Probing RAS Function Using Monobody and NanoBiT Technologies.

Missense mutations in the RAS family of oncogenes (HRAS, KRAS, and NRAS) are present in approximately 20% of human cancers, making RAS a valuable therapeutic target (Prior et al., Cancer Res 80:2969-2974, 2020). Although decades of research efforts to develop therapeutic inhibitors of RAS were unsuccessful, there has been success in recent years with the entrance of FDA-approved KRASG12C-specific inhibitors to the clinic (Skoulidis et al., N Engl J Med 384:2371-2381, 2021; Jänne et al., N Engl J Med 387:120-131, 2022). Additionally, KRASG12D-specific inhibitors are presently undergoing clinical trials (Wang et al., J Med Chem 65:3123-3133, 2022). The advent of these allele specific inhibitors has disproved the previous notion that RAS is undruggable. Despite these advancements in RAS-targeted therapeutics, several RAS mutants that frequently arise in cancers remain without tractable drugs. Thus, it is critical to further understand the function and biology of RAS in cells and to develop tools to identify novel therapeutic vulnerabilities for development of anti-RAS therapeutics. To do this, we have exploited the use of monobody (Mb) technology to develop specific protein-based inhibitors of selected RAS isoforms and mutants (Spencer-Smith et al., Nat Chem Biol 13:62-68, 2017; Khan et al., Cell Rep 38:110322, 2022; Wallon et al., Proc Natl Acad Sci USA 119:e2204481119, 2022; Khan et al., Small GTPases 13:114-127, 2021; Khan et al., Oncogene 38:2984-2993, 2019). Herein, we describe our combined use of Mbs and NanoLuc Binary Technology (NanoBiT) to analyze RAS protein-protein interactions and to screen for RAS-binding small molecules in live-cell, high-throughput assays.

Calcia magnesia alumino silicate (CMAS) corrosion attack on thermally sprayed thermal barrier coatings: a comprehensive review.

Calcia-Magnesia-Alumino Silicate (CMAS) is a form of molten siliceous residue generated at elevated temperatures within aeroengines. CMAS adheres to the surface of thermal barrier coatings (TBCs) and has the potential to cause significant damage to engine components, resulting in TBC failures. The aviation industry has long recognized CMAS as a substantial threat to aircraft engines, and this threat persists today. A substantial amount of research has been carried out, primarily focusing on gaining a fundamental understanding of the degradation mechanism of traditional TBCs manufactured using air plasma spraying (APS) and electron beam physical vapor deposition (EB-PVD) technologies after CMAS attack. A thorough understanding of why CMAS forms, its role in causing severe spallation, and how to prevent it is of significant concern both academically and industrially. This review article provides a detailed examination of the chemistry of CMAS and the resulting degradation mechanisms that the TBC may encounter throughout the aeroengine service life. This article also explores recent research, incorporating case studies, on the impact of CMAS attack on the resulting chemical and structural modifications of the ceramic topcoats. Current strategies designed to mitigate CMAS infiltration and perspectives for enhanced mitigation are discussed.

Impact of Insurance Type on Access to Pain Management Specialists for the Treatment of Lower Back Pain.

