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The purpose of this study was to examine reactions to the Centers for Disease Control and Prevention revised recommendations for HIV testing by women attending community health clinics. A total of 30 women attending three community clinics completed semistructured individual interviews containing three questions about the recommendations. Thematic content analysis of responses was conducted. Results were that all agreed with the recommendation for universal testing. Most viewed opt-out screening as an acceptable approach to HIV testing. Many emphasized the importance of provision of explicit verbal informed consent. The majority strongly opposed the elimination of the requirement for pretest prevention counseling and spontaneously talked about the ongoing importance of posttest counseling. The conclusion was that there was strong support for universal testing of all persons 13 to 64 years old but scant support for the elimination of pretest prevention counseling. In general, respondents believed that verbal informed consent for testing as well as provision of HIV-related information before and after testing were crucial. [ABSTRACT FROM AUTHOR]

BACKGROUND: Somatoform disorders may have their roots in childhood through processes that involve an enhanced parental focus on health. The aim of this study was to test the hypothesis that somatizing mothers will show less joint involvement than other mothers during play but greater responsiveness when this play involves a 'medical' theme. METHOD: Cross-sectional observational study of 42 chronic somatizers, 44 organically ill and 50 healthy mothers and their 4-8 year-old children during structured play and a meal. Tasks comprised boxes containing tea-set items, 'medical' items and a light snack. RESULTS: Somatizing mothers were emotionally flatter and showed lower rates of joint attention than other mothers during both play tasks. While the three groups had similar rate of bids for attention, somatizing mothers were more responsive to their child's bids during play with the medical box than at other times. In contrast, the children of somatizing mothers ignored a greater proportion of their mother's bids during play with the medical box than did children of other mothers or during play with a non-medical theme. CONCLUSION: The study has demonstrated tentative evidence in support of the hypothesis. [ABSTRACT FROM AUTHOR]

The prevalence of maternal depression was investigated in the mothers of 96 children: 30 premature infants at risk for the development of cerebral palsy; 35 premature infants considered not to be at risk for the development of cerebral palsy; and 31 healthy fullterm infants. There were equally high levels of depression in all three groups of mothers, regardless of birth status, prediction of disability, or presence of actual disability, throughout the first year of the children's lives. Depressed mothers were, however, found to have significantly more psychosocial stress. An early physiotherapy intervention had no effect on the prevalence of depression in mothers whose children were at risk for the development of cerebral palsy. [ABSTRACT FROM AUTHOR]

The three RAS genes ( HRAS , KRAS , and NRAS ) comprise the most frequently mutated oncogene family in cancer. KRAS is the predominant isoform mutated in cancer and is most prevalently mutated in major causes of cancer deaths including lung, colorectal, and pancreatic cancers. Despite extensive academic and industry efforts to target KRAS, it would take nearly four decades before approval of the first clinically effective KRAS inhibitors for the treatment of KRAS mutant lung cancer. We revisit past anti-KRAS strategies and painful lessons learned and then focus on the rapidly evolving landscape of direct RAS inhibitors, resistance mechanisms, and potential combination treatments., (© 2025 Cox and Der; Published by Cold Spring Harbor Laboratory Press.)

This review evaluated single-case experimental design research that examined challenging behavior interventions utilizing punishment elements. Thirty articles published between 2013 and 2022 met study inclusion criteria. Study quality was also assessed. Through multiple levels of analysis (e.g., descriptive statistics, non-parametric statistics), we examined (a) participant and study trends, (b) differential outcomes related to temporal reinforcement approaches (antecedent, consequent, or combined reinforcement) applied alongside punishment element(s), (c) differential outcomes related to the punishment type (negative, positive) applied alongside reinforcement, and (d) effect sizes associated with study rigor across peer-reviewed and gray literature. Our results may tentatively suggest that, for certain situations, concurrently applying punishment with antecedent reinforcement approaches may coincide with significantly larger effect sizes compared to combined temporal reinforcement approaches, while positive punishment applied concurrently with reinforcement may coincide with larger but non-significant intervention effects. Most featured articles met rigor criteria, but larger effects were seen in peer-reviewed literature., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

A single small molecule degrades numerous KRAS variants involved in cancer.

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.

How the KRAS oncogene drives cancer growth remains poorly understood. Therefore, we established a systemwide portrait of KRAS- and extracellular signal-regulated kinase (ERK)-dependent gene transcription in KRAS-mutant cancer to delineate the molecular mechanisms of growth and of inhibitor resistance. Unexpectedly, our KRAS-dependent gene signature diverges substantially from the frequently cited Hallmark KRAS signaling gene signature, is driven predominantly through the ERK mitogen-activated protein kinase (MAPK) cascade, and accurately reflects KRAS- and ERK-regulated gene transcription in KRAS-mutant cancer patients. Integration with our ERK-regulated phospho- and total proteome highlights ERK deregulation of the anaphase promoting complex/cyclosome (APC/C) and other components of the cell cycle machinery as key processes that drive pancreatic ductal adenocarcinoma (PDAC) growth. Our findings elucidate mechanistically the critical role of ERK in driving KRAS-mutant tumor growth and in resistance to KRAS-ERK MAPK targeted therapies.

