolerance and rejection of the graft is orchestrated by different cell types, including both donor and recipient-derived lymphocytes, macrophages, and other immune and donor-derived tissue cells (e.g., endothelium). Here, we delineate the role of different cell and tissue types during VCA rejection. Rejection of VCA grafts and the necessity of life-long multidrug immunosuppression remains one of the major challenges in this field. This review sheds light on recent developments in decoding the cellular signature of graft rejection in VCA and how these may, ultimately, influence the clinical management of VCA patients by way of novel therapies that target specific cellular processes.

Insights from immunoproteomic profiling of a rejected full-face transplant.

Vascularized composite allografts (VCAs) of faces and extremities are subject to chronic rejection that is incompletely understood. Here we report on immunoproteomic evaluation of a full facial VCA removed 88 months after transplantation due to chronic rejection. CD8-positive T cells of donor (graft) origin infiltrate deep intragraft arteries in apposition to degenerating endothelium of chimeric recipient origin in association with arteriosclerotic alterations. Digital spatial proteomic profiling highlighted proteins expressed by activated cytotoxic T cells and macrophages as well as pathway components involved in atherogenic responses, including Indoleamine 2,3-Dioxygenase 1 (IDO1) and Stimulator of Interferon Response CGAMP Interactor (STING). Chronic facial VCA rejection thus involves T cell/macrophage-mediated accelerated arteriosclerosis not normally represented in punch biopsies and potentially driven by persistent graft-resident effector T cells and recipient target endothelium that chimerically repopulates graft arteries.

Epigenetic Regulation of Corneal Epithelial Differentiation by TET2.

Epigenetic DNA modification by 5-hydroxymethylcytosine (5hmC), generated by the Ten-eleven translocation (TET) dioxygenases, regulates diverse biological functions in many organ tissues, including the mammalian eye. For example, 5hmC has been shown to be involved in epigenetic regulation of retinal gene expression. However, a functional role of 5hmC in corneal differentiation has not been investigated to date. Here, we examined 5hmC and TET function in the human cornea. We found 5hmC highly expressed in MUC16-positive terminally differentiated cells that also co-expressed the 5hmC-generating enzyme TET2. TET2 knockdown (KD) in cultured corneal epithelial cells led to significant reductions of 5hmC peak distributions and resulted in transcriptional repression of molecular pathways involved in corneal differentiation, as evidenced by downregulation of MUC4, MUC16, and Keratin 12. Additionally, integrated TET2 KD RNA-seq and genome-wide Reduced Representation Hydroxymethylation Profiling revealed novel epigenetically regulated genes expressed by terminally differentiated cells, including KRT78, MYEOV, and MAL. In aggregate, our findings reveal a novel function of TET2 in the epigenetic regulation of corneal epithelial gene expression and identify novel TET2-controlled genes expressed in differentiated corneal epithelial cells. These results point to potential roles for TET2 induction strategies to enhance treatment of corneal diseases associated with abnormal epithelial maturation.

Limbal BCAM expression identifies a proliferative progenitor population capable of holoclone formation and corneal differentiation.

The corneal epithelium is renowned for high regenerative potential, which is dependent on the coordinated function of its diverse progenitor subpopulations. However, the molecular pathways governing corneal epithelial progenitor differentiation are incompletely understood. Here, we identify a highly proliferative limbal epithelial progenitor subpopulation characterized by expression of basal cell adhesion molecule (BCAM) that is capable of holocone formation and corneal epithelial sheet generation. BCAM-positive cells can be found among ABCB5-positive limbal stem cells (LSCs) as well as among ABCB5-negative limbal epithelial cell populations. Mechanistically, we show that BCAM is functionally required for cellular migration and differentiation and that its expression is regulated by the transcription factor p63. In aggregate, our study identifies limbal BCAM expression as a marker of highly proliferative corneal epithelial progenitor cells and defines the role of BCAM as a critical molecular mediator of corneal epithelial differentiation.

High expression of SARS-CoV2 viral entry-related proteins in human limbal stem cells.