Background Low back pain is known to be one of the leading causes of disability among the young and elderly population. Low back pain can stem from multiple sources, including spinal degeneration, injury, herniated discs, sciatica, and other contributing causes. This symptom significantly influences the quality of life of affected individuals. Its implications include extensive social and economic costs. Economic considerations arise from the fact that not all healthcare facilities accept the insurance plans available to retired individuals under Medicare. This places an additional burden on patients who must bear the financial responsibility for healthcare services not covered by their insurance plan. Florida, renowned as a favored state for retirement, consists of a demographic composition wherein 21% of its residents are aged 65 or older. A significant proportion of this demographic qualifies for Traditional Medicare (TM) and/or Medicare Advantage (MA) plans. Thus, understanding the disparities in healthcare access between Medicare and Medicare Advantage plans is crucial. This study aims to evaluate different Medicare insurances available in the market and their impact on the ease of accessibility to pain management specialists for the treatment of lower back pain in Florida patients. Methods We analyzed the Florida Department of Health database to identify the four counties in Florida with the highest Medicare enrollment rates in 2022: Miami-Dade, Palm Beach, Broward, and Pinellas County. Using the U.S. News and Report directory, 25 Pain Management-trained anesthesiologists were randomly selected from each of the four counties. Each office was contacted four times via telephone by four different team members to assess appointment availability for a fictional 65-year-old grandfather seeking treatment for chronic low back pain. The study examined appointment availability and accepted insurance types, including Cigna (commercial insurance), TM, Humana Gold Plus HMO (Medicare Advantage plan), and Blue Medicare Select PPO (Medicare Advantage plan). Practices without contact information or retired physicians were excluded from the analysis. Time to appointment was measured in business days. Results Of the 100 Pain Management Physicians contacted, 44 fit the inclusion criteria of being non-retired physicians, still practicing in one of the four counties with open offices and valid contact information. Blue Medicare Select PPO was accepted by 47.73%, Humana Gold Plus HMO by 56.82%, TM by 93.18%, and Cigna by 93.18% of the encounters. Blue Medicare select PPO and Humana Gold Plus HMO were accepted at significantly lower rates when compared to Traditional Medicare and Cigna with P values of P < .00001 and P < .000176, respectively. There was no significant difference found in the time to appointment between insurances with P value < 7. Conclusion The study found that patients enrolled in Medicare Advantage plans have significantly decreased access to care when compared to those enrolled in TM or commercial insurance. Further research is needed to elucidate the reasons behind differences in access to care across different insurances, as identified in the study.

TrkA expression directs the anti-neoplastic activity of MLK3 inhibitors in triple-negative breast cancer.

Mixed Lineage Kinase 3 (MLK3) is a viable target for neoplastic diseases; however, it is unclear whether its activators or inhibitors can act as anti-neoplastic agents. We reported that the MLK3 kinase activity was higher in triple-negative (TNBC) than in hormone receptor-positive human breast tumors, where estrogen inhibited MLK3 kinase activity and provided a survival advantage to ER+ breast cancer cells. Herein, we show that in TNBC, the higher MLK3 kinase activity paradoxically promotes cancer cell survival. Knockdown of MLK3 or MLK3 inhibitors, CEP-1347 and URMC-099, attenuated tumorigenesis of TNBC cell line and Patient-Derived (PDX) xenografts. The MLK3 kinase inhibitors decreased both the expression and activation of MLK3, PAK1, and NF-kB protein and caused cell death in TNBC breast xenografts. RNA-seq analysis identified several genes downregulated by MLK3 inhibition, and the NGF/TrkA MAPK pathway was significantly enriched in tumors sensitive to growth inhibition by MLK3 inhibitors. The TNBC cell line unresponsive to kinase inhibitor had substantially lower TrkA, and overexpression of TrkA restored the sensitivity to MLK3 inhibition. These results suggest that the functions of MLK3 in breast cancer cells depend on downstream targets in TNBC tumors expressing TrkA, and MLK3 kinase inhibition may provide a novel targeted therapy.

Evaluation of the safety and efficacy of the fully automated active robotic system in robotic assisted total knee arthroplasty.