The cellular junctional architecture remodeling by Listeria adhesion protein-heat shock protein 60 (LAP-Hsp60) interaction for Listeria monocytogenes ( Lm ) passage through the epithelial barrier is incompletely understood. Here, using the gerbil model, permissive to internalin (Inl) A/B-mediated pathways like in humans, we demonstrate that Lm crosses the intestinal villi at 48 h post-infection. In contrast, the single isogenic ( lap - or Δ inlA ) or double ( lap - Δ inlA ) mutant strains show significant defects. LAP promotes Lm translocation via endocytosis of cell-cell junctional complex in enterocytes that do not display luminal E-cadherin. In comparison, InlA facilitates Lm translocation at cells displaying apical E-cadherin during cell extrusion and mucus expulsion from goblet cells. LAP hijacks caveolar endocytosis to traffic integral junctional proteins to the early and recycling endosomes. Pharmacological inhibition in a cell line and genetic knockout of caveolin-1 in mice prevents LAP-induced intestinal permeability, junctional endocytosis, and Lm translocation. Furthermore, LAP-Hsp60-dependent tight junction remodeling is also necessary for InlA access to E-cadherin for Lm intestinal barrier crossing in InlA-permissive hosts., Importance: Listeria monocytogenes ( Lm ) is a foodborne pathogen with high mortality (20%-30%) and hospitalization rates (94%), particularly affecting vulnerable groups such as pregnant women, fetuses, newborns, seniors, and immunocompromised individuals. Invasive listeriosis involves Lm 's internalin (InlA) protein binding to E-cadherin to breach the intestinal barrier. However, non-functional InlA variants have been identified in Lm isolates, suggesting InlA-independent pathways for translocation. Our study reveals that Listeria adhesion protein (LAP) and InlA cooperatively assist Lm entry into the gut lamina propria in a gerbil model, mimicking human listeriosis in early infection stages. LAP triggers caveolin-1-mediated endocytosis of critical junctional proteins, transporting them to early and recycling endosomes, facilitating Lm passage through enterocytes. Furthermore, LAP-Hsp60-mediated junctional protein endocytosis precedes InlA's interaction with basolateral E-cadherin, emphasizing LAP and InlA's cooperation in enhancing Lm intestinal translocation. This understanding is vital in combating the severe consequences of Lm infection, including sepsis, meningitis, encephalitis, and brain abscess., Competing Interests: A patent on LAP use as a tight junction modulator has been issued.

Visual inspection of single-subject data is the primary method for behavior analysts to interpret the effect of an independent variable on a dependent variable; however, there is no consensus on the most suitable method for teaching graph construction for single-subject designs. We systematically replicated and extended Tyner and Fienup (2015) using a repeated-measures between-subjects design to compare the effects of instructor-led, video-model, and no-instruction control tutorials on the graphing performance of 81 master's students with some reported Microsoft Excel experience. Our mixed-design analysis revealed a statistically significant main effect of pretest, tutorial, and posttest submissions for each tutorial group and a nonsignificant main effect of tutorial group. Tutorial group significantly interacted with submissions, suggesting that both instructor-led and video-model tutorials may be superior to providing graduate students with a written list of graphing conventions (i.e., control condition). Finally, training influenced performance on an untrained graph type (multielement) for all tutorial groups., (© 2024 Society for the Experimental Analysis of Behavior (SEAB).)

It is considered best practice to conduct a functional analysis and visually inspect data collected to determine the function of problem behavior, which then informs the intervention approaches applied. Visual inspection has been described as a "subjective" process that may be affected by factors unrelated to the data. Structured decision-making guidelines have been established to address some of these shortcomings. The current paper is a follow-up to earlier work describing positive outcomes related to the viability of a decision support system based on structured criteria from Roane et al. Here, we demonstrate important improvements in a computer script's interpretation of functional analysis data, including improvement in agreement between the updated computer script version and experienced human raters (89%) compared to our original agreement outcomes (81%). This paper further supports the use of decision support systems for functional analysis interpretation., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Cancer-associated mutations in the guanosine triphosphatase (GTPase) RHOA are found at different locations from the mutational hotspots in the structurally and biochemically related RAS. Tyr 42 -to-Cys (Y42C) and Leu 57 -to-Val (L57V) substitutions are the two most prevalent RHOA mutations in diffuse gastric cancer (DGC). RHOA Y42C exhibits a gain-of-function phenotype and is an oncogenic driver in DGC. Here, we determined how RHOA L57V promotes DGC growth. In mouse gastric organoids with deletion of Cdh1 , which encodes the cell adhesion protein E-cadherin, the expression of RHOA L57V , but not of wild-type RHOA, induced an abnormal morphology similar to that of patient-derived DGC organoids. RHOA L57V also exhibited a gain-of-function phenotype and promoted F-actin stress fiber formation and cell migration. RHOA L57V retained interaction with effectors but exhibited impaired RHOA-intrinsic and GAP-catalyzed GTP hydrolysis, which favored formation of the active GTP-bound state. Introduction of missense mutations at KRAS residues analogous to Tyr 42 and Leu 57 in RHOA did not activate KRAS oncogenic potential, indicating distinct functional effects in otherwise highly related GTPases. Both RHOA mutants stimulated the transcriptional co-activator YAP1 through actin dynamics to promote DGC progression; however, RHOA L57V additionally did so by activating the kinases IGF1R and PAK1, distinct from the FAK-mediated mechanism induced by RHOA Y42C . Our results reveal that RHOA L57V and RHOA Y42C drive the development of DGC through distinct biochemical and signaling mechanisms.