PURPOSE: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). While the ocular surface is considered one of the major SARS-CoV2 transmission routes, the specific cellular tropism of SARS-CoV2 is not fully understood. In the current study, we evaluated the expression and regulation of two SARS-CoV2 viral entry proteins, TMPRSS2 and ACE2, in human ocular epithelial cells and stem cells. METHODS: TMPRSS2 and ACE2 expression in ABCB5-positive limbal stem cells (LSCs) were assessed by RNAseq, flow cytometry and immunohistochemistry. PAX6, TMPRSS2, and ACE2 mRNA expression values were obtained from the GSE135455 and DRA002960 RNA-seq datasets. siRNA-mediated PAX6 knockdown (KD) was performed in limbal and conjunctival epithelial cells. TMPRSS2 and ACE2 expression in the PAX6 KD cells was analyzed by qRT-PCR and Western blot. RESULTS: We found that ABCB5-positive LSCs express high levels of TMPRSS2 and ACE2 compared to ABCB5-negative limbal epithelial cells. Mechanistically, gene knockout and overexpression models revealed that the eye transcription factor PAX6 negatively regulates TMPRSS2 expression. Therefore, low levels of PAX6 in ABCB5-positive LSCs promote TMPRSS2 expression, and high levels of TMPRSS2 and ACE2 expression by LSCs indicate enhanced susceptibility to SARS-CoV2 infection in this stem cell population. CONCLUSIONS: Our study points to a need for COVID-19 testing of LSCs derived from donor corneas before transplantation to patients with limbal stem cell deficiency. Furthermore, our findings suggest that expandable human ABCB5+ LSC cultures might represent a relevant novel model system for studying cellular SARS-CoV2 viral entry mechanisms and evaluating related targeting strategies.

At home and online during the early months of the COVID-19 pandemic and the relationship to alcohol consumption in a national sample of U.S. adults.

INTRODUCTION: The current study aimed to understand the links between social media use and alcohol consumption during the early months of the COVID-19 pandemic. METHOD: Data were from the national Understanding American Study, a probability-based Internet panel weighted to represent the U.S. population. Subjects (N = 5874; 51% female) were adults, 18 years and older, who completed a March survey (wave 1) and a follow-up survey one month later (wave 3). Analyses assessed the relationships of social media use at wave 1 with wave 3 alcohol use frequency, accounting for wave 1 alcohol use frequency and the sociodemographic characteristics of the sample. Two alcohol use change variables were also assessed as outcomes-increased and decreased alcohol use between waves. We considered the effect of work status changes (working/studying from home and job loss) as potential moderators. RESULTS: Twitter and Instagram users and users of multiple social media platforms, but not Facebook users, drank more frequently at wave 3. The results were similar when assessing relationships between social media use and increased alcohol use between waves. For Instagram users, more frequent alcohol use at wave 3 was at least partially attributed to drinking frequency at wave 1. Additionally, working/studying from home at wave 3 and employment (rather than job loss) were associated with greater consumption. The interaction effect between Twitter use and working/studying from home was statistically significant in association with alcohol use frequency at wave 3, as was the interaction effect between using multiple platforms and working/studying from home in association with decreased alcohol use between waves. DISCUSSION: Exposure to content about COVID-19 and increased alcohol consumption during the pandemic may have contributed to more frequent alcohol use for some social media users. The study of public health messaging via social media to change alcohol use behaviors during traumatic events is warranted.

ATF-3 expression inhibits melanoma growth by downregulating ERK and AKT pathways.

Activating transcription factor 3 (ATF-3), a cyclic AMP-dependent transcription factor, has been shown to play a regulatory role in melanoma, although its function during tumor progression remains unclear. Here, we demonstrate that ATF-3 exhibits tumor suppressive function in melanoma. Specifically, ATF-3 nuclear expression was significantly diminished with melanoma progression from nevi to primary to metastatic patient melanomas, correlating low expression with poor prognosis. Significantly low expression of ATF-3 was also found in cultured human metastatic melanoma cell lines. Importantly, overexpression of ATF-3 in metastatic melanoma cell lines significantly inhibited cell growth, migration, and invasion in vitro; as well as abrogated tumor growth in a human melanoma xenograft mouse model in vivo. RNA sequencing analysis revealed downregulation of ERK and AKT pathways and upregulation in apoptotic-related genes in ATF-3 overexpressed melanoma cell lines, which was further validated by Western-blot analysis. In summary, this study demonstrated that diminished ATF-3 expression is associated with melanoma virulence and thus provides a potential target for novel therapies and prognostic biomarker applications.

Targeting the ABC transporter ABCB5 sensitizes glioblastoma to temozolomide-induced apoptosis through a cell-cycle checkpoint regulation mechanism.