Background: Use of semi-active and active robotic system for performing Total Knee Arthroplasty (TKA) is increasing. The novel fully automated active robotic system performs milling of the bone surfaces with a high speed burr. The aim of the current study was to assess the safety and efficacy of the system in robotic assisted TKA (RA-TKA). Materials and methods: A single center clinical trial was conducted following 30 knees undergoing active RA-TKA for 6 months. Inclusion criteria were patients undergoing RA-TKA for end stage arthritis. Patients undergoing conventional TKA and revision TKA were excluded from the study. Sample size was estimated to be 28 patients with α error of 0.05 and ß error of 0.2 with power of study being 80. A pre-defined list of RA-TKA adverse events was employed to study the safety of the system. Efficacy was judged by comparing the planned versus achieved Implant size, alignment and limb alignment on post-operative radiographs. The post-operative clinical evaluation was done by an independent observer who was not part of the operating team. The primary safety and efficacy hypothesis was tested using a one sided Exact binomial test. The p value < 0.05 was considered significant. Results: Pre-defined adverse events did not occur in any of the 30 RA-TKA (statistically significant p value < 0.001). The implant size accuracy was 100% (30 out of 30 knees) for femoral component and 96.67% (29 out of 30 knees) for tibial component (statistically significant, Chi-squared test, p value 0.0105 and 0.0461 respectively). The implant position and limb alignment was accurate in 100% of patients (p value < 0.001). Conclusion: Early experience of the use of fully automated active robotic system in TKA shows that it is safe and also is effective in achieving accurate implant size and implant/limb alignment.

Efficacy of the Pre-operative Three-Dimensional (3D) CT Scan Templating in Predicting Accurate Implant Size and Alignment in Robot Assisted Total Knee Arthroplasty.

Background: Nearly 20% of Total knee Arthroplasty patients remain dissatisfied. This is a major concern in twenty-first century arthroplasty practice. Accurate implant sizing is shown to improve the implant survival, knee balance and patient reported outcome. Aim of the current study is to assess the efficacy of pre-operative three-dimensional (3D) CT scan templating in a robot-assisted TKA in predicting the correct implant sizes and alignment. Materials and methods: Prospectively collected data in a single center from 30 RA-TKAs was assessed. Inclusion criterion was patients with end stage arthritis (both osteoarthritis and rheumatoid arthritis) undergoing primary TKA. Patients undergoing revision TKA and patients not willing to participate in the study were excluded. Preliminary study of ten patients had indicated almost 100% accuracy in determining the implant size and position. Sample size was estimated to be 28 for 90% reduction in implant size and position error with α error of 0.05 and beta error of 0.20 with power of study being 80. Post-operative radiographs were assessed by an independent observer with respect to implant size and position. The accuracy of femoral and tibial component sizing in the study was compared with the historic control with Chi-squared test. The p value < 0.05 was considered significant. Results: The pre-operative CT scan 3D templating accuracy was 100% (30 out of 30 knees) for femoral component and 96.67% (29 out of 30 knees) for tibial component. The implant position and limb alignment was accurate in 100% of patients. The accuracy of femoral component and tibial component sizing is statistically significant (Chi-squared test, p value 0.0105 and 0.0461, respectively). Conclusion: The study results show the effectiveness of pre-operative 3 D CT scan planning in predicting the implant sizes and implant positioning. This may have a potential to improve the implant longevity, clinical outcomes and patient satisfaction.

Improvement of Poly(lactic acid)-Poly(hydroxy butyrate) Blend Properties for Use in Food Packaging: Processing, Structure Relationships.

Poly(lactic acid)-poly(hydroxybutyrate) (PLA-PHB)-based nanocomposite films were prepared with bio-based additives (CNCs and ChNCs) and oligomer lactic acid (OLA) compatibilizer using extrusion and then blown to films at pilot scale. The aim was to identify suitable material formulations and nanocomposite production processes for film production at a larger scale targeting food packaging applications. The film-blowing process for both the PLA-PHB blend and CNC-nanocomposite was unstable and led to non-homogeneous films with wrinkles and creases, while the blowing of the ChNC-nanocomposite was stable and resulted in a smooth and homogeneous film. The optical microscopy of the blown nanocomposite films indicated well-dispersed chitin nanocrystals while the cellulose crystals were agglomerated to micrometer-size particles. The addition of the ChNCs also resulted in the improved mechanical performance of the PLA-PHB blend due to well-dispersed crystals in the nanoscale as well as the interaction between biopolymers and the chitin nanocrystals. The strength increased from 27 MPa to 37 MPa compared to the PLA-PHB blend and showed almost 36 times higher elongation at break resulting in 10 times tougher material. Finally, the nanocomposite film with ChNCs showed improved oxygen barrier performance as well as faster degradation, indicating its potential exploitation for packaging applications.