Primary/intrinsic and treatment-induced acquired resistance limit the initial response rate to and long-term efficacy of direct inhibitors of the KRASG12C mutant in cancer. To identify potential mechanisms of resistance, we applied a CRISPR/Cas9 loss-of-function screen and observed loss of multiple components of the Hippo tumor suppressor pathway, which acts to suppress YAP1/TAZ-regulated gene transcription. YAP1/TAZ activation impaired the antiproliferative and proapoptotic effects of KRASG12C inhibitor (G12Ci) treatment in KRASG12C-mutant cancer cell lines. Conversely, genetic suppression of YAP1/WWTR1 (TAZ) enhanced G12Ci sensitivity. YAP1/TAZ activity overcame KRAS dependency through two distinct TEAD transcription factor-dependent mechanisms, which phenocopy KRAS effector signaling. First, TEAD stimulated ERK-independent transcription of genes normally regulated by ERK (BIRC5, CDC20, ECT2, FOSL1, and MYC) to promote progression through the cell cycle. Second, TEAD caused activation of PI3K-AKT-mTOR signaling to overcome apoptosis. G12Ci treatment-induced acquired resistance was also caused by YAP1/TAZ-TEAD activation. Accordingly, concurrent treatment with pharmacologic inhibitors of TEAD synergistically enhanced KRASG12C inhibitor antitumor activity in vitro and prolonged tumor suppression in vivo. In summary, these observations reveal YAP1/TAZ-TEAD signaling as a crucial driver of primary and acquired resistance to KRAS inhibition and support the use of TEAD inhibitors to enhance the antitumor efficacy of KRAS-targeted therapies., Significance: YAP1/TAZ-TEAD activation compensates for loss of KRAS effector signaling, establishing a mechanistic basis for concurrent inhibition of TEAD to enhance the efficacy of KRASG12C-selective inhibitor treatment of KRASG12C-mutant cancers. See related commentary by Johnson and Haigis, p. 4005., (©2023 American Association for Cancer Research.)

Background: Research on training direct support professionals to address challenging behaviour exhibited by adults with intellectual disabilities is essential in generating effective training approaches for this sector. This systematic review's objective was to evaluate the effects of training types and whether specific training delivery components influenced outcomes., Methods: Following PRISMA (2020) guidelines, 16 single-case design studies were included that directly evaluated behaviour change of service providers training for adults with intellectual disabilities in community settings. Study quality was assessed using Horner et al. (Exceptional Children, 2005, 71(2), 165-180) criteria. The database searched included Academic Search Complete, CINAHL, Embase, ERIC, Psych Info, and Web of Science., Results: Descriptive analysis of effect size outcomes suggested that all training types were associated with improved outcomes. Further, in situ training was associated with improved service provider performance. Interestingly, feedback was associated with poorer service provider performance., Conclusions: We provide possible explanations for this surprising outcome and propose future research., (© 2023 The Authors. Journal of Applied Research in Intellectual Disabilities published by John Wiley & Sons Ltd.)

The rising incidence of inflammatory bowel disease (IBD) has necessitated the search for safe and effective novel therapeutic strategies. Dietary flavonoids exhibited antioxidant, antiproliferative, and anticarcinogenic activities in several model systems with proven abilities to reduce inflammation and oxidative stress, thus they could be promising therapeutic agents for IBD prevention/treatment. However, understanding the role of a specific class of compounds in foods that promote health is difficult because of the chemically complex food matrices. This study aimed to utilize four maize near-isogenic lines to determine the anti-colitis effects of specific classes of flavonoids, anthocyanins and/or phlobaphenes, in a whole-food matrix. Results showed that the intake of anthocyanin and phlobaphene-enriched maize diets effectively alleviated dextran sodium sulfate (DSS)-induced colitis in mice via reducing the intestinal permeability and restoring the barrier function. Anthocyanin diets were more effective in maintaining the crypt structure and muc2 protein levels and reducing inflammation. Bacterial communities of mice consuming diets enriched with anthocyanins and phlobaphenes were more similar to the healthy control compared to the DSS control group, suggesting the role of flavonoids in modulating the gut microbiota to retrieve intestinal homeostasis. Microbiota depletion rendered these compounds ineffective against colitis. Lower serum concentrations of several phenolic acids were detected in the microbiota-depleted mice, indicating that gut microbiota plays a role in flavonoid metabolism and bioavailability.

Sea squirts' or tunicates' bodies are composed of cellulose nanofibers and gallol- functionalized proteins. These sea creatures are known to heal their injuries under seawater by forming crosslinks between gallols and functional groups from other proteins in their bodies. Inspired by their wound healing mechanism, herein, we have developed a tissue sealant using zein (a plant-based protein) and tannic acid (gallol-containing polyphenol). Except for fibrin- based sealants, most commercial surgical adhesives, and sealants available today are derived from petroleum products that compromise their biodegradability. They often have complicated and multi-step synthesis processes that ultimately affect their affordability. To overcome this challenge, we ensured that these sea squirt-inspired tissue sealants are bio-based, easily synthesized, and low-cost. The sealants were studied on their own and with a food-grade enzyme transglutaminase. The adhesion performances of the sealants were found to be higher than physiological pressures in seven out of nine different tissue substrates studied here. Their performance was also better than or on par with the FDA-approved fibrin sealant Tisseel. Ex vivo models demonstrate instant sealing of leaking wounds in less than a minute. The sealants were not only cytocompatible but also showed complete wound healing on par with sutures and Tisseel when applied in vivo on skin incisions in rats. Overall, these sea squirt-inspired bio-based sealants show great potential to replace currently available wound closure methods.