Glioblastoma multiforme (GBM) is a malignant brain tumor with a poor prognosis resulting from tumor resistance to anticancer therapy and a high recurrence rate. Compelling evidence suggests that this is driven by subpopulations of cancer stem cells (CSCs) with tumor-initiating potential. ABC subfamily B member 5 (ABCB5) has been identified as a molecular marker for distinct subsets of chemoresistant tumor-initiating cell populations in diverse human malignancies. In the current study, we examined the potential role of ABCB5 in growth and chemoresistance of GBM. We found that ABCB5 is expressed in primary GBM tumors, in which its expression was significantly correlated with the CSC marker protein CD133 and with overall poor survival. Moreover, ABCB5 was also expressed by CD133-positive CSCs in the established human U-87 MG, LN-18, and LN-229 GBM cell lines. Antibody- or shRNA-mediated functional ABCB5 blockade inhibited proliferation and survival of GBM cells and sensitized them to temozolomide (TMZ)-induced apoptosis in vitro Likewise, in in vivo human GBM xenograft experiments with immunodeficient mice, mAb treatment inhibited growth of mutant TP53, WT PTEN LN-229 tumors, and sensitized LN-229 tumors to TMZ therapy. Mechanistically, we demonstrate that ABCB5 blockade inhibits TMZ-induced G2/M arrest and augments TMZ-mediated cell death. Our results identify ABCB5 as a GBM chemoresistance marker and point to the potential utility of targeting ABCB5 to improve current GBM therapies.

Loss of the Epigenetic Mark 5-hmC in Psoriasis: Implications for Epidermal Stem Cell Dysregulation.

Epigenetic regulation has a profound influence on stem cell fate during normal development in maintenance of physiologic tissue homeostasis. Here we report diminished ten-eleven translocation (TET) methylcytosine dioxygenase expression and loss of the DNA hydroxymethylation mark 5-hydroxymethylcytosine (5-hmC) in keratinocyte stem cells and transit amplifying cells in human psoriasis and in imiquimod-induced murine psoriasis. Loss of 5-hmC was associated with dysregulated keratinocyte stem cell kinetics, resulting in accumulation of nestin and FABP5-expressing transit amplifying cells to produce classic psoriatic epidermal architecture. Moreover, 5-hmC loss was accompanied by diminished TET1 and TET2 mRNA expression. Genome-wide mapping of epidermal 5-hmC in murine psoriasis revealed loci-specific loss of 5-hmC in genes regulating stem cell homeostasis, including MBD1, RTN1, STRN4, PRKD2, AKT1, and MAPKAP2, as well as those associated with RAR and Wnt/ß-catenin signaling pathways. In vitro restoration of TET expression by ascorbic acid was accomplished in cultured human keratinocyte stem cells to show similar Ca++-induced differentiation, resulting in increased 5-hmC levels and reduced nestin expression. To our knowledge, an epigenetic deficiency in psoriasis with relevance to stem cell dysregulation has not been previously reported. This observation raises the possibility that epigenetic modifiers that impact on the TET-5-hmC pathway may be a relevant approach of heretofore unappreciated therapeutic utility.

Modeling and rescue of defective blood-brain barrier function of induced brain microvascular endothelial cells from childhood cerebral adrenoleukodystrophy patients.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene. 40% of X-ALD patients will convert to the deadly childhood cerebral form (ccALD) characterized by increased permeability of the brain endothelium that constitutes the blood-brain barrier (BBB). Mutation information and molecular markers investigated to date are not predictive of conversion. Prior reports have focused on toxic metabolic byproducts and reactive oxygen species as instigators of cerebral inflammation and subsequent immune cell invasion leading to BBB breakdown. This study focuses on the BBB itself and evaluates differences in brain endothelium integrity using cells from ccALD patients and wild-type (WT) controls. METHODS: The blood-brain barrier of ccALD patients and WT controls was modeled using directed differentiation of induced pluripotent stem cells (iPSCs) into induced brain microvascular endothelial cells (iBMECs). Immunocytochemistry and PCR confirmed characteristic expression of brain microvascular endothelial cell (BMEC) markers. Barrier properties of iBMECs were measured via trans-endothelial electrical resistance (TEER), sodium fluorescein permeability, and frayed junction analysis. Electron microscopy and RNA-seq were used to further characterize disease-specific differences. Oil-Red-O staining was used to quantify differences in lipid accumulation. To evaluate whether treatment with block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO) could mitigate defective properties, ccALD-iBMECs were treated with PEO-PPO block copolymers and their barrier properties and lipid accumulation levels were quantified. RESULTS: iBMECs from patients with ccALD had significantly decreased TEER (2592 ± 110 Ω cm2) compared to WT controls (5001 ± 172 Ω cm2). They also accumulated lipid droplets to a greater extent than WT-iBMECs. Upon treatment with a PEO-PPO diblock copolymer during the differentiation process, an increase in TEER and a reduction in lipid accumulation were observed for the polymer treated ccALD-iBMECs compared to untreated controls. CONCLUSIONS: The finding that BBB integrity is decreased in ccALD and can be rescued with block copolymers opens the door for the discovery of BBB-specific molecular markers that can indicate the onset of ccALD and has therapeutic implications for preventing the conversion to ccALD.

