Angiopoietin-like 4 is upregulated by amphiregulin and activates cell proliferation and migration through p38 kinase in head and neck squamous cell carcinoma.

BACKGROUND: Angiopoietin-like 4 is a molecular hallmark that correlates with the growth and metastasis of head and neck squamous cell carcinoma, one of the most prevalent cancers worldwide. However, the molecular mechanisms by which angiopoietin-like 4 promotes head and neck squamous cell carcinoma tumorigenesis are unclear. METHODS: Using well-characterized cell lines of head and neck squamous cell carcinoma development, including human normal oral keratinocytes, dysplastic oral keratinocytes, oral leukoplakia-derived oral keratinocytes, and head and neck squamous cell carcinoma cell lines, HN13, HN6, HN4, HN12, and CAL27, we investigated the signaling pathways upstream and downstream of angiopoietin-like 4-induced head and neck squamous cell carcinoma tumorigenesis. RESULTS: We found that both epidermal growth factor receptor ligands, epithelial growth factor, and amphiregulin led to angiopoietin-like 4 upregulation in normal oral keratinocytes and dysplastic oral keratinocytes and cooperated with the activation of hypoxia-inducible factor-1 in this effect. Interestingly, amphiregulin and angiopoietin-like 4 were increased in dysplastic oral keratinocytes and head and neck squamous cell carcinoma cell lines, and amphiregulin-induced activation of cell proliferation was dependent on angiopoietin-like 4. Although both p38 mitogen-activated protein kinases (p38 MAPK) and protein kinase B (AKT) were activated by angiopoietin-like 4, only pharmacological inhibition of p38 MAPK was sufficient to prevent head and neck squamous cell carcinoma cell proliferation and migration. We further observed that angiopoietin-like 4 promoted the secretion of interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin-1 alpha (IL-1α), vascular endothelial growth factor, platelet-derived growth factor (PDGF), and tumour necrosis factor alpha (TNF-α), cytokines and chemokines previously implicated in head and neck squamous cell carcinoma pathogenesis. CONCLUSION: Our results demonstrate that angiopoietin-like 4 is a downstream effector of amphiregulin and promotes head and neck squamous cell carcinoma development both through direct activation of p38 kinase as well as paracrine mechanisms.

Feasibility of using bacteriophage therapy to treat Staphylococcal aureus fracture-related infections.

OBJECTIVE: Staphylococcus aureus fracture-related infections (FRIs) are associated with significant morbidity in part because conventional antibiotic therapies have limited ability to eradicate S. aureus in sessile states. Therefore, the objective of this study was to assess the feasibility of using Staphylococcal bacteriophages for FRI by testing the activity of a library of Staphylococcal bacteriophage therapeutics against historically preserved S. aureus FRI clinical isolates. METHODS: Current Procedural Terminology codes were used to identify patients with FRI from January 1, 2021 to December 31, 2021. Preserved S. aureus FRI isolates from the cases were then tested against a library of 51 Staphylococcal bacteriophages from an American company. This was conducted by assessing the ability of bacteriophages to reduce bacterial growth over time. Growth inhibition greater than 16 h was considered adequate for this study. RESULTS: All of the S. aureus preserved clinical isolates had at least one bacteriophage with robust lytic activity and six bacteriophages (11.8 %) had robust lytic activity to seven or more of the clinical isolates. However, 41 of the bacteriophages (80.4 %) had activity to less than three of the clinical isolates and no bacteriophage had activity to all the clinical isolates. CONCLUSION: Our findings show that Staphylococcal bacteriophage therapeutics are readily available for S. aureus FRI clinical isolates. However, when correlated with the current barriers to using bacteriophages to treat FRI, designated Staphylococcal bacteriophage cocktails with broad spectrum activity should be created.

Hormonal control of promoter activities of Cannabis sativa prenyltransferase 1 and 4 and salicylic acid mediated regulation of cannabinoid biosynthesis.