Impact of intracardiac echocardiography on readmission morbidity and mortality following atrial fibrillation ablation.

INTRODUCTION: The use of intracardiac echocardiography (ICE) is beneficial during the ablation of atrial fibrillation (AF). Evidence is conflicting regarding the clinical impact of using ICE on arrhythmia recurrence and mortality. METHODS: Patients undergoing catheter ablation of AF during 2010-2017 were identified using the International Classification of Diseases-9th and 10th Revision-Clinical Modification (ICD-9-CM and ICD-10-CM) from the Nationwide Readmissions Database. Propensity matching was used to generate a control group. Patient demographics, Charlson comorbidity indexes, time from discharge to readmission, and the reason of readmission were extracted. RESULTS: From 2010 to 2017, 51 129 patients were included in the analysis out of which ICE was used in 8005 (15.7%) patients. The in-hospital mortality at readmission was significantly higher in the patients without ICE use (2.9% vs. 1.7%, p = .02). The length of stay (LOS) at readmission was significantly higher in non-ICE arm (median [interquartile range, IQR]: 3 [2-6] vs. 2 [3-5] days, p < .0001) with similar healthcare-associated cost (HAC) in both the groups (median [IQR]: US$7507.3 [4057.8-15 474.2] vs. 7339.4 [4024.8-15 191.6], p = .43). Freedom from readmission was 12% higher (hazard ratio [HR] [95% confidence interval, CI]: 0.88 [0.83-0.94], p < .0001) with the use of ICE at 90-day follow-up, which was driven by 24% reduction in heart failure (HF) at follow-up (HR [95% CI]: 0.76 [0.60-0.96], p = .02). CONCLUSIONS: ICE use during AF ablation procedure reduces readmissions at 90 days by 12%, driven by a 24% decrease in HF-related admissions. The non-ICE arm showed a significantly higher LOS which offsets marginally higher HAC in the ICE arm.

Mixed lineage kinase 3 and CD70 cooperation sensitize trastuzumab-resistant HER2+ breast cancer by ceramide-loaded nanoparticles.

Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2+ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2+ human breast tumors. Our in vitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2+ breast cancer.

Calcium channel blockers potentiate gemcitabine chemotherapy in pancreatic cancer.

There is currently no effective treatment for pancreatic ductal adenocarcinoma (PDAC). While palliative chemotherapy offers a survival benefit to most patients, nearly all will eventually progress on treatment and long-term survivability remains poor. Given the lack of subsequent line treatment options, in this study, we sought to identify novel strategies to prevent, delay, or overcome resistance to gemcitabine, one of the most widely used medications in PDAC. Using a combination of single-cell RNA sequencing and high-throughput proteomic analysis, we identified a subset of gemcitabine-resistant tumor cells enriched for calcium/calmodulin signaling. Pharmacologic inhibition of calcium-dependent calmodulin activation led to the rapid loss of drug-resistant phenotypes in vitro, which additional single-cell RNA sequencing identified was due to impaired activation of the RAS/ERK signaling pathway. Consistent with these observations, calcium chelation or depletion of calcium in the culture media also impaired ERK activation in gemcitabine-resistant cells, and restored therapeutic responses to gemcitabine in vitro. We observed similar results using calcium channel blockers (CCBs) such as amlodipine, which inhibited prosurvival ERK signaling in vitro and markedly enhanced therapeutic responses to gemcitabine in both orthotopic xenografts and transgenic models of PDAC. Combined, these results offer insight into a potential means of gemcitabine resistance and suggest that select CCBs may provide a clinical benefit to PDAC patients receiving gemcitabine-based chemotherapy.