Wound infections with antibiotic-resistant bacteria, particularly the Gram-negative strains, pose a substantial health risk for patients with limited treatment options. Recently topical administration of gaseous ozone and its combination with antibiotics through portable systems has been demonstrated to be a promising approach to eradicate commonly found Gram-negative strains of bacteria in wound infections. However, despite the significant impact of ozone in treating the growing number of antibiotic-resistant infections, uncontrolled and high concentrations of ozone can cause damage to the surrounding tissue. Hence, before such treatments could advance into clinical usage, it is paramount to identify appropriate levels of topical ozone that are effective in treating bacterial infections and safe for use in topical administration. To address this concern, we have conducted a series of in vivo studies to evaluate the efficacy and safety of a portable and wearable adjunct ozone and antibiotic wound therapy system. The concurrent ozone and antibiotics are applied through a wound interfaced gas permeable dressing coated with water-soluble nanofibers containing vancomycin and linezolid (traditionally used to treat Gram-positive infections) and connected to a portable ozone delivery system. The bactericidal properties of the combination therapy were evaluated on an ex vivo wound model infected with Pseudomonas aeruginosa , a common Gram-negative strain of bacteria found in many skin infections with high resistance to a wide range of currently available antibiotics. The results indicated that the optimized combination delivery of ozone (4 mg h -1 ) and topical antibiotic (200 μg cm -2 ) provided complete bacteria eradication after 6 h of treatment while having minimum cytotoxicity to human fibroblast cells. Furthermore, in vivo local and systemic toxicity studies (e.g., skin monitoring, skin histopathology, and blood analysis) on pig models showed no signs of adverse effects of ozone and antibiotic combination therapy even after 5 days of continuous administration. The confirmed efficacy and biosafety profile of the adjunct ozone and antibiotic therapy places it as a strong candidate for treating wound infection with antimicrobial-resistant bacteria and further pursuing human clinical trials.

Aortic dissection occurs when a weakened portion of the intima tears, and a separation of layers propagates along the aortic wall to form a false lumen filled with active blood flow or intramural thrombus. The unpredictable nature of aortic dissection formation and need for immediate intervention leaves limited serial human image data to study the formation and morphological changes that follow dissection. We used volumetric ultrasound examination, histology, and scanning electron microscopy (SEM) to examine intramural thrombi at well-defined timepoints after dissection occurs in apolipoprotein E-deficient mice infused with angiotensin II (n = 71). Stratification of red blood cell (RBC) morphologies (biconcave, intermediate biconcave, intermediate polyhedrocyte, and polyhedrocyte) in the thrombi with scanning electron microscopy (n = 5) was used to determine degree of thrombus deposition/contraction. Very few biconcave RBCs (1.2 ± 0.6%) were in the thrombi, and greater amounts of intermediate biconcave RBCs (25.8 ± 6.7%) were located in the descending thoracic portion of the dissection while more polyhedrocytes (14.6 ± 5.1%) and fibrin (42.3 ± 4.5%; P < .05) were found in the distal suprarenal aorta. Thrombus deposition likely plays some role in patient outcomes, and this multimodality technique can help investigate thrombus deposition and characteristics in experimental animal models and human tissue samples., (© 2023 by the Society for Vascular Surgery. Published by Elsevier Inc.)

We and others have recently shown that proteins involved in the DNA damage response (DDR) are critical for KRAS -mutant pancreatic ductal adenocarcinoma (PDAC) cell growth in vitro . However, the CRISPR-Cas9 library that enabled us to identify these key proteins had limited representation of DDR-related genes. To further investigate the DDR in this context, we performed a comprehensive, DDR-focused CRISPR-Cas9 loss-of-function screen. This screen identified valosin-containing protein ( VCP ) as an essential gene in KRAS -mutant PDAC cell lines. We observed that genetic and pharmacologic inhibition of VCP limited cell growth and induced apoptotic death. Addressing the basis for VCP-dependent growth, we first evaluated the contribution of VCP to the DDR and found that loss of VCP resulted in accumulation of DNA double-strand breaks. We next addressed its role in proteostasis and found that loss of VCP caused accumulation of polyubiquitinated proteins. We also found that loss of VCP increased autophagy. Therefore, we reasoned that inhibiting both VCP and autophagy could be an effective combination. Accordingly, we found that VCP inhibition synergized with the autophagy inhibitor chloroquine. We conclude that concurrent targeting of autophagy can enhance the efficacy of VCP inhibitors in KRAS -mutant PDAC., Competing Interests: CONFLICTS OF INTEREST KLB has received research funds from SpringWorks Therapeutics. CJD is an advisory board member for Deciphera Pharmaceuticals, Mirati Therapeutics, Reactive Biosciences, Revolution Medicines and SHY Therapeutics, has received research funding support from Deciphera Pharmaceuticals, Mirati Therapeutics, Reactive Biosciences, Revolution Medicines and SpringWorks Therapeutics, and has consulted for Day One Biotherapeutics, Eli Lilly, Jazz Therapeutics, Ribometrix, Sanofi, and Turning Point Therapeutics. ADC has consulted for Eli Lilly and Mirati Therapeutics. All other authors have no conflicts to declare.