Cannabis sativa aromatic prenyltransferase 4 (CsPT4) and 1 (CsPT1) have been shown to catalyze cannabigerolic acid (CBGA) biosynthesis, a step that rate-limits the cannabinoid biosynthetic pathway; both genes are highly expressed in flowers. CsPT4 and CsPT1 promoter driven ß-glucuronidase (GUS) activities were detected in leaves of cannabis seedlings, and strong CsPT4 promoter activities were associated with glandular trichomes. Hormonal regulation of cannabinoid biosynthetic genes is poorly understood. An in silico analysis of the promoters identified putative hormone responsive elements. Our work examines hormone-responsive elements in the promoters of CsPT4 and CsPT1 in the context of physiological responses of the pathway to the hormone in planta. Dual luciferase assays confirmed the regulation of promoter activities by the hormones. Further studies with salicylic acid (SA) demonstrated that SA pretreatment increased the expression of genes located downstream of the cannabinoid biosynthetic pathway. The results from all aspects of this study demonstrated an interaction between certain hormones and cannabinoid synthesis. The work provides information relevant to plant biology, as we present evidence demonstrating correlations between molecular mechanisms that regulate gene expression and influence plant chemotypes.

Transport-Associated Vibrational Stress Triggers Drug-Reversible Apoptosis and Cardiac Allograft Failure in Mice.

Increasingly complex and long-range donor organ allocation routes coupled with implementation of unmanned aerial vehicles (UAVs) have prompted investigations of the conditions affecting organs once packaged for shipment. Our group has previously demonstrated that different modes of organ transport exert unique environmental stressors, in particular vibration. Using a mouse heart transplant model, we demonstrated that vibrational forces exert tangible, cellular effects in the form of cardiomyocyte apoptosis and cytoskeletal derangement. Functionally, these changes translated into accelerated allograft loss. Notably, administration of an apoptosis inhibitor, Z-VAD-FMK, helped to ameliorate the detrimental cellular and functional effects of mechanical vibration in a dose-dependent manner. These findings constitute one of the first reports of the negative impact of transit environment on transplant outcomes, a contributing mechanism underpinning this effect, and a potential agent to prophylax against this process. Given current limitations in measuring donor organ transit environments in situ, further study is required to better characterize the impact of transport environment and to potentially improve the care of donor organs during shipment. Clinical and Translational Impact Statement: We show that apoptosis inhibitor, Z-VAD-FMK, ameliorated transport-related vibrational stress in murine heart transplants, which presents a potential therapeutic or preservation solution additive for future use in transporting donor organs.

Agreement between cardiac output estimation by multi-beat analysis of arterial blood pressure waveforms and continuous thermodilution in post cardiac surgery intensive care unit patients.

We sought to assess agreement of cardiac output estimation between continuous pulmonary artery catheter (PAC) guided thermodilution (CO-CTD) and a novel pulse wave analysis (PWA) method that performs an analysis of multiple beats of the arterial blood pressure waveform (CO-MBA) in post-operative cardiac surgery patients. PAC obtained CO-CTD measurements were compared with CO-MBA measurements from the Argos monitor (Retia Medical; Valhalla, NY, USA), in prospectively enrolled adult cardiac surgical intensive care unit patients. Agreement was assessed via Bland-Altman analysis. Subgroup analysis was performed on data segments identified as arrhythmia, or with low CO (less than 5 L/min). 927 hours of monitoring data from 79 patients was analyzed, of which 26 had arrhythmia. Mean CO-CTD was 5.29 ± 1.14 L/min (bias ± precision), whereas mean CO-MBA was 5.36 ± 1.33 L/min, (4.95 ± 0.80 L/min and 5.04 ± 1.07 L/min in the arrhythmia subgroup). Mean of differences was 0.04 ± 1.04 L/min with an error of 38.2%. In the arrhythmia subgroup, mean of differences was 0.14 ± 0.90 L/min with an error of 35.4%. In the low CO subgroup, mean of differences was 0.26 ± 0.89 L/min with an error of 40.4%. In adult patients after cardiac surgery, including those with low cardiac output and arrhythmia CO-MBA is not interchangeable with the continuous thermodilution method via a PAC, when using a 30% error threshold.