Influence of Chitin Nanocrystals on the Crystallinity and Mechanical Properties of Poly(hydroxybutyrate) Biopolymer.

This study focuses on the use of pilot-scale produced polyhydroxy butyrate (PHB) biopolymer and chitin nanocrystals (ChNCs) in two different concentrated (1 and 5 wt.%) nanocomposites. The nanocomposites were compounded using a twin-screw extruder and calendered into sheets. The crystallization was studied using polarized optical microscopy and differential scanning calorimetry, the thermal properties were studied using thermogravimetric analysis, the viscosity was studied using a shear rheometer, the mechanical properties were studied using conventional tensile testing, and the morphology of the prepared material was studied using optical microscopy and scanning electron microscopy. The results showed that the addition of ChNCs significantly affected the crystallization of PHB, resulting in slower crystallization, lower overall crystallinity, and smaller crystal size. Furthermore, the addition of ChNCs resulted in increased viscosity in the final formulations. The calendering process resulted in slightly aligned sheets and the nanocomposites with 5 wt.% ChNCs evaluated along the machine direction showed the highest mechanical properties, the strength increased from 24 to 33 MPa, while the transversal direction with lower initial strength at 14 MPa was improved to 21 MPa.

Bioplastics and Carbon-Based Sustainable Materials, Components, and Devices: Toward Green Electronics.

The continuously growing number of short-life electronics equipment inherently results in a massive amount of problematic waste, which poses risks of environmental pollution, endangers human health, and causes socioeconomic problems. Hence, to mitigate these negative impacts, it is our common interest to substitute conventional materials (polymers and metals) used in electronics devices with their environmentally benign renewable counterparts, wherever possible, while considering the aspects of functionality, manufacturability, and cost. To support such an effort, in this study, we explore the use of biodegradable bioplastics, such as polylactic acid (PLA), its blends with polyhydroxybutyrate (PHB) and composites with pyrolyzed lignin (PL), and multiwalled carbon nanotubes (MWCNTs), in conjunction with processes typical in the fabrication of electronics components, including plasma treatment, dip coating, inkjet and screen printing, as well as hot mixing, extrusion, and molding. We show that after a short argon plasma treatment of the surface of hot-blown PLA-PHB blend films, percolating networks of single-walled carbon nanotubes (SWCNTs) having sheet resistance well below 1 kΩ/□ can be deposited by dip coating to make electrode plates of capacitive touch sensors. We also demonstrate that the bioplastic films, as flexible dielectric substrates, are suitable for depositing conductive micropatterns of SWCNTs and Ag (1 kΩ/□ and 1 Ω/□, respectively) by means of inkjet and screen printing, with potential in printed circuit board applications. In addition, we exemplify compounded and molded composites of PLA with PL and MWCNTs as excellent candidates for electromagnetic interference shielding materials in the K-band radio frequencies (18.0-26.5 GHz) with shielding effectiveness of up to 40 and 46 dB, respectively.

MAP4K4 promotes pancreatic tumorigenesis via phosphorylation and activation of mixed lineage kinase 3.

MAP4K4 is a Ste20 member and reported to play important roles in various pathologies, including in cancer. However, the mechanism by which MAP4K4 promotes pancreatic cancer is not fully understood. It is suggested that MAP4K4 might function as a cancer promoter via specific downstream target(s) in an organ-specific manner. Here we identified MLK3 as a direct downstream target of MAP4K4. The MAP4K4 and MLK3 associates with each other, and MAP4K4 phosphorylates MLK3 on Thr738 and increases MLK3 kinase activity and downstream signaling. The phosphorylation of MLK3 by MAP4K4 promotes pancreatic cancer cell proliferation, migration, and colony formation. Moreover, MAP4K4 is overexpressed in human pancreatic tumors and directly correlates with the disease progression. The MAP4K4-specific pharmacological inhibitor, GNE-495, impedes pancreatic cancer cell growth, migration, induces cell death, and arrests cell cycle progression. Additionally, the GNE-495 reduced the tumor burden and extended survival of the KPC mice with pancreatic cancer. The MAP4K4 inhibitor also reduced MAP4K4 protein expression, tumor stroma, and induced cell death in murine pancreatic tumors. These findings collectively suggest that MLK3 phosphorylation by MAP4K4 promotes pancreatic cancer, and therefore therapies targeting MAP4K4 might alleviate the pancreatic cancer tumor burden in patients.