Computer-aided behavior observation is gradually supplanting paper-and-pencil approaches to behavior observation, but there is a dearth of evidence on the relative accuracy of paper-and-pencil versus computer-aided behavior observation formats in the literature. The current study evaluated the accuracy resulting from paper-and-pencil observation and from two computer-aided behavior observation methods: The Observer XT® desktop software and the Big Eye Observer® smartphone application. Twelve postgraduate students without behavior observation experience underwent a behavior observation training protocol. As part of a multi-element design, participants recorded 60 real clinical sessions randomly assigned to one of the three observation methods. All three methods produced high levels of accuracy (paper-and-pencil, .88 ± .01; The Observer XT, .84 ± .01; Big Eye Observer, .84 ± .01). A mixed linear model analysis indicated that paper-and-pencil observation produced marginally superior accuracy values, whereas the accuracy produced by The Observer XT and Big Eye Observer did not differ. The analysis suggests that accuracy of recording was mediated by the number of recordable events in the observation videos. The implications of these findings for research and practice are discussed., (© 2022. The Author(s).)

Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK-MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K-AKT-mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor-based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC., Significance: CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer., (©2022 American Association for Cancer Research.)

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the structure of the lipopolysaccharide O-chain (OPS) of three strains of F. nucleatum HM-994, HM-995, and HM-997, isolated from cancerous tissues: -3-β-D-ManNAc4Lac-4-β-D-Glc6OAc-3-β-D-FucNAc4N- HM-994. -4-α-L-GalNHBuA-3-α-D-QuiNAc4NHBu-3-α-L-Rha-6-α-D-GalN- HM-995. -3-[α-L-GulNAcA-4-]-β-D-Glc-4-β-D-ManNAcAN-3-β-D-FucNAc4N-3- HM-997. where HBu is 3-hydroxybutyryl, ManNAc4Lac is 4-O-(1-carboxyethyl)-2-acetamido-2-deoxy-mannose. All monosaccharides are in the pyranose form. The structures were determined using standard NMR (2D homo- and hetero-nuclear techniques), MS and chemical methods following gtypical LPS isolation and purification methods. In some cases polymeric material was further degraded in order to produce compounds that gave improved NMR spectra that were easier to be fully interpreted. Structure of the OPS from strain HM-994 was identical to the OPS from F. nucleatum strain MJR 7757 B. Structures of the OPS from HM-995 and HM-997 are novel and to our knowledge have not been previously reported and include the often observed 6-deoxy- sugars found in several F. nucleatum strains and butyrate rather than acetate modifications in the HM-995 strain. This structural knowledge adds to the ever increasing variation found in LPS O-antigen structures from F. nucleatum strain from both oral and cancerous origin and suggests that there may be a multitude of different LPS O-antigen structures elaborated by this organism that may present challenges to any serotyping efforts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Previous research showed that canary seed ( Phalaris canariensis L.) peptides (CSP) possess robust in vitro antiobesity properties via inhibition of pancreatic lipase (PL). Nevertheless, no studies have yet explored their antiobesity properties in vivo. Consequently, we investigated the effects of CSP in C57BL/6J mice under a Western diet (WD). Mice were assigned into groups and fed a normal diet (ND) or a WD accompanied by an oral dose of CSP (250 or 500 mg/kg/day), orlistat (40 mg/kg/day), or distilled water. The results showed that consuming CSP can provide metabolic benefits, including preventing weight gain by up to 20%, increasing glucose tolerance, and reducing insulin, leptin, and LDL/VLDL levels in plasma. Conversely, total ghrelin was unaffected by CSP-500, but decreased by CSP-250, and amplified by orlistat. Surprisingly, CSP-250 was more effective in preventing weight gain and promoting satiety than CSP-500. Parallel to this, protein absorption in CSP-500 was decreased, supported by a rise in fecal crude protein (+3.5%). Similarly, fecal fat was increased by orlistat (38%) and was unaffected by CSP-250 (3.0%) and CSP (3.0%), comparatively to WD (2.5%). Despite this, both CSP treatments were equally effective in decreasing hepatic steatosis and avoiding hyperlipidemia. Furthermore, the enzymatic analysis showed that CSP-PL complexes dissociated faster (15 min) than orlistat-PL complexes (41 min). Lastly, CSP did not affect expression of hepatic lipid oxidation genes ACO and PPAR-α, but reduced the expression of the hydrolase gene LPL, and lipogenesis related genes FAS and ACC. Taken together, these results suggest that CSP antiobesity mechanism relies on lipid metabolism retardation to increase fat transit time and subsequently suppress hunger.

