Novel molecular requirements for CRISPR RNA-guided transposition.

CRISPR-associated transposases (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system (VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CAST systems.

Morphological and Elemental Evaluation of Investigative Mouthwashes to Repair Acid-Eroded Tooth Surface.

Purpose: Erosive tooth wear (ETW) is characterized by subsurface demineralization and tooth substance loss with crater formation. Remineralization of subsurface demineralization has previously been demonstrated; however, repair of the eroded surface is still under investigation. This study investigated the effectiveness of mouthwashes containing hydrolyzed wheat protein (HWP) in repairing ETW through promotion of organized crystal growth. Methods: Enamel Erosion was created on 210 enamel blocks by 10-minute demineralization in 1% Citric Acid (pH 3.5). Then, blocks were randomly assigned to seven groups (30/group); (A) 0.2% HWP, B) 1% HWP, (C) 2% HWP, (D) 1% HWP + 0.05% NaF, (E) Listerine™ mouthwash, (F) 0.02% NaF Crest™ Pro-health mouthwash and (G) artificial saliva (AS) only. Groups were subjected to daily pH-cycling consisting of one 5-minute erosive challenge with citric acid, three 1-minute mouthwash treatment periods, and then storage in AS for the rest of the time for 28 days. Treatment effects were assessed using SEM-EDX. Statistical analysis was by ANOVA and Tukey's multiple comparison. Results: In groups exposed to HWP-containing mouthwashes, there was growth of fiber-like crystals that increased in packing density in a dose-dependent manner (0.2%, 1%, 2%) on the eroded enamel surfaces, with increased calcium and phosphate contents on the treated surfaces. The non-HWP-containing groups had the eroded surfaces covered by structureless deposit layer firmly attached to the surface. Conclusion: Treating eroded enamel surface with HWP-containing mouthwash resulted in repair of the damaged tissue by formation of a protective layer of crystal deposits within and on the eroded enamel tissue.

Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration.

CRISPR-associated transposons (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system. On the donor DNA, large mutagenic libraries identified core binding sites recognized by the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications of CAST systems.

Characterization of white spot lesions formed on human enamel under microcosm biofilm for different experimental periods.

The initial characteristics of white spot lesion (WSLs), such as the degree of integrated mineral loss (ΔZ), depth and pattern of mineral distribution, have an impact on further demineralization and remineralization. However, these lesion parameters have not been evaluated in WSLs produced from microcosm biofilms. OBJECTIVE: This study characterized artificial white spot lesions produced on human enamel under microcosm biofilm for different experimental periods. METHODOLOGY: In total, 100 human enamel specimens (4x4mm) were assigned to 5 distinct groups (n=20/group) differing according to the period of biofilm formation (2, 4, 6, 8 or 10 days). Microcosm biofilm was produced on the specimens from a mixture of human and McBain saliva at the first 8h. Enamel samples were then exposed to McBain saliva containing 0.2% sucrose. WSLs formed were characterized by quantitative light-induced fluorescence (QLF) and transverse microradiography (TMR). Data were analyzed by ANOVA/Tukey or Kruskal-Wallis/Dunn tests (p<0.05). RESULTS: A clear time-response pattern was observed for both analyses, but TMR was able to better discriminate among the lesions. Regarding QLF analysis, median (95%CI; %) changes in fluorescence ∆Z were -7.74(-7.74:-6.45)a, -8.52(-8.75:-8.00)ab, -9.17(-10.00:-8.71)bc, -9.58(-10.53:-8.99)bc and -10.01(-11.44:-9.72)c for 2, 4, 6, 8, and 10 days, respectively. For TMR, median (95%CI; vol%.µm) ∆Z were 1410(1299-1479)a, 2420(2327-2604)ab, 2775(2573-2899)bc, 3305(3192-3406)cd and 4330(3972-4465)d, whereas mean (SD; µm) lesion depth were 53.7(12.3)a, 71.4(12.0)a, 103.8(24.8)b, 130.5(27.2)bc, 167.2(39.3)c for 2, 4, 6, 8 and 10 days, respectively. CONCLUSION: The progression of WSLs formed on human enamel under microcosm biofilm can be characterized over 2-10 days, both by QLF and TMR analyses, although the latter provides better discrimination among the lesions.

The Effectiveness of Cannabis and Cannabis Derivatives in Treating Lower Back Pain in the Aged Population: A Systematic Review.