A multitask clustering approach for single-cell RNA-seq analysis in Recessive Dystrophic Epidermolysis Bullosa.

Single-cell RNA sequencing (scRNA-seq) has been widely applied to discover new cell types by detecting sub-populations in a heterogeneous group of cells. Since scRNA-seq experiments have lower read coverage/tag counts and introduce more technical biases compared to bulk RNA-seq experiments, the limited number of sampled cells combined with the experimental biases and other dataset specific variations presents a challenge to cross-dataset analysis and discovery of relevant biological variations across multiple cell populations. In this paper, we introduce a method of variance-driven multitask clustering of single-cell RNA-seq data (scVDMC) that utilizes multiple single-cell populations from biological replicates or different samples. scVDMC clusters single cells in multiple scRNA-seq experiments of similar cell types and markers but varying expression patterns such that the scRNA-seq data are better integrated than typical pooled analyses which only increase the sample size. By controlling the variance among the cell clusters within each dataset and across all the datasets, scVDMC detects cell sub-populations in each individual experiment with shared cell-type markers but varying cluster centers among all the experiments. Applied to two real scRNA-seq datasets with several replicates and one large-scale droplet-based dataset on three patient samples, scVDMC more accurately detected cell populations and known cell markers than pooled clustering and other recently proposed scRNA-seq clustering methods. In the case study applied to in-house Recessive Dystrophic Epidermolysis Bullosa (RDEB) scRNA-seq data, scVDMC revealed several new cell types and unknown markers validated by flow cytometry. MATLAB/Octave code available at https://github.com/kuanglab/scVDMC.

Critical Components of the Conjugation Machinery of the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

UNLABELLED: Conjugation, or mating, plays a profound role in bacterial evolution by spreading genes that allow bacteria to adapt to and colonize new niches. ICEBs1, an integrative and conjugative element of Bacillus subtilis, can transfer itself and mobilize resident plasmids. DNA transfer is mediated by a type IV secretion system (T4SS). Characterized components of the ICEBs1 T4SS include the conserved VirB4-like ATPase ConE, the bifunctional cell wall hydrolase CwlT, and the presumed VirD4-like coupling protein ConQ. A fusion of ConE to green fluorescent protein (GFP) localizes to the membrane preferentially at the cell poles. One or more ICEBs1 proteins are required for ConE's localization at the membrane, as ConE lacks predicted transmembrane segments and ConE-GFP is found dispersed throughout the cytoplasm in cells lacking ICEBs1. Here, we analyzed five ICEBs1 genes to determine if they are required for DNA transfer and/or ConE-GFP localization. We found that conB, conC, conD, and conG, but not yddF, are required for both ICEBs1 transfer and plasmid mobilization. All four required genes encode predicted integral membrane proteins. conB and, to some extent, conD were required for localization of ConE-GFP to the membrane. Using an adenylate cyclase-based bacterial two-hybrid system, we found that ConE interacts with ConB. We propose a model in which the ICEBs1 conjugation machinery is composed of ConB, ConC, ConD, ConE, ConG, CwlT, ConQ, and possibly other ICEBs1 proteins, and that ConB interacts with ConE, helping to recruit and/or maintain ConE at the membrane. IMPORTANCE: Conjugation is a major form of horizontal gene transfer and has played a profound role in bacterial evolution by moving genes, including those involved in antibiotic resistance, metabolism, symbiosis, and infectious disease. During conjugation, DNA is transferred from cell to cell through the conjugation machinery, a type of secretion system. Relatively little is known about the conjugation machinery of Gram-positive bacteria. Here, we analyzed five genes of the integrative and conjugative element ICEBs1 of Bacillus subtilis. Our research identifies four new components of the ICEBs1 conjugation machinery (ConB, ConC, ConD, and ConG) and shows an interaction between ConB and ConE that is required for ConE to associate with the cell membrane.

A conserved helicase processivity factor is needed for conjugation and replication of an integrative and conjugative element.

Integrative and conjugative elements (ICEs) are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT) by the ICE-encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP), encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.

Astringency of foods may not be directly related to salivary lubricity.

UNLABELLED: Astringency, a tactile sensation felt in the mouth after exposure to various foods, is poorly understood. Our objective was to investigate the mechanism of astringency by determining if it was related to a loss of saliva's ability to lubricate mouth surfaces. We used 2 complementary approaches: (1) instrumentally determining if astringents decrease salivary lubricity and (2) determining if human subjects could perceive these changes. Instrumentally measured friction of saliva-tannin mixtures was greater than that of saliva-water mixtures, but these differences were not detectable by human subjects. Adding alum or acid to saliva did not increase friction measured either instrumentally or by human subjects rubbing the mixture between their thumb and fingers. Thus a loss of salivary lubricity is likely not a central mechanism of astringency. PRACTICAL APPLICATIONS: If astringency's mechanism can be elucidated, food producers will gain knowledge that will enable them to manufacture, alter, or treat the food in a way to retain its healthful attributes while minimizing the astringency. This knowledge will also benefit sensory scientists interested in the intensity perceptions of the sensations and will allow them to devise improved methods for the assessment of the attribute.

The Bacillus subtilis conjugative transposon ICEBs1 mobilizes plasmids lacking dedicated mobilization functions.

Integrative and conjugative elements (ICEs, also known as conjugative transposons) are mobile elements that are found integrated in a host genome and can excise and transfer to recipient cells via conjugation. ICEs and conjugative plasmids are found in many bacteria and are important agents of horizontal gene transfer and microbial evolution. Conjugative elements are capable of self-transfer and also capable of mobilizing other DNA elements that are not able to self-transfer. Plasmids that can be mobilized by conjugative elements are generally thought to contain an origin of transfer (oriT), from which mobilization initiates, and to encode a mobilization protein (Mob, a relaxase) that nicks a site in oriT and covalently attaches to the DNA to be transferred. Plasmids that do not have both an oriT and a cognate mob are thought to be nonmobilizable. We found that Bacillus subtilis carrying the integrative and conjugative element ICEBs1 can transfer three different plasmids to recipient bacteria at high frequencies. Strikingly, these plasmids do not have dedicated mobilization-oriT functions. Plasmid mobilization required conjugation proteins of ICEBs1, including the putative coupling protein. In contrast, plasmid mobilization did not require the ICEBs1 conjugative relaxase or cotransfer of ICEBs1, indicating that the putative coupling protein likely interacts with the plasmid replicative relaxase and directly targets the plasmid DNA to the ICEBs1 conjugation apparatus. These results blur the current categorization of mobilizable and nonmobilizable plasmids and indicate that conjugative elements play a role in horizontal gene transfer even more significant than previously recognized.

The role of salivary proteins in the mechanism of astringency.

Understanding astringency has focused on the interaction of tannins with the salivary proline-rich proteins (PRPs), although it remains unclear if other astringents precipitate the PRPs or how this interaction relates to sensory perceptions of astringency. We used 2 approaches to compare how distinct classes of astringent compounds interacted with the salivary PRPs and mucins. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis, we evaluated protein patterns and characterized the salivary proteins present in the supernatants and pellets of pooled saliva assayed with tannin, alum, and hydrochloric acid solutions. Tannins and alum precipitated many of the PRPs, but acid did not. Mucins were precipitated by both the acid and alum, but not by the tannins. From our research, it appears that the precipitation of salivary proteins may be involved in the mechanism of astringency, but the precipitation of PRPs is not requisite for the development of astringency. We also measured mucin and deoxyribonucleic acid content of expectorated solutions of astringents that panelists swished in their mouths to determine if astringency was associated with a loss of oral lubricating films.