Longitudinal map of transcriptome changes in the Lyme pathogen Borrelia burgdorferi during tick-borne transmission.

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, adapts to vastly different environments as it cycles between tick vector and vertebrate host. During a tick bloodmeal, Bb alters its gene expression to prepare for vertebrate infection; however, the full range of transcriptional changes that occur over several days inside of the tick are technically challenging to capture. We developed an experimental approach to enrich Bb cells to longitudinally define their global transcriptomic landscape inside nymphal Ixodes scapularis ticks during a transmitting bloodmeal. We identified 192 Bb genes that substantially change expression over the course of the bloodmeal from 1 to 4 days after host attachment. The majority of upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 outer surface lipoproteins embedded in the unusual protein-rich coat of Bb. As these proteins may facilitate Bb interactions with the host, we utilized mass spectrometry to identify candidate tick proteins that physically associate with Bb. The Bb enrichment methodology along with the ex vivo Bb transcriptomes and candidate tick interacting proteins presented here provide a resource to facilitate investigations into key determinants of Bb priming and transmission during the tick stage of its unique transmission cycle.

A Narrative Review of Mental Illness Stigma Reduction Interventions Among African Americans in The United States.

Among African Americans, the chronicity and severity of mental illness correlates with worse health outcomes and widens health disparities. Stigma related to mental illness compounds mental health disparities by creating barriers to help-seeking behavior. We examine the current tools designed to reduce mental illness stigma and promote improved mental health outcomes among African Americans. The authors reviewed the current evidence in the literature for such stigma reduction interventions. The review team developed a focused search across four databases: PubMed, Embase, Scopus, and APA PsycINFO. Initial searches identified 120 articles, from which six studies were included as reporting on mental illness stigma reduction interventions among African Americans. We describe these four quantitative and two qualitative studies. There have been various interventions used among African Americans to reduce mental illness stigma, and the level of efficacy and effectiveness is not well studied. Our review demonstrated a need for more robust studies to yield strong evidence on effectiveness among stigma reduction interventions in this target population. The evidence does support tailoring intervention studies to this population. Effectively engaging and partnering with key stakeholders, including schools, community organizations, and faith-based institutions enhances the acceptance and delivery of stigma reduction interventions.

Mobile element warfare via CRISPR and anti-CRISPR in Pseudomonas aeruginosa.

Bacteria deploy multiple defenses to prevent mobile genetic element (MGEs) invasion. CRISPR-Cas immune systems use RNA-guided nucleases to target MGEs, which counter with anti-CRISPR (Acr) proteins. Our understanding of the biology and co-evolutionary dynamics of the common Type I-C CRISPR-Cas subtype has lagged because it lacks an in vivo phage-host model system. Here, we show the anti-phage function of a Pseudomonas aeruginosa Type I-C CRISPR-Cas system encoded on a conjugative pKLC102 island, and its Acr-mediated inhibition by distinct MGEs. Seven genes with anti-Type I-C function (acrIC genes) were identified, many with highly acidic amino acid content, including previously described DNA mimic AcrIF2. Four of the acr genes were broad spectrum, also inhibiting I-E or I-F P. aeruginosa CRISPR-Cas subtypes. Dual inhibition comes at a cost, however, as simultaneous expression of Type I-C and I-F systems renders phages expressing the dual inhibitor AcrIF2 more sensitive to targeting. Mutagenesis of numerous acidic residues in AcrIF2 did not impair anti-I-C or anti-I-F function per se but did exacerbate inhibition defects during competition, suggesting that excess negative charge may buffer DNA mimics against competition. Like AcrIF2, five of the Acr proteins block Cascade from binding DNA, while two function downstream, likely preventing Cas3 recruitment or activity. One such inhibitor, AcrIC3, is found in an 'anti-Cas3' cluster within conjugative elements, encoded alongside bona fide Cas3 inhibitors AcrIF3 and AcrIE1. Our findings demonstrate an active battle between an MGE-encoded CRISPR-Cas system and its diverse MGE targets.

Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/ß-Catenin Signaling.

Wnt/ß-catenin signaling is necessary for lymphatic vascular development. Oscillatory shear stress (OSS) enhances Wnt/ß-catenin signaling in cultured lymphatic endothelial cells (LECs) to induce expression of the lymphedema-associated transcription factors GATA2 and FOXC2. However, the mechanisms by which OSS regulates Wnt/ß-catenin signaling and GATA2 and FOXC2 expression are unknown. We show that OSS activates autocrine Wnt/ß-catenin signaling in LECs in vitro. Tissue-specific deletion of Wntless, which is required for the secretion of Wnt ligands, reveals that LECs and vascular smooth muscle cells are complementary sources of Wnt ligands that regulate lymphatic vascular development in vivo. Further, the LEC master transcription factor PROX1 forms a complex with ß-catenin and the TCF/LEF transcription factor TCF7L1 to enhance Wnt/ß-catenin signaling and promote FOXC2 and GATA2 expression in LECs. Thus, our work defines Wnt sources, reveals that PROX1 directs cell fate by acting as a Wnt signaling component, and dissects the mechanisms of PROX1 and Wnt synergy.

Discovery of widespread type I and type V CRISPR-Cas inhibitors.

Bacterial CRISPR-Cas systems protect their host from bacteriophages and other mobile genetic elements. Mobile elements, in turn, encode various anti-CRISPR (Acr) proteins to inhibit the immune function of CRISPR-Cas. To date, Acr proteins have been discovered for type I (subtypes I-D, I-E, and I-F) and type II (II-A and II-C) but not other CRISPR systems. Here, we report the discovery of 12 acr genes, including inhibitors of type V-A and I-C CRISPR systems. AcrVA1 inhibits a broad spectrum of Cas12a (Cpf1) orthologs-including MbCas12a, Mb3Cas12a, AsCas12a, and LbCas12a-when assayed in human cells. The acr genes reported here provide useful biotechnological tools and mark the discovery of acr loci in many bacteria and phages.

