Metabolic perturbations associated with hIAPP-induced insulin resistance in skeletal muscles: Implications to the development of type 2 diabetes.

The human islet amyloid polypeptide (hIAPP) tends to misfold and self-assemble to form amyloid fibrils, which has been associated with the loss of function and viability of pancreatic ß-cells in type 2 diabetes mellitus (T2DM). The role of hIAPP in the development of insulin resistance (a hallmark of T2DM) in skeletal muscles - the major sites for glucose utilization - needs further investigation. Even though, insulin-resistant conditions have been known to stimulate hIAPP aggregation, the events that lead to the development of insulin resistance due to hIAPP aggregation in skeletal muscles remain unidentified. Here, we have attempted to identify metabolic perturbations in L6 myotubes that were exposed to increasing concentrations of recombinant hIAPP for different time durations. It was observed that hIAPP exposure was associated with increased mitochondrial and cellular ROS levels, loss in mitochondrial membrane potential and viability of the myotubes. Metabolomic investigations of hIAPP-treated myotubes revealed significant perturbations in o-phosphocholine, sn-glycero-3-phosphocholine and dimethylamine levels (p < 0.05). Therefore, we anticipate that defects in glycerophospholipid metabolism and the associated oxidative stress and membrane damage may play key roles in the development of insulin resistance due to protein misfolding in skeletal muscles. In summary, the perturbed metabolites and their pathways have not only the potential to be used as early biomarkers to predict the onset of insulin resistance and T2DM but also as therapeutic targets for the effective management of the same.

Correlation of pre-operative and post-operative retinal nerve fibre layer thickness with visual outcome following decompression of pituitary macroadenoma.

OBJECTIVE: To (i) correlate preoperative retinal nerve fibre layer (RNFL) thickness with visual parameters in patients with pituitary macroadenomas. (ii) study the predictive role of preoperative RNFL in visual outcome following surgery for pituitary macroadenomas (iii) correlate change in postoperative RNFL thickness (RNFLT) with visual outcome. METHODS: Preoperative and post-operative RNFL thickness of thirty-three consecutive patients operated for pituitary macro adenoma between June 2022 and May 2023 were measured using Optical Coherence Tomography (OCT) and compared with standard visual examination findings and Magnetic Resonance Imaging (MRI) measurements. RESULTS: A total of 66 eyes of 33 patients who underwent surgical excision of pituitary macro adenoma between June 2022 and May 2023 were studied. The mean age in years of the study group was 44.36 ± 13.77 and both sexes were equally represented (Male: Female = 16:17). RNFL thinning predominantly involved the temporal (51.21+/-15.19 µm) followed by nasal quadrants (62.67+/- 17.03 µm) and correlated well with the visual field (VF) deficit (p <0.001). Patients with severe disc pallor had extremely thin RNFL (less than 67 +/- 8.68 µm). Patients with moderate to severe visual acuity (VA) deficits had significantly thinner RNFLs (65.08±7.09) compared to patients with normal to mild impairment in vision. (83.185±1.2) (p<0.05). RNFL values were significantly thinner for patients with Wilson Grade C, D and E tumours (66.13 ±12.19 µm) compared to those in Grade A and B (77.67±22.12 µm). The mean preop RNFL of patients who showed post-operative improvement in vision was 87.025± 15.02 µm, of patients in whom vision remained static was 74.58 ±18.31 µm. The mean VA (Decimal) increased from a minimum of 0.60 at the pre-operative timepoint to a maximum of 0.68 at the post-operative timepoint. (Wilcoxon Test: V = 42.5, p = <0.001). The mean RNFLT (µm) increased from 77.14 µm at the pre-operative timepoint to 83.77 µm at the post-operative timepoint. (Wilcoxon Test: V = 218.0, p = <0.001). The mean change of RNFL in patients in whom vision improved was 3.6 µm and the mean change of RNFL in patients in whom vision remained static was 9.51 µm. Absence of postoperative visual improvement was noted despite postoperative RNFL thickness improvement in eyes which showed significant preoperative thinning of the nasal (<65 µm) and temporal (<52µm) quadrants. CONCLUSION: RNFL thinning corelates directly with visual acuity, visual field, and optic disc pallor. Patients with pituitary adenoma have preferential thinning of temporal and nasal quadrants. Visual outcome is better in patients with preserved RNFLT of values more than 82 +/- 5 µm. Reversal of RNFL thinning postoperatively need not necessarily correlate with visual improvement especially in patients who showed significant preoperative thinning of nasal and temporal quadrants.

A Noncanonical CD56dimCD16dim/- NK Cell Subset Indicative of Prior Cytotoxic Activity Is Elevated in Patients with Autoantibody-Mediated Neurologic Diseases.

Neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein Ab disease, and autoimmune myasthenia gravis (MG) are autoantibody-mediated neurologic conditions where autoantibodies can induce Ab-dependent cellular cytotoxicity (ADCC), a NK cell-mediated effector function. However, whether ADCC is a pathogenic mechanism in patients with these conditions has not been confirmed. We sought to characterize circulatory NK cells using functional assays, phenotyping, and transcriptomics to elucidate their role in pathology. NK cells from NMOSD patients and MG patients with elevated disease burden exhibited reduced ADCC and CD56dimCD16hi NK cells, along with an elevated frequency of CD56dimCD16dim/- NK cells. We determined that ADCC induces a similar phenotypic shift in vitro. Bulk RNA sequencing distinguished the CD56dimCD16dim/- population from the canonical CD56dimCD16hi cytotoxic and CD56hiCD16- immunomodulatory subsets, as well as CD56hiCD16+ NK cells. Multiparameter immunophenotyping of NK cell markers, functional proteins, and receptors similarly showed that the CD56dimCD16dim/- subset exhibits a unique profile while still maintaining expression of characteristic NK markers CD56, CD94, and NKp44. Notably, expression of perforin and granzyme is reduced in comparison with CD56dimCD16hi NK cells. Moreover, they exhibit elevated trogocytosis capability, HLA-DR expression, and many chemokine receptors, including CCR7. In contrast with NMOSD and MG, myelin oligodendrocyte glycoprotein Ab disease NK cells did not exhibit functional, phenotypic, or transcriptomic perturbations. In summary, CD56dimCD16dim/- NK cells are a distinct peripheral blood immune cell population in humans elevated upon prior cytotoxic activity by the CD56dimCD16hi NK cell subset. The elevation of this subset in NMOSD and MG patients suggests prior ADCC activity.

Practical Clinical Consensus Guidelines for the Management of Cancer Associated Anemia in Low- and Middle-Income Countries.

Purvish M. ParikhCancer-associated anemia (CAA) remains a major unmet need that compromises overall survival (OS) and quality of life (QoL). Currently, available guidelines do not take into consideration the unique challenges in low- and middle-income countries (LMIC). Our CAA patients have to battle preexisting impaired nutritional status, depleted body iron stores, financial limitations, and difficulty in having easily accessible affordable healthcare. Hence, we fulfilled the need of guidelines for LMIC. A group of subject experts were put together, given background literature, met in a face-to-face discussion, voted using Delphi process, and finally agreed on the contents of this guideline document. As many as 50% of cancer patients will have significant anemia (hemoglobin < 10 g/dL) at initial diagnosis. It is most commonly seen with gastrointestinal malignancies, head and neck cancers, and acute leukemias. The hemoglobin falls further after initiation of cancer directed therapy, due to chemotherapy itself or heightened nutritional deficiency. Its evaluation should include tests for complete blood count, red blood cell morphology, reticulocyte count, Coombs test, and levels of vitamin B12 and folic acid. Iron status should be monitored using test to measure serum iron, total iron binding capacity, transferring saturation, and serum ferritin levels. A minimum of 50% of cancer patients with anemia require iron supplements. The preferred mode of therapy is with intravenous (IV) iron using ferric carboxymaltose (FCM). Most patients respond satisfactorily to single dose of 1000 mg. It is also safe and does not require use of a test dose. Significant anemia is found in at least half of all cancer patients in India, South Asian Association for Regional Cooperation region, and other LMIC countries. Its awareness among healthcare professionals will prevent it from remaining undiagnosed (in up to 70% of all cancer patients) and adversely affecting OS and QoL. The benefits of treating them with IV iron therapy are quick replenishment of iron stores, hemoglobin returning to normal, better QoL, and avoiding risk of infections/reactions with blood transfusions. Many publications have proven the value of single-dose FCM in such clinical situations. CAA has been proven to be an independent prognostic factor that adversely affects both QoL and OS in cancer patients. Use of FCM as single IV dose of 1000 mg is safe and effective in the majority of patients with CAA.

Remission of severe myasthenia gravis after autologous stem cell transplantation.

OBJECTIVE: Myasthenia gravis (MG) is an autoantibody-mediated neuromuscular junction disorder involving the acetylcholine receptors on the motor endplate. The safety and response to high-dose chemotherapy (HDIT) and autologous hematopoietic cell transplantation (HCT) were assessed in a patient with severe refractory MG. METHODS: As part of a pilot study of HDIT/HCT for patients with treatment-resistant autoimmune neurological disorders, a patient with severe refractory MG underwent treatment. After mobilization of hematopoietic stem cells with rituximab, prednisone, and G-CSF, the patient had HDIT consisting of carmustine, etoposide, cytarabine, melphalan, and rabbit antithymocyte globulin, followed by autologous HCT. The effect of treatment on the autoantibody to the acetylcholine receptor (AChR) was assessed. RESULTS: The patient had been diagnosed with AChR antibody-positive MG 14 years before HDIT/HCT and had failed thymectomy, therapeutic plasma exchange, and multiple immunomodulatory agents. The Myasthenia Gravis Foundation of America (MGFA) clinical classification was IVb before HDIT/HCT. She tolerated HDIT/HCT well and started to improve clinically within days of treatment. At both 1 and 2 years after HDIT/HCT, patients remained symptom-free. After HDIT/HCT, AChR-binding autoantibodies persisted, and the relative frequency of immune cell subtypes shifted. INTERPRETATION: HDIT/HCT induced a complete response of disease activity in a patient with severe refractory MG. This response may suggest that a cell-mediated etiology may be a significant contributing factor in refractory MG cases. A phase 2 clinical trial is warranted to establish if HDIT/HCT can be an effective therapy for severe refractory MG and to gain a further understanding of disease pathogenesis.

Discovery and Optimization of a STING Agonist Platform for Application in Antibody Drug Conjugates.

While STING agonists have proven to be effective preclinically as anti-tumor agents, these promising results have yet to be translated in the clinic. A STING agonist antibody-drug conjugate (ADC) could overcome current limitations by improving tumor accessibility, allowing for systemic administration as well as tumor-localized activation of STING for greater anti-tumor activity and better tolerability. In line with this effort, a STING agonist ADC platform was identified through systematic optimization of the payload, linker, and scaffold based on multiple factors including potency and specificity in both in vitro and in vivo evaluations. The platform employs a potent non-cyclic dinucleotide STING agonist, a cleavable ester-based linker, and a hydrophilic PEG8-bisglucamine scaffold. A tumor-targeted ADC built with the resulting STING agonist platform induced robust and durable anti-tumor activity and demonstrated high stability and favorable pharmacokinetics in nonclinical species.

Study of foetomaternal outcomes in COVID-19 afflicted parturients who underwent operative delivery - A retrospective analysis.

Background and Aims: Parturients with coronavirus disease (COVID)-19 are increasingly presenting for operative delivery. The aim of this study was to outline the foetomaternal outcome in COVID-19-afflicted pregnant women who underwent lower segment caeserean section (LSCS). Methods: Data of all COVID-19 positive pregnant females who underwent caesarean section surgery between 1 April and 30 June 2021 was collected. Clinical parameters, including oxygen requirement, laboratory investigations, treatment measures, complications, length of hospital and intensive care unit/neonatal intensive care unit stay, and outcome of parturients and neonates, were collected and analysed. All patients were followed up either during their visits to the obstetric outpatient department or by making phone calls between 1 and 2 months of discharge. Statistical Package for the Social Sciences statistical software 16.0 was used for analysis. Independent group t-test or Mann-Whitney test was used for mean of continuous data. Chi-square test or Fisher's test was used for proportion of categorical data. A P value of <0.05 was considered significant. Results: A total of 71 parturients delivered by caesarean section. 36.51% had mild COVID-19, and 87.5% had moderate COVID-19 at admission. One each with mild and moderate disease expired. The median (interquartile range) length of hospital stay was 7 (5-5.9) days for those with mild disease, and it was significantly longer for those with the moderate disease at 14 (9.5-17.5) days. Our study found that after a mean of 41.72 days of follow-up, of the 69 surviving mothers, 17 complained of fatigue, five complained of myalgia and one needed intermittent supplemental oxygen. Out of 74 babies born, seven died, which is 94.6 per 1000 live births. Conclusion: COVID-19 parturients delivered by LSCS stand a higher risk of maternal and neonatal mortality and adverse effects, including more hospital stay and increased mortality.

MOGAD patient autoantibodies induce complement, phagocytosis, and cellular cytotoxicity.

Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an inflammatory demyelinating CNS condition characterized by the presence of MOG autoantibodies. We sought to investigate whether human MOG autoantibodies are capable of mediating damage to MOG-expressing cells through multiple mechanisms. We developed high-throughput assays to measure complement activity (CA), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC) of live MOG-expressing cells. MOGAD patient sera effectively mediate all of these effector functions. Our collective analyses reveal that (a) cytotoxicity is not incumbent on MOG autoantibody quantity alone; (b) engagement of effector functions by MOGAD patient serum is bimodal, with some sera exhibiting cytotoxic capacity while others did not; (c) the magnitude of CDC and ADCP is elevated closer to relapse, while MOG-IgG binding is not; and (d) all IgG subclasses can damage MOG-expressing cells. Histopathology from a representative MOGAD case revealed congruence between lesion histology and serum CDC and ADCP, and we identified NK cells, mediators of ADCC, in the cerebrospinal fluid of relapsing patients with MOGAD. Thus, MOGAD-derived autoantibodies are cytotoxic to MOG-expressing cells through multiple mechanisms, and assays quantifying CDC and ADCP may prove to be effective tools for predicting risk of future relapses.

Development and characterization of a patient­derived orthotopic xenograft of therapy­resistant breast cancer.

Numerous years of cell line­based studies have enhanced the current understanding of cancer and its treatment. However, limited success has been achieved in treating hormone receptor­positive, HER2­negative metastatic breast cancers that are refractory to treatment. The majority of cancer cell lines are unsuitable for use as pre­clinical models that mimic this critical and often fatal clinical type, since they are derived from treatment­naive or non­metastatic breast cancer cases. The aim of the present study was to develop and characterize patient­derived orthotopic xenografts (PDOXs) from patients with endocrine hormone receptor­positive, HER2­negative metastatic breast cancer who had relapsed on therapy. A patient who progressed on endocrine hormone therapy provided her tumor via a biobank. This tumor was implanted in mice. It was then serially passaged by implanting PDOX tumor fragments into another set of mice to develop further generations of PDOXs. These tissues were characterized using various histological and biochemical techniques. Histological, immunofluorescence and western blot analyses indicated that the PDOX tumors retained a similar morphology, histology and subtype­specific molecular features to that of the patient's tumor. The present study successfully established PDOXs of hormone­resistant breast cancer and characterized them in comparison with those derived from the original breast cancer tissue of the patient. The data highlight the reliability and usefulness of PDOX models for studies of biomarker discovery and preclinical drug screening. The present study was registered with the clinical trial registry of India (CTRI; registration no. CTRI/2017/11/010553; registered on 17/11/2017).

Groundwater characterization and non-carcinogenic and carcinogenic health risk assessment of nitrate exposure in the Mahanadi River Basin of India.

Agriculture is the mainstay of India's economy and chemical fertilizers have been extensively used to meet increasing demands. Anthropogenic interventions at the soil surface, especially the application of nitrogenous fertilizers in agricultural fields, provide essential nutrients but become major pollutant sources in terrestrial ecosystems and aquatic environments. Groundwater samples from phreatic aquifers of the Mahanadi River Basin, Chhattisgarh, India, showed that the Ca2+-Mg2+-HCO3- freshwater type dominates, followed by the Ca2+-Mg2+-Cl- and Na+-HCO3- types. Increasing trends in the ionic ratios of (NO3-+Cl-)/HCO3- over TDS and of NO3-/Cl- over Cl- indicated the significant impact of anthropogenic pollution on groundwater contamination. Deterministic and probabilistic approaches were used to assess the non-carcinogenic and carcinogenic health risks of nitrate to children and adults. Both approaches produced the same results and indicated children were more prone to non-carcinogenic health risk than adults. An excess gastric cancer risk (ER) exposure model showed that approximately 42% of the groundwater samples had a non-negligible ER (1.00 × 10-4 to 1.00 × 10-5). Sensitivity analysis indicated groundwater nitrate concentration, ingestion rate, and the percentage of nitrite from nitrate were the most significant variables in determining HI and ER. It is suggested to adopt proper management of control policies for reducing the elevated groundwater nitrate concentration in the present study area.

Lady with Long Eyelashes !

A sixty years postmenopausal lady presented with growing longer eyelashes for 8 years. She had thick, dark, curly eyelashes measuring 23 mm and 15 mm at the centre and periphery respectively suggesting marked trichomegaly. Increased vellus hair was noticed above lips and chin suggesting hypertrichosis. An important clue in history was the use of chemotherapeutic agent Erlotinib after bronchoscopic surgery for non-small cell lung carcinoma for the past 8 years. Erlotinib competitively binds to the tyrosine kinase domain of Epidermal Growth Factor Receptor inhibiting receptor activation and blocking the signal transduction. Thus, disrupting the transition of hair growth from anagen to telogen phase, leading to aberrant anagen phase and consequently abnormal hair growth. Trichomegaly is seen after 2-5 months of treatment. Mostly innocuous, it can lead to eyelid infections and rarely corneal ulceration. EGFR inhibitors are associated with hypertrichosis in other areas, as was the case in this patient. This case highlights the significance of detailed history including drugs, thus abating additional work-up for trichomegaly.

SABRE hyperpolarized anticancer agents for use in 1 H MRI.

PURPOSE: Enabling drug tracking (distribution/specific pathways) with magnetic resonance spectroscopy requires manipulation (via hyperpolarization) of spin state populations and targets with sufficiently long magnetic lifetimes to give the largest possible window of observation. Here, we demonstrate how the proton resonances of a group of thienopyridazines (with known anticancer properties), can be amplified using the para-hydrogen (p-H2 ) based signal amplification by reversible exchange (SABRE) hyperpolarization technique. METHODS: Thienopyridazine isomers, including a 2 H version, were synthesized in house. Iridium-based catalysts dissolved in a methanol-d4 solvent facilitated polarization transfer from p-H2 gas to the target thienopyridazines. Subsequent SABRE 1 H responses of hyperpolarized thienopyridazines were completed (400 MHz NMR). Pseudo-singlet state approaches were deployed to extend magnetic state lifetimes. Proof of principle spectral-spatial images were acquired across a range of field strengths (7T-9.4T MRI). RESULTS: 1 H-NMR signal enhancements of -10,130-fold at 9.4T (~33% polarization) were achieved on thieno[2,3-d]pyridazine (T[2,3-d]P), using SABRE under optimal mixing/field transfer conditions. 1 H T1 lifetimes for the thienopyridazines were ~18-50 s. Long-lived state approaches extended the magnetic lifetime of target proton sites in T[2,3-d]P from an average of 25-40 seconds. Enhanced in vitro imaging (spatial and chemical shift based) of target T[2,3-d]P was demonstrated. CONCLUSION: Here, we demonstrate the power of SABRE to deliver a fast and cost-effective route to hyperpolarization of important chemical motifs of anticancer agents. The SABRE approach outlined here lays the foundations for realizing continuous flow, hyperpolarized tracking of drug delivery/pathways.

Targeted Degradation of the Oncogenic Phosphatase SHP2.

SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras-MAPK pathway upon stimulation of receptor tyrosine kinases, which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at submicromolar concentrations, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.

Impact of titanium dioxide particle size on macrophage production of intracellular reactive oxygen species.

OBJECTIVES: The aim of this study was to investigate the response of THP-1 monocyte-derived macrophages following exposure to titanium dioxide nanoparticles (TiO2 NPs) and microparticles (TiO2 MPs) in an in vitro system. DESIGN: THP-1 monocytes were maintained in RPMI medium and transformed into M0 macrophages using Phorbol 12-myristate 13-acetate (PMA). TiO2 particle size characterization was performed using scanning electron microscopy (SEM) and dynamic light scattering (DLS) technology. A viability study using an XTT assay was performed by treating THP-1-derived macrophages with TiO2 NPs (<100 nm) and TiO2 MPs (<5 µm) at concentrations ranging from 100, 50, 25, 12.5, 6.25 and 3.125 µg/mL. Macrophages were then treated with three different concentrations of NPs and MPs (5, 20 or 100 µg/mL) for 24 h, and ROS production and TiO2 particle cellular uptake were measured using ROS assays and flow cytometry, respectively. RESULTS: There was no significant change in the viability of THP-1 monocytes after treatment with TiO2 NPs and MPs. The uptake of both particles was confirmed and showed an increase in ROS generation, and the MPs produced more ROS than NPs. The increase in ROS generation with NPs was concentration-dependent. CONCLUSION: Uptake of TiO2 NPs and MPs in macrophages at subcytotoxic levels generate ROS in a size- and dose-dependent manner.

A comparative study evaluating the dose volume parameters in 3D conformal radiation of left sided whole breast irradiation including regional lymphnodes - a need of resource constrained countries.

BACKGROUND: The purpose of this study was to compare four 3D conformal radiation techniques in treatment of left breast cancer patients. MATERIALS AND METHODS: Radiation was planned for 20 patients to the left breast and regional lymph nodes using four techniques: partially wide tangents, photon-photon mix, photon-electron mix and 30/70 photon-electron mix. All plans were evaluated for internal mammary nodes (IMN) coverage, hotspot and normal tissue constraints. RESULT: The 85% of planning target volume (PTV) coverage was lesser for upper IMN than the lower IMN (below the lower border of the clavicular head) for all four techniques. The lower IMN coverage was better for partially wide tangent (80.46