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report four new structures of the lipopolysaccharide O-chain (OPS) from five strains of F. nucleatum CTX47T, CC2&#95;6JVN3, CC2&#95;3FMU1, CC2&#95;1JVN3, HM-996, isolated from cancerous tissues. Three of the four structures have a common sequence of hexose-diaminofucose-hexitol-phosphate in the main chain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Missense mutations at the three hotspots in the guanosine triphosphatase (GTPase) RAS-Gly 12 , Gly 13 , and Gln 61 (commonly known as G12, G13, and Q61, respectively)-occur differentially among the three RAS isoforms. Q61 mutations in KRAS are infrequent and differ markedly in occurrence. Q61H is the predominant mutant (at 57%), followed by Q61R/L/K (collectively 40%), and Q61P and Q61E are the rarest (2 and 1%, respectively). Probability analysis suggested that mutational susceptibility to different DNA base changes cannot account for this distribution. Therefore, we investigated whether these frequencies might be explained by differences in the biochemical, structural, and biological properties of KRAS Q61 mutants. Expression of KRAS Q61 mutants in NIH 3T3 fibroblasts and RIE-1 epithelial cells caused various alterations in morphology, growth transformation, effector signaling, and metabolism. The relatively rare KRAS Q61E mutant stimulated actin stress fiber formation, a phenotype distinct from that of KRAS Q61H/R/L/P , which disrupted actin cytoskeletal organization. The crystal structure of KRAS Q61E was unexpectedly similar to that of wild-type KRAS, a potential basis for its weak oncogenicity. KRAS Q61H/L/R -mutant pancreatic ductal adenocarcinoma (PDAC) cell lines exhibited KRAS-dependent growth and, as observed with KRAS G12 -mutant PDAC, were susceptible to concurrent inhibition of ERK-MAPK signaling and of autophagy. Our results uncover phenotypic heterogeneity among KRAS Q61 mutants and support the potential utility of therapeutic strategies that target KRAS Q61 mutant-specific signaling and cellular output.

Pseudomonas aeruginosa produces a variety of cell surface glycans. Previous studies identified a common polysaccharide (PS) antigen often termed A-band PS that was composed of a neutral d-rhamnan trisaccharide repeating unit as a relatively conserved cell surface carbohydrate. However, nuclear magnetic resonance (NMR) spectra and chemical analysis of A-PS preparations showed the presence of several additional components. Here, we report the characterization of the carbohydrate component responsible for these signals. The carbohydrate antigen consists of an immunogenic methylated rhamnan oligosaccharide at the nonreducing end of the A-band PS. Initial studies performed with the isolated antigen permitted the production of conjugates that were used to immunize mice and rabbits and generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains in our collection, and three monoclonal antibodies were generated, one of which was able to recognize and facilitate opsonophagocytic killing of a majority of P. aeruginosa strains. This monoclonal antibody was able to recognize all P. aeruginosa strains in our collection that includes clinical and serotype strains. Synthetic oligosaccharides (mono- to pentasaccharides) representing the terminal 3- O -methyl d-rhamnan were prepared, and the trisaccharide was identified as the antigenic determinant required to effectively mimic the natural antigen recognized by the broadly cross-reactive monoclonal antibody. These data suggest that there is considerable promise in this antigen as a vaccine or therapeutic target.

Pseudomonas aeruginosa was added to the World Health Organization's priority pathogen list for research and development of new antibiotics in 2017. Alongside the development of new antibiotics to fight antimicrobial-resistant P. aeruginosa , vaccines would be an appealing addition to the toolbox health professionals have against this bacteria, which causes life-threatening respiratory infections. Recently, the structure of a novel immunogenic terminal carbohydrate moiety on the cell surface of P. aeruginosa was elucidated, consisting of a 3- O -methyl (1→4)-α-d-rhamnan pentasaccharide. As isolating this oligosaccharide from P. aeruginosa in sufficient amounts for producing a conjugate vaccine is challenging, herein we describe the synthesis of 3- O- methyl d-rhamnose oligosaccharide. We also report the conjugation of the synthetic pentasaccharide to human serum albumin and its resulting immunogenicity.

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the structures of the lipopolysaccharide O-chain (OPS) of two strains of F. nucleatum, SB-106CP and HM-992, both isolated from cancerous tissues. These strains elaborate the same sugar chain, differing only by their N-acylation pattern: -6-α-D-GlcNAc-4-β-D-GlcNHBu3NABuA-3-β-D-QuiNAc4NABuAc- SB-106CP -6-α-D-GlcNAc-4-β-D-GlcNHBu3NABuA-3-β-D-QuiNAc4NAc- HM-992 ABu = (R)-3-amino-butyryl AbuAc = (R)-3-N-acetyl-3-aminobutyryl HBu = (R)-3-hydroxy-butyryl All monosaccharides are in the pyranose form. Previously we published the structure of the OPS from F. nucleatum 12230, a transtracheal isolate, which had similar sugar chain, differing by replacement of GlcNAc with Glc and a different acylation pattern: -6-α-d-Glc-4-β-d-GlcNHBu3NHBuA-3-β-d-QuiNAc4NABu- A mouse monoclonal antibody specific for the 12230 O-antigen did not cross react with the LPS of strains SB-106CP and HM-992 confirming the structural differentiation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Trace metals have been an important ingredient for life throughout Earth's history. Here, we describe the genome-guided cultivation of a member of the elusive archaeal lineage Caldarchaeales (syn. Aigarchaeota), Wolframiiraptor gerlachensis, and its growth dependence on tungsten. A metagenome-assembled genome (MAG) of W. gerlachensis encodes putative tungsten membrane transport systems, as well as pathways for anaerobic oxidation of sugars probably mediated by tungsten-dependent ferredoxin oxidoreductases that are expressed during growth. Catalyzed reporter deposition-fluorescence in-situ hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) show that W. gerlachensis preferentially assimilates xylose. Phylogenetic analyses of 78 high-quality Wolframiiraptoraceae MAGs from terrestrial and marine hydrothermal systems suggest that tungsten-associated enzymes were present in the last common ancestor of extant Wolframiiraptoraceae. Our observations imply a crucial role for tungsten-dependent metabolism in the origin and evolution of this lineage, and hint at a relic metabolic dependence on this trace metal in early anaerobic thermophiles., (© 2022. The Author(s).)