BACKGROUND/AIMS: Cannabis is increasingly used in the management of pain, though minimal research exists to support its use since approval. Reduction in stigma has led to a growing interest in pharmaceutical cannabinoids as a possible treatment for lower back pain (LBP). The objective of this review was to assess the role and efficacy of cannabis and its derivatives in the management of LBP and compile global data related to the role of cannabis in the management of LBP in an aging population. METHODS: A systematic review was conducted using predetermined keywords by 3 independent researchers. Predetermined inclusion and exclusion criteria were applied, and 23 articles were selected for further analysis. RESULTS: Studies identified both significant and insignificant impacts of cannabis on LBP. Contradicting evidence was noted on the role of cannabis in the management of anxiety and insomnia, 2 common comorbidities with LBP. The existing literature suggests that cannabis may be used in the management of LBP and comorbid symptoms. CONCLUSIONS: Further research is needed to consider cannabis as an independent management option. There is a lack of evidence pertaining to the benefits of cannabis in an aged population, and thus, additional research is warranted to support its use in the aged population.

Characterization of white spot lesions formed on human enamel under microcosm biofilm for different experimental periods

Abstract The initial characteristics of white spot lesion (WSLs), such as the degree of integrated mineral loss (ΔZ), depth and pattern of mineral distribution, have an impact on further demineralization and remineralization. However, these lesion parameters have not been evaluated in WSLs produced from microcosm biofilms. Objective: This study characterized artificial white spot lesions produced on human enamel under microcosm biofilm for different experimental periods. Methodology: In total, 100 human enamel specimens (4x4mm) were assigned to 5 distinct groups (n=20/group) differing according to the period of biofilm formation (2, 4, 6, 8 or 10 days). Microcosm biofilm was produced on the specimens from a mixture of human and McBain saliva at the first 8h. Enamel samples were then exposed to McBain saliva containing 0.2% sucrose. WSLs formed were characterized by quantitative light-induced fluorescence (QLF) and transverse microradiography (TMR). Data were analyzed by ANOVA/Tukey or Kruskal-Wallis/Dunn tests (p<0.05). Results: A clear time-response pattern was observed for both analyses, but TMR was able to better discriminate among the lesions. Regarding QLF analysis, median (95%CI; %) changes in fluorescence ∆Z were -7.74(-7.74:-6.45)a, -8.52(-8.75:-8.00)ab, -9.17(-10.00:-8.71)bc, -9.58(-10.53:-8.99)bc and -10.01(-11.44:-9.72)c for 2, 4, 6, 8, and 10 days, respectively. For TMR, median (95%CI; vol%.µm) ∆Z were 1410(1299-1479)a, 2420(2327-2604)ab, 2775(2573-2899)bc, 3305(3192-3406)cd and 4330(3972-4465)d, whereas mean (SD; µm) lesion depth were 53.7(12.3)a, 71.4(12.0)a, 103.8(24.8)b, 130.5(27.2)bc, 167.2(39.3)c for 2, 4, 6, 8 and 10 days, respectively. Conclusion: The progression of WSLs formed on human enamel under microcosm biofilm can be characterized over 2-10 days, both by QLF and TMR analyses, although the latter provides better discrimination among the lesions.

Benzodiazepine Use and Morbidity-Mortality Outcomes in a Geriatric Palliative Care Unit: A Retrospective Review.

INTRODUCTION: Patients often experience delirium at the end of life. Benzodiazepine use may be associated with an increased risk of developing delirium. Alternate medications used in conjunction with benzodiazepines may serve as an independent precipitant of delirium. The aim is to understand the role of benzodiazepines in precipitating delirium and advanced mortality in palliative care population at the end of life. METHODS: A retrospective medical chart review was conducted at a hospice and palliative care inpatient unit between the periods of June 2017-December 2017 and October 2017-November 2018. It included patients in hospice and palliative care inpatient units who received a benzodiazepine and those who did not. Patient characteristics, as well as Palliative Performance Scale score, diagnosis, and occurrence of admission, terminal, and/or recurrent delirium, were collected and analyzed. RESULTS: Use of a benzodiazepine was not significantly associated with overall mortality nor cause-specific death without terminal delirium rate. However, it was significantly associated with higher cause-specific death with terminal delirium rate and a higher recurrent delirium rate. DISCUSSION: This retrospective chart review suggests an association between benzodiazepine use and specific states of delirium and cause-specific death. However, it does not provide strong evidence on the use of this drug, especially at the end of life, as it pertains to the overall mortality rate. Suggested is a contextual approach to the use of benzodiazepines and the need to consider Palliative Performance Scale score and goals of care in the administration of this drug at varying periods during patient length of stay.

Sinophobic Epidemics in America: Historical Discontinuity in Disease-related Yellow Peril Imaginaries of the Past and Present.

Modern scholarship has drawn hasty and numerous parallels between the Yellow Peril discourses of the 19th- and 20th-century plagues and the recent racialization of infectious disease in the 21st-century. While highlighting these similarities is politically useful against Sinophobic epidemic narratives, Michel Foucault argues that truly understanding the past's continuity in the present requires a more rigorous genealogical approach. Employing this premise in a comparative analysis, this work demonstrates a critical discontinuity in the epidemic imaginary that framed the Chinese as pathogenic. Consequently, those seeking to prevent future disease racialization must understand modern Sinophobia as fundamentally distinct from that of the past.

Microfluidic production and characterization of biofunctionalized giant unilamellar vesicles for targeted intracellular cargo delivery.

Lipid-based vesicles have found widespread applications in the life sciences, allowing for fundamental insights into membrane-based processes in cell biology and as carrier systems for drug delivery purposes. So far, mostly small unilamellar vesicles (SUVs) with diameters of ~100 nm have been applied as carrier systems for biomedical applications. Despite this progress, several systematic limitations have arisen due to SUV dimensions, e.g., the size and total amount of applicable cargo is limited. Giant unilamellar vesicles (GUVs) might offer a pragmatic alternative for efficient cargo delivery. However, due to the lack of reliable high-throughput production technologies for GUV-carrier systems, only little is known about their interaction with cells. Here we present a microfluidic-based mechanical droplet-splitting pipeline for the production of carrier-GUVs with diameters of ~2 µm. The technology developed allows for highly efficient cargo loading and unprecedented control over the biological and physicochemical properties of GUV membranes. By generating differently charged (between -31 and + 28 mV), bioligand-conjugated (e.g. with E-cadherin, NrCam and antibodies) and PEG-conjugated GUVs, we performed a detailed investigation of attractive and repulsive GUV-cell interactions. Fine-tuning of these interactions allowed for targeted cellular GUV delivery. Moreover, we evaluated strategies for intracellular GUV cargo release by lysosomal escape mediated by the pH sensitive lipid DOBAQ, enabling cytoplasmic transmission. The presented GUV delivery technology and the systematic characterization of associated GUV-cell interactions could provide a means for more efficient drug administration and will pave the way for hitherto impossible approaches towards a targeted delivery of advanced cargo such as microparticles, viruses or macromolecular DNA-robots.

PYK2 Is Involved in Premalignant Acinar Cell Reprogramming and Pancreatic Ductal Adenocarcinoma Maintenance by Phosphorylating ß-CateninY654.

BACKGROUND & AIMS: Identification and validation of new functionally relevant and pharmacologically actionable targets for pancreatic ductal adenocarcinoma (PDAC) remains a great challenge. Premalignant acinar cell reprogramming (acinar-to-ductal metaplasia [ADM]) is a precursor of pancreatic intraepithelial neoplasia (PanIN) lesions that can progress to PDAC. This study investigated the role of proline-rich tyrosine kinase 2 (PYK2) in mutant Kras-induced and pancreatitis-associated ADM and PanIN formation, as well as in PDAC maintenance. METHODS: Genetically engineered mouse models of mutant Kras (glycine 12 to aspartic acid) and Pyk2 deletion were used for investigating the role of PYK2 in PDAC genesis in mice. In vitro ADM assays were conducted using primary pancreatic acinar cells isolated from mice. Immunohistochemistry, immunofluorescence, and a series of biochemical experiments were used to investigate upstream regulators/downstream targets of PYK2 in pancreatic carcinogenesis. PDAC cell line xenograft experiments were performed to study the role of PYK2 and its downstream target in PDAC maintenance. RESULTS: PYK2 was increased substantially in ADM lesions induced by mutant Kras or inflammatory injury. Pyk2 deletion remarkably suppressed ADM and PanIN formation in a mutant Kras-driven and pancreatitis-associated PDAC model, whereas PYK2 knockdown substantially inhibited PDAC cell growth in vitro and in nude mice. This study uncovered a novel yes-associated protein 1/transcriptional co-activator with PDZ binding motif/signal transducer and activator of transcription 3/PYK2/ß-catenin regulation axis in PDAC. Our results suggest that PYK2 contributes to PDAC genesis and maintenance by activating the Wnt/ß-catenin pathway through directly phosphorylating ß-cateninY654. CONCLUSIONS: The current study uncovers PYK2 as a novel downstream effector of mutant KRAS signaling, a previously unrecognized mediator of pancreatitis-induced ADM and a novel intervention target for PDAC.

Wnt/ß-Catenin Pathway Activation Mediates Adaptive Resistance to BRAF Inhibition in Colorectal Cancer.

One of the most encouraging developments in oncology has been the success of BRAF inhibitors in BRAF-mutant melanoma. However, in contrast to its striking efficacy in BRAF-mutant melanomas, BRAF inhibitor monotherapy is ineffective in BRAF-mutant colorectal cancer. Although many studies on BRAF inhibitor resistance in colorectal cancer have focused on mechanisms underlying the reactivation of the EGFR/RAS/RAF/MEK/ERK pathway, the current study focuses on identifying novel adaptive signaling mechanisms, a fresh angle on colorectal cancer resistance to BRAF inhibition. We found that treatment with BRAF inhibitors (both current and next-generation BRAF inhibitors) upregulated the Wnt/ß-catenin pathway in BRAFV600E-mutant colorectal cancer cell lines through activating the cytoplasmic tyrosine kinase focal adhesion kinase (FAK). The results showed that FAK activation upon BRAF inhibitor treatment did not require EGFR or ERK1/2 activation, implying that BRAF inhibitor treatment-induced hyperactivation of Wnt signaling is "pathway reactivation"-independent. BRAF inhibition-induced Wnt pathway activation was further validated in preclinical models of BRAFV600E-mutant colorectal cancer, including cell line xenograft model and a patient-derived xenograft model. Combined inhibition of BRAF/Wnt pathways or BRAF/FAK pathways exerted strong synergistic antitumor effects in cell culture model and mouse xenograft model. Overall, the current study has identified activation of the Wnt/ß-catenin pathway as a novel fundamental cause of colon cancer resistance to BRAF inhibition. Our results suggest that although complete vertical pathway blockade is pivotal for effective and durable control of BRAF-mutant colorectal cancer, cotargeting parallel adaptive signaling-the Wnt/ß-catenin pathway-is also essential. Mol Cancer Ther; 17(4); 806-13. ©2017 AACR.

MAP4K4 is a novel MAPK/ERK pathway regulator required for lung adenocarcinoma maintenance.

About 76% of patients with lung adenocarcinoma harbor activating mutations in the receptor tyrosine kinase (RTK)/RAS/RAF pathways, leading to aberrant activation of the mitogen-activated protein kinase (MAPK) pathways particularly the MAPK/ERK pathway. However, many lung adenocarcinomas lacking these genomic mutations also display significant MAPK pathway activation, suggesting that additional MAPK pathway alterations remain undetected. This study has identified serine/threonine kinase mitogen-activated protein 4 kinase 4 (MAP4K4) as a novel positive regulator of MAPK/ERK signaling in lung adenocarcinoma. The results showed that MAP4K4 was drastically elevated in lung adenocarcinoma independently of KRAS or EGFR mutation status. Knockdown of MAP4K4 inhibited proliferation, anchorage-independent growth and migration of lung adenocarcinoma cells, and also inhibited human lung adenocarcinoma xenograft growth and metastasis. Mechanistically, we found that MAP4K4 activated ERK through inhibiting protein phosphatase 2 activity. Our results further showed that downregulation of MAP4K4 prevented ERK reactivation in EGFR inhibitor erlotinib-treated lung adenocarcinoma cells. Together, our findings identify MAP4K4 as a novel MAPK/ERK pathway regulator in lung adenocarcinoma that is required for lung adenocarcinoma maintenance.

FAK/PYK2 promotes the Wnt/ß-catenin pathway and intestinal tumorigenesis by phosphorylating GSK3ß.

Aberrant activation of Wnt/ß-catenin signaling plays an unequivocal role in colorectal cancer, but identification of effective Wnt inhibitors for use in cancer remains a tremendous challenge. New insights into the regulation of this pathway could reveal new therapeutic point of intervention, therefore are greatly needed. Here we report a novel FAK/PYK2/GSK3ß(Y216)/ß-catenin regulation axis: FAK and PYK2, elevated in adenomas in APC(min/+) mice and in human colorectal cancer tissues, functioned redundantly to promote the Wnt/ß-catenin pathway by phosphorylating GSK3ß(Y216) to reinforce pathway output-ß-catenin accumulation and intestinal tumorigenesis. We previously showed that Wnt-induced ß-catenin accumulation requires Wnt-induced GSK3ß/ß-TrCP interaction; the current study revealed that phosphorylation of GSK3ß(Y216) was a molecular determinant of GSK3ß recruitment of ß-TrCP. Pharmacological inhibition of FAK/PYK2 suppressed adenoma formation in APC(min/+) mice accompanied with reduced intestinal levels of phospho-GSK3ß(Y216) and ß-catenin, indicating that FAK/PYK2/GSK3ß(Y216) axis is critical for the activation of Wnt/ß-catenin signaling in APC driven intestinal tumorigenesis.