Delineating genetic regulation of cannabinoid biosynthesis during female flower development in Cannabis sativa.

Cannabinoids are predominantly produced in the glandular trichomes on cannabis female flowers. There is little known on how cannabinoid biosynthesis is regulated during female flower development. We aim to understand the rate-limiting step(s) in the cannabinoid biosynthetic pathway. We investigated the transcript levels of cannabinoid biosynthetic genes together with cannabinoid contents during 7 weeks of female flower development. We demonstrated that the enzymatic steps for producing cannabigerol (CBG), which involve genes GPPS, PT, TKS, and OAC, could rate limit cannabinoid biosynthesis. Our findings further suggest that upregulation of cannabinoid synthases, CBDAS and THCAS in a commercial hemp and medical marijuana variety, respectively, is not critical for cannabinoid biosynthesis. The cannabinoid biosynthetic genes are generally upregulated during flower maturation; increased expression occurs coincident with glandular trichome development and cannabinoid production in the maturing flower. The results also suggest that different cannabis varieties may experience discrete transcriptional regulation of cannabinoid biosynthetic genes. In addition, we showed that methyl jasmonate (MeJA) can potentially increase cannabinoid production. We propose that biweekly applications of 100 µM MeJA starting from flower initiation would be efficacious for promoting cannabinoid biosynthesis. Our findings provide important genetic information for cannabis breeding to generate new varieties with favorable traits.

Effects of Harpin and Flg22 on Growth Enhancement and Pathogen Defense in Cannabis sativa Seedlings.

Pathogen-associated molecular patterns, PAMPs, are a diverse group of molecules associated with pathogenic microbes and are known to activate immune response and in some cases enhance growth in plants. Two PAMPs, harpin and flg22, have shown these affects in various plant species. PAMPs are known to activate basal immunity, the ethylene signaling pathway, alter gene expression and change plant composition. Pretreatment with harpin enhanced hemp seedling resistance to Pythium aphanidermatum, while flg22 failed to induce the defense mechanism towards P. aphanidermatum. In the absence of the pathogen, both harpin and flg22 enhanced seedling growth when compared to the water control. Ethylene is a hormone involved in both plant defense signaling and growth. Both harpin and flg22 pretreatment induced certain ethylene responsive genes but not all the genes examined, indicating that harpin and flg22 act differently in ethylene and potentially defense signaling. In addition, both harpin and flg22 induced CsFRK1 and CsPR1, two marker genes for plant innate immunity. Both PAMPs can enhance growth but likely induce different defense signaling pathways.

Prevalence of human papillomavirus types in invasive vulvar cancers and vulvar intraepithelial neoplasia 3 in the United States before vaccine introduction.

OBJECTIVE: The study aimed to determine the baseline prevalence of human papillomavirus (HPV) types in invasive vulvar cancer (IVC) and vulvar intraepithelial neoplasia 3 (VIN 3) cases using data from 7 US cancer registries. MATERIALS AND METHODS: Registries identified eligible cases diagnosed in 1994 to 2005 and requested pathology laboratories to prepare 1 representative block for HPV testing on those selected. Hematoxylin-eosin-stained sections preceding and following those used for extraction were reviewed to confirm representation. Human papillomavirus was detected using L1 consensus polymerase chain reaction (PCR) with PGMY9/11 primers and type-specific hybridization, with retesting of samples with negative and inadequate results with SPF10 primers. For IVC, the confirmatory hematoxylin-eosin slides were re-evaluated to determine histological type. Descriptive analyses were performed to examine distributions of HPV by histology and other factors. RESULTS: Human papillomavirus was detected in 121/176 (68.8%) cases of IVC and 66/68 (97.1%) cases of VIN 3 (p < .0001). Patients with IVC and VIN 3 differed by median age (70 vs 55 y, p = .003). Human papillomavirus 16 was present in 48.6% of IVC cases and 80.9% of VIN 3 cases; other high-risk HPV was present in 19.2% of IVC cases and 13.2% of VIN 3 cases. Prevalence of HPV differed by squamous cell carcinoma histological subtype (p < .0001) as follows: keratinizing, 49.1% (n = 55); nonkeratinizing, 85.7% (n = 14), basaloid, 92.3% (n = 14), warty 78.2% (n = 55), and mixed warty/basaloid, 100% (n = 7). CONCLUSIONS: Nearly all cases of VIN 3 and two thirds of IVC cases were positive for high-risk HPV. Prevalence of HPV ranged from 49.1% to 100% across squamous cell carcinoma histological subtypes. Given the high prevalence of HPV in IVC and VIN 3 cases, prophylactic vaccines have the potential to decrease the incidence of vulvar neoplasia.

Pain symptomatology and pain medication use in civilian PTSD.

The comorbidity of pain syndromes and trauma-related syndromes has been shown to be high. However, there have been limited data, especially in civilian medical populations, on the role of trauma-related disorders such as posttraumatic stress disorder (PTSD) on chronic pain and pain medication use. We analyzed 647 general hospital patients in primary care and obstetrics and gynecological waiting rooms for the experience of trauma and PTSD-related stress disorders. PTSD symptoms were found to be significantly positively correlated with pain ratings (r=.282, P<0.001) and pain-related functional impairment (r=0.303, P<0.001). Those with a current PTSD diagnosis had significantly higher subjective pain and pain-related impairment ratings than those with no PTSD. Furthermore, those with a current diagnosis of PTSD were significantly more likely to have used opioid analgesics for pain control compared to those without a diagnosis of PTSD (χ(2)=8.98, P=0.011). When analyzing the separate PTSD symptom subclusters (re-experiencing, avoidance, and hyperarousal), all symptom clusters were significantly related to pain and pain-related impairment ratings, but only the avoidance cluster was significantly related to prior opioid pain medication use. We conclude that PTSD and trauma-related disorders are common in impoverished medical populations and that their presence should be examined in patients with pain syndromes. Furthermore, these data suggest that PTSD and pain may share a vulnerability pathway, including the endogenous opioid neurotransmission systems.

Fear extinction in traumatized civilians with posttraumatic stress disorder: relation to symptom severity.

BACKGROUND: The symptoms of posttraumatic stress disorder (PTSD) can be explained, at least in part, as an inability to inhibit learned fear during conditions of safety. Our group has shown that fear inhibition is impaired in both combat and civilian PTSD populations. On the basis of our earlier findings, we employed an established fear extinction paradigm to further explore fear dysregulation in a civilian traumatized population. METHODS: Fear-potentiated startle (FPS) was examined in 127 trauma-exposed individuals with and without PTSD. We used a protocol in which conditioned fear was first acquired through the presentation of one colored shape (reinforced conditioned stimulus, [CS+]) that was paired with an aversive air blast to the larynx (unconditioned stimulus) and a different colored shape that was not paired to the air blast (nonreinforced condition stimulus). Fear was extinguished 10 min later through repeated presentations of the CSs without reinforcement. RESULTS: Both groups demonstrated successful fear conditioning on the basis of startle and unconditioned stimulus-expectancy ratings; however, participants with PTSD displayed greater FPS responses to the CS+ and nonreinforced conditioned stimulus compared with the group without PTSD. During fear extinction, the PTSD group showed elevated FPS responses to the previously reinforced CS+ during the early and middle stages of extinction. During the acquisition and extinction phases, PTSD participants with higher levels of reexperiencing symptoms exhibited greater potentiated startle responses to the CS+ compared with PTSD participants with lower reexperiencing symptoms. CONCLUSIONS: These results suggest that PTSD is associated with enhanced fear learning and a greater "fear load" to extinguish after conditioned fear is acquired.