Lyme disease (LD) due to Borrelia burgdorferi is the most prevalent vector-borne disease in the United States. There is a poor understanding of how immunity contributes to bacterial control, pathology, or both during LD. Dogs in an area of endemicity were screened for B. burgdorferi and Anaplasma exposure and stratified according to seropositivity, presence of LD symptoms, and doxycycline treatment. Significantly elevated serum interleukin-21 (IL-21) and increased circulating CD3 + CD94 + lymphocytes with an NK-like CD8 + T cell phenotype were predominant in asymptomatic dogs exposed to B. burgdorferi. Both CD94 + T cells and CD3 - CD94 + lymphocytes, corresponding to NK cells, from symptomatic dogs expressed gamma interferon (IFN-γ) at a 3-fold-higher frequency upon stimulation with B. burgdorferi than the same subset among endemic controls. Surface expression of activating receptor NKp46 was reduced on CD94 + T cells from LD, compared to cells after doxycycline treatment. A higher frequency of NKp46-expressing CD94 + T cells correlated with significantly increased peripheral blood mononuclear cell (PBMC) cytotoxic activity via calcein release assay. PBMCs from dogs with symptomatic LD showed significantly reduced killing ability compared with endemic control PBMCs. An elevated NK-like CD8 + T cell response was associated with protection against development of clinical LD, while excess IFN-γ was associated with clinical disease.

Human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is a common cancer worldwide with an unmet need for more effective, less toxic treatments. Currently, both the disease and the treatment of HNSCC cause significant mortality and morbidity. Targeted therapies hold new promise for patients with HPV-negative status whose tumors harbor oncogenic HRAS mutations. Recent promising clinical results have renewed interest in the development of farnesyltransferase inhibitors (FTIs) as a therapeutic strategy for HRAS-mutant cancers. With the advent of clinical evaluation of the FTI tipifarnib for the treatment of HRAS-mutant HNSCC, we investigated the activity of tipifarnib and inhibitors of HRAS effector signaling in HRAS-mutant HNSCC cell lines. First, we validated that HRAS is a cancer driver in HRAS-mutant HNSCC lines. Second, we showed that treatment with the FTI tipifarnib largely phenocopied HRAS silencing, supporting HRAS as a key target of FTI antitumor activity. Third, we performed reverse-phase protein array analyses to profile FTI treatment-induced changes in global signaling, and conducted CRISPR/Cas9 genetic loss-of-function screens to identify previously unreported genes and pathways that modulate sensitivity to tipifarnib. Fourth, we determined that concurrent inhibition of HRAS effector signaling (ERK, PI3K, mTORC1) increased sensitivity to tipifarnib treatment, in part by overcoming tipifarnib-induced compensatory signaling. We also determined that ERK inhibition could block tipifarnib-induced epithelial-to-mesenchymal transition, providing a potential basis for the effectiveness of this combination. Our results support future investigations of these and other combination treatments for HRAS mutant HNSCC., (©2022 American Association for Cancer Research.)

Ischemic heart disease is the leading cause of death in the United States, Canada, and worldwide. Severe disease is characterized by coronary artery occlusion, loss of blood flow to the myocardium, and necrosis of tissue, with subsequent remodeling of the heart wall, including fibrotic scarring. The current study aims to demonstrate the efficacy of quantitating infarct size via two-dimensional (2-D) echocardiographic akinetic length and four-dimensional (4-D) echocardiographic infarct volume and surface area as in vivo analysis techniques. We further describe and evaluate a new surface area strain analysis technique for estimating myocardial infarction (MI) size after ischemic injury. Experimental MI was induced in mice via left coronary artery ligation. Ejection fraction and infarct size were measured through 2-D and 4-D echocardiography. Infarct size established via histology was compared with ultrasound-based metrics via linear regression analysis. Two-dimensional echocardiographic akinetic length ( r = 0.76, P = 0.03), 4-D echocardiographic infarct volume ( r = 0.85, P = 0.008), and surface area ( r = 0.90, P = 0.002) correlate well with histology. Although both 2-D and 4-D echocardiography were reliable measurement techniques to assess infarct, 4-D analysis is superior in assessing asymmetry of the left ventricle and the infarct. Strain analysis performed on 4-D data also provides additional infarct sizing techniques, which correlate with histology (surface strain: r = 0.94, P < 0.001, transmural thickness: r = 0.76, P = 0.001). Two-dimensional echocardiographic akinetic length, 4-D echocardiography ultrasound, and strain provide effective in vivo methods for measuring fibrotic scarring after MI. NEW & NOTEWORTHY Our study supports that both 2-D and 4-D echocardiographic analysis techniques are reliable in quantifying infarct size though 4-D ultrasound provides a more holistic image of LV function and structure, especially after myocardial infarction. Furthermore, 4-D strain analysis correctly identifies infarct size and regional LV dysfunction after MI. Therefore, these techniques can improve functional insight into the impact of pharmacological interventions on the pathophysiology of cardiac disease.

Rationale: As acquired brain injury rates continue to increase, the ongoing need for efficient and effective treatment within neurobehavioral rehabilitation settings is clear. Some evidence suggests certain treatment components may be very important to incorporate into service delivery models (e.g., multidisciplinary). However, program evaluation literature and the uptake of complementary intervention strategies, like applied behavior analysis (ABA), in existing neurobehavioral settings remains largely unexplored., Primary Objective: The purpose of this project was to: (1) develop and implement a simple, systematic program evaluation informed by best-practices (i.e., research) to assess service delivery models of several neurobehavioral rehabilitation settings, and (2) survey the current use of ABA by participating neurobehavioral agencies., Methodology: The program evaluation tool was applied to the charts of randomly selected past and current clients (referred to as participants). A secondary research assistant independently reviewed 29% of the charts to conduct interobserver agreement, which s. was 80% (range, 53%-100%)., Results: Average program evaluation total percentage score was 33% (range, 4% - 63%), and program evaluation items describing ABA-uptake suggested the incorporation of ABA was low., Discussion: We discuss service model areas of strengths and areas for improvement as specified by tool outcomes, as well as in relation to quality improvement implications.

The aggressive nature of pancreatic ductal adenocarcinoma (PDAC) mandates the development of improved therapies. As KRAS mutations are found in 95% of PDAC and are critical for tumor maintenance, one promising strategy involves exploiting KRAS-dependent metabolic perturbations. The macrometabolic process of autophagy is upregulated in KRAS-mutant PDAC, and PDAC growth is reliant on autophagy. However, inhibition of autophagy as monotherapy using the lysosomal inhibitor hydroxychloroquine (HCQ) has shown limited clinical efficacy. To identify strategies that can improve PDAC sensitivity to HCQ, we applied a CRISPR-Cas9 loss-of-function screen and found that a top sensitizer was the receptor tyrosine kinase (RTK) insulin-like growth factor 1 receptor (IGF1R). Additionally, reverse phase protein array pathway activation mapping profiled the signaling pathways altered by chloroquine (CQ) treatment. Activating phosphorylation of RTKs, including IGF1R, was a common compensatory increase in response to CQ. Inhibition of IGF1R increased autophagic flux and sensitivity to CQ-mediated growth suppression both in vitro and in vivo. Cotargeting both IGF1R and pathways that antagonize autophagy, such as ERK-MAPK axis, was strongly synergistic. IGF1R and ERK inhibition converged on suppression of glycolysis, leading to enhanced dependence on autophagy. Accordingly, concurrent inhibition of IGF1R, ERK, and autophagy induced cytotoxicity in PDAC cell lines and decreased viability in human PDAC organoids. In conclusion, targeting IGF1R together with ERK enhances the effectiveness of autophagy inhibitors in PDAC., Significance: Compensatory upregulation of IGF1R and ERK-MAPK signaling limits the efficacy of autophagy inhibitors chloroquine and hydroxychloroquine, and their concurrent inhibition synergistically increases autophagy dependence and chloroquine sensitivity in pancreatic ductal adenocarcinoma., (©2021 American Association for Cancer Research.)

Psychopharmacological and behavioral interventions are often combined in the treatment of problem behavior in people with intellectual and developmental disability (IDD). However, little is known about the interaction between medication pharmacodynamics and behavior function. A better understanding of these mechanisms could serve as the conceptual foundation for combined interventions. The current analysis is a systematic replication of Valdovinos et al. (2009). We conducted continuous functional analyses within analogue reversal and parametric analyses monitoring the impact of various dosages of primarily antipsychotic medications on problem behavior and its function. Four individuals with IDD and problem behavior who were also receiving psychotropic medications participated. Medication adjustments produced small to negligible decreases in problem behavior, and behavior function remained largely unchanged through the 14 medication adjustments evaluated. The continuous functional analysis helped to identify what could be delayed medication effects on problem behavior. The clinical and methodological implications of this replication are discussed., (© 2021 The Authors. Journal of Applied Behavior Analysis published by Wiley Periodicals LLC on behalf of Society for the Experimental Analysis of Behavior (SEAB).)

Background and Aims: Many epidemiological studies suggest an association between antibiotic exposure and the development of inflammatory bowel disease [IBD]. However, the majority of these studies are observational and still the question remains, "Does the specific antibiotic administration regimen play a role in the development of colitis?" This study aimed to compare the possible effects of continuous and intermittent antibiotic exposure on the development of colitis using a colitis-susceptible IL-10 knockout [IL-10 -/- ] mouse model., Methods: IL-10-/- mice [C57BL/6] were randomly assigned to a non-antibiotic group, continuous antibiotic group and intermittent antibiotic group, and observed for 30 weeks. The antibiotic cocktail was given via the drinking water. The differential response to antibiotics was assessed., Results: Intermittent antibiotic treatment resulted in severe colitis with early disease onset in IL-10-/- mice. Higher unit colon weight and spleen weight were observed in intermittent antibiotic-treated mice but not in the continuous antibiotic group. Moreover, intermittent antibiotic treatment aggravated epithelial damage and colonic inflammation, mucosal barrier dysfunction and colonic allergic sensitization in IL-10-/- mice, whereas continuous antibiotic treatment ameliorated these symptoms. Male IL-10-/- mice with intermittent antibiotic exposure were more susceptible to colonic inflammation and allergic response than females., Conclusions: In summary, intermittent antibiotic exposure accelerated the development of severe colitis more than continuous antibiotic exposure in IL-10-/- male mice. In addition to the colonic damage and impaired barrier function, stimulation of allergic response may play a role in accelerating the development of colitis in genetically susceptible mice., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)