A longitudinal study to evaluate the bleeding pattern of patients on low dose aspirin therapy following dental extraction.

BACKGROUND AND AIMS: Antiplatelet dugs are often interrupted preceding invasive dental extraction because of concern of bleeding complications. The fear of uncontrolled bleeding often prompts medical and dental practitioners to stop aspirin intake for 7 to 10 days before any surgical procedure, which puts the patient at risk from adverse thrombotic events. The aim of the study conducted was to evaluate the bleeding pattern after routine dental extraction among patients on low dose long term aspirin therapy. METHODS: A total of 104 subjects in the age group of 30-65 years, who continued to have aspirin intake during extraction were included in the study. Dental extraction was performed without stopping aspirin therapy under local anesthesia. The post-operative blood loss was quantified by weighing the gauze pre and post operatively and adding total volume of fluid in the suction jar. RESULTS: Of these 104 patients treated, 87% of patients had mild bleeding (<20 ml) and 13% of patients had moderate bleeding (20-30 ml). The total study population showed a mean blood loss of 16.15 ± 3.5 ml. CONCLUSION: Within in the limitations, our study concluded that the routine dental extraction in patients under low dose aspirin therapy did not cause clinically significant post extraction hemorrhage. Aspirin intake can be continued during routine dental extraction as post extraction bleeding encountered will be negligible.

Mixed Lineage Kinase 3 phosphorylates prolyl-isomerase PIN1 and potentiates GLI1 signaling in pancreatic cancer development.

The transcription factor Glioma-Associated Oncogene Homolog 1 (GLI1) is activated by sonic hedgehog (SHH) cascade and is an established driver of pancreatic ductal adenocarcinoma (PDAC). However, therapies targeting upstream hedgehog signaling have shown little to no efficacy in clinical trials. Here, we identify Mixed Lineage Kinase 3 (MLK3) as a druggable regulator of oncogenic GLI1. Earlier, we reported that MLK3 phosphorylated a peptidyl-prolyl isomerase PIN1 on the S138 site, and the PIN1-pS138 translocated to the nucleus. In this report, we identify GLI1 as one of the targets of PIN1-pS138 and demonstrate that PIN1-pS138 is upregulated in human PDAC and strongly associates with the upregulation of GLI1 and MLK3 expression. Moreover, we also identified two new phosphorylation sites on GLI1, T394, and S1089, which are directly phosphorylated by MLK3 to promote GLI1 nuclear translocation, transcriptional activity, and cell proliferation. Additionally, pharmacological inhibition of MLK3 by CEP-1347 promoted apoptosis in PDAC cell lines, reduced tumor burden, extended survival, and reduced GLI1 expression in the Pdx1-Cre x LSL-KRASG12D x LSL-TP53R172H (KPC) mouse model of PDAC. These findings collectively suggest that MLK3 is an important regulator of oncogenic GLI1 and that therapies targeting MLK3 warrant consideration in the management of PDAC patients.

Comparative Evaluation of Classical Inferior Alveolar Nerve Block and Gow-gates Nerve Block for Surgical Removal of Mandibular Third Molar: A Prospective Study.