Bacteriophage Cooperation Suppresses CRISPR-Cas3 and Cas9 Immunity.

Bacteria utilize CRISPR-Cas adaptive immune systems for protection from bacteriophages (phages), and some phages produce anti-CRISPR (Acr) proteins that inhibit immune function. Despite thorough mechanistic and structural information for some Acr proteins, how they are deployed and utilized by a phage during infection is unknown. Here, we show that Acr production does not guarantee phage replication when faced with CRISPR-Cas immunity, but instead, infections fail when phage population numbers fall below a critical threshold. Infections succeed only if a sufficient Acr dose is contributed to a single cell by multiple phage genomes. The production of Acr proteins by phage genomes that fail to replicate leave the cell immunosuppressed, which predisposes the cell for successful infection by other phages in the population. This altruistic mechanism for CRISPR-Cas inhibition demonstrates inter-virus cooperation that may also manifest in other host-parasite interactions.

A Novel l-Asparaginase with low l-Glutaminase Coactivity Is Highly Efficacious against Both T- and B-cell Acute Lymphoblastic Leukemias In Vivo.

Acute lymphoblastic leukemia (ALL) is the most common type of pediatric cancer, although about 4 of every 10 cases occur in adults. The enzyme drug l-asparaginase serves as a cornerstone of ALL therapy and exploits the asparagine dependency of ALL cells. In addition to hydrolyzing the amino acid l-asparagine, all FDA-approved l-asparaginases also have significant l-glutaminase coactivity. Since several reports suggest that l-glutamine depletion correlates with many of the side effects of these drugs, enzyme variants with reduced l-glutaminase coactivity might be clinically beneficial if their antileukemic activity would be preserved. Here we show that novel low l-glutaminase variants developed on the backbone of the FDA-approved Erwinia chrysanthemi l-asparaginase were highly efficacious against both T- and B-cell ALL, while displaying reduced acute toxicity features. These results support the development of a new generation of safer l-asparaginases without l-glutaminase activity for the treatment of human ALL.Significance: A new l-asparaginase-based therapy is less toxic compared with FDA-approved high l-glutaminase enzymes Cancer Res; 78(6); 1549-60. ©2018 AACR.

Regulation of Tcf7l1 DNA binding and protein stability as principal mechanisms of Wnt/ß-catenin signaling.

Wnt/ß-catenin signal transduction requires direct binding of ß-catenin to Tcf/Lef proteins, an event that is classically associated with stimulating transcription by recruiting coactivators. This molecular cascade plays critical roles throughout embryonic development and normal postnatal life by affecting stem cell characteristics and tumor formation. Here, we show that this pathway utilizes a fundamentally different mechanism to regulate Tcf7l1 (formerly named Tcf3) activity. ß-catenin inactivates Tcf7l1 without a switch to a coactivator complex by removing it from DNA, which leads to Tcf7l1 protein degradation. Mouse genetic experiments demonstrate that Tcf7l1 inactivation is the only required effect of the Tcf7l1-ß-catenin interaction. Given the expression of Tcf7l1 in pluripotent embryonic and adult stem cells, as well as in poorly differentiated breast cancer, these findings provide mechanistic insights into the regulation of pluripotency and the role of Wnt/ß-catenin in breast cancer.

CT features of exophthalmos in Chinese subjects with thyroid-associated ophthalmopathy.

AIM: : To investigate computed tomography (CT) features of exophthalmos in Chinese subjects with thyroid-associated ophthalmopathy (TAO). METHODS: A total of 605 eyes of 325 patients with exophthalmos due to TAO were classified as grade I (mild exophthalmos) or II (severe exophthalmos) based on orbital CT imaging. The increased orbital volume features, such as changes from extraocular muscles, orbital fat, or both, were analyzed. RESULTS: A total of 605 eyes were analyzed, among them 62.98% presented grade I exophthalmos, while 36.02% showed grade II exophthalmos. In grade I, 56.69% showed orbital fat change, and in grade II, 89.29% exhibited extraocular muscle enlargement. CONCLUSION: Orbital fat and extraocular muscle enlargement are likely to be observed on CTs of subjects with mild and severe exophthalmos, respectively. Our results suggest that CT findings may guide TAO clinical therapy recommendations and prognosis.

Induced pluripotent stem cell-derived hepatocytes have the functional and proliferative capabilities needed for liver regeneration in mice.

The ability to generate induced pluripotent stem (iPS) cells from a patient's somatic cells has provided a foundation for organ regeneration without the need for immune suppression. However, it has not been established that the differentiated progeny of iPS cells can effectively reverse failure of a vital organ. Here, we examined whether iPS cell-derived hepatocytes have both the functional and proliferative capabilities needed for liver regeneration in mice with fumarylacetoacetate hydrolase deficiency. To avoid biases resulting from random genomic integration, we used iPS cells generated without viruses. To exclude compensation by hepatocytes not derived from iPS cells, we generated chimeric mice in which all hepatocytes were iPS cell derived. In vivo analyses showed that iPS cells were intrinsically able to differentiate into fully mature hepatocytes that provided full liver function. The iPS cell-derived hepatocytes also replicated the unique proliferative capabilities of normal hepatocytes and were able to regenerate the liver after transplantation and two-thirds partial hepatectomy. Thus, our results establish the feasibility of using iPS cells generated in a clinically acceptable fashion for rapid and stable liver regeneration.