BACKGROUND: The most commonly used nerve block procedure to anesthetize the mandibular arch is the classical inferior alveolar nerve block (IANB). In 1973, Gow-Gates developed a new procedure known as the Gow Gates nerve block, to achieve anesthesia in the same area with fewer complications. METHODOLOGY: The study comprised 80 patients who reported for the surgical removal of impacted third molar. The patients were randomly assigned into two groups- Group I received Gow-gates nerve block and Group II were administered classical IANB. Positive aspiration, meantime for the onset of anesthesia, mouth opening before and after each block and pain during the surgical procedure were compared. RESULTS: Group 1 yielded positive aspiration in 2.5% of the cases (one patient) and 15% had positive aspiration in Group 2 (six patient). The mean time taken for onset of anesthesia was 6.16 min in Group 1 as compared to 2.78 min in Group 2. While comparing the quality of anesthesia between the blocks, 87.5% of the patients in Group 1 and Group 2 had successful anesthesia equally i.e., 35 of the 40 patients fell into category 1 and 2 of the eight-point category rating scale in both the groups and the remaining five patients (12.5%) in both the groups had unsuccessful anesthesia. CONCLUSION: Both approaches offer quality anesthesia in the posterior mandibular area when meticulously followed. The percentage of unsuccessful anesthesia in the Gow-Gates group could be attributed to the inexperience of the operator. Postoperative comfort and patient satisfaction were greater in the other group.

Study of Patterns of Maxillofacial Injuries: An Institution-based Observational Study.

Maxillofacial fractures are one of the common presentations in an emergency department. They are considered to be one of the significant and dominant conditions that requires treatment as the fractures can result in morbidity, mortality, psychological, functional disability, and facial mutilation. The incidence, patterns, and etiology of maxillofacial fractures vary from one country to another due to the geographical, cultural, social, and economic differences. The present study included 176 patients from January 2019 to September 2020 that aimed to evaluate the pattern of maxillofacial fractures and to learn the etiology for the same.

Berberine Represses ß-Catenin Translation Involving 4E-BPs in Hepatocellular Carcinoma Cells.

Aberrant activation of Wnt/ß-catenin axis occurs in several gastrointestinal malignancies due to inactivating mutations of adenomatous polyposis coli (in colorectal cancer) or activating mutations of ß-catenin itself [in hepatocellular carcinoma (HCC)]. These lead to ß-catenin stabilization, increase in ß-catenin/T-cell factor (TCF)-mediated transcriptional activation, and target gene expression, many of which are involved in tumor progression. While studying pharmaceutical agents that can target ß-catenin in cancer cells, we observed that the plant compound berberine (BBR), a potent activator of AMP-activated protein kinase (AMPK), can reduce ß-catenin expression and downstream signaling in HCC cells in a dose-dependent manner. More in-depth analyses to understand the mechanism revealed that BBR-induced reduction of ß-catenin occurs independently of AMPK activation and does not involve transcriptional or post-translational mechanisms. Pretreatment with protein synthesis inhibitor cycloheximide antagonized BBR-induced ß-catenin reduction, suggesting that BBR affects ß-catenin translation. BBR treatment also antagonized mammalian target of rapamycin (mTOR) activity and was associated with increased recruitment of eukaryotic translation initiation factor 4E-binding protein (4E-BP) 1 in the translational complex, which was revealed by 7-methyl-cap-binding assays, suggesting inhibition of cap-dependent translation. Interestingly, knocking down 4E-BP1 and 4E-BP2 significantly attenuated BBR-induced reduction of ß-catenin levels and expression of its downstream target genes. Moreover, cells with 4E-BP knockdown were resistant to BBR-induced cell death and were resensitized to BBR after pharmacological inhibition of ß-catenin. Our findings indicate that BBR antagonizes ß-catenin pathway by inhibiting ß-catenin translation and mTOR activity and thereby reduces HCC cell survival. These also suggest that BBR could be used for targeting HCCs that express mutated/activated ß-catenin variants that are currently undruggable. SIGNIFICANCE STATEMENT: ß-catenin signaling is aberrantly activated in different gastrointestinal cancers, including hepatocellular carcinoma, which is currently undruggable. In this study we describe a novel mechanism of targeting ß-catenin translation via utilizing a plant compound, berberine. Our findings provide a new avenue of targeting ß-catenin axis in cancer, which can be utilized toward the designing of effective therapeutic strategies to combat ß-catenin-dependent cancers.

Rationalized inhibition of mixed lineage kinase 3 and CD70 enhances life span and antitumor efficacy of CD8+ T cells.

BACKGROUND: The mitogen-activated protein kinases (MAPKs) are important for T cell survival and their effector function. Mixed lineage kinase 3 (MLK3) (MAP3K11) is an upstream regulator of MAP kinases and emerging as a potential candidate for targeted cancer therapy; yet, its role in T cell survival and effector function is not known. METHODS: T cell phenotypes, apoptosis and intracellular cytokine expressions were analyzed by flow cytometry. The apoptosis-associated gene expressions in CD8+CD38+ T cells were measured using RT2 PCR array. In vivo effect of combined blockade of MLK3 and CD70 was analyzed in 4T1 tumor model in immunocompetent mice. The serum level of tumor necrosis factor-α (TNFα) was quantified by enzyme-linked immunosorbent assay. RESULTS: We report that genetic loss or pharmacological inhibition of MLK3 induces CD70-TNFα-TNFRSF1a axis-mediated apoptosis in CD8+ T cells. The genetic loss of MLK3 decreases CD8+ T cell population, whereas CD4+ T cells are partially increased under basal condition. Moreover, the loss of MLK3 induces CD70-mediated apoptosis in CD8+ T cells but not in CD4+ T cells. Among the activated CD8+ T cell phenotypes, CD8+CD38+ T cell population shows more than five fold increase in apoptosis due to loss of MLK3, and the expression of TNFRSF1a is significantly higher in CD8+CD38+ T cells. In addition, we observed that CD70 is an upstream regulator of TNFα-TNFRSF1a axis and necessary for induction of apoptosis in CD8+ T cells. Importantly, blockade of CD70 attenuates apoptosis and enhances effector function of CD8+ T cells from MLK3-/- mice. In immune-competent breast cancer mouse model, pharmacological inhibition of MLK3 along with CD70 increased tumor infiltration of cytotoxic CD8+ T cells, leading to reduction in tumor burden largely via mitochondrial apoptosis. CONCLUSION: Together, these results demonstrate that MLK3 plays an important role in CD8+ T cell survival and effector function and MLK3-CD70 axis could serve as a potential target in cancer.

Mixed lineage kinase 3 inhibition induces T cell activation and cytotoxicity.

Mixed lineage kinase 3 (MLK3), also known as MAP3K11, was initially identified in a megakaryocytic cell line and is an emerging therapeutic target in cancer, yet its role in immune cells is not known. Here, we report that loss or pharmacological inhibition of MLK3 promotes activation and cytotoxicity of T cells. MLK3 is abundantly expressed in T cells, and its loss alters serum chemokines, cytokines, and CD28 protein expression on T cells and its subsets. MLK3 loss or pharmacological inhibition induces activation of T cells in in vitro, ex vivo, and in vivo conditions, irrespective of T cell activating agents. Conversely, overexpression of MLK3 decreases T cell activation. Mechanistically, loss or inhibition of MLK3 down-regulates expression of a prolyl-isomerase, Ppia, which is directly phosphorylated by MLK3 to increase its isomerase activity. Moreover, MLK3 also phosphorylates nuclear factor of activated T cells 1 (NFATc1) and regulates its nuclear translocation via interaction with Ppia, and this regulates T cell effector function. In an immune-competent mouse model of breast cancer, MLK3 inhibitor increases Granzyme B-positive CD8+ T cells and decreases MLK3 and Ppia gene expression in tumor-infiltrating T cells. Likewise, the MLK3 inhibitor in pan T cells, isolated from breast cancer patients, also increases cytotoxic CD8+ T cells. These results collectively demonstrate that MLK3 plays an important role in T cell biology, and targeting MLK3 could serve as a potential therapeutic intervention via increasing T cell cytotoxicity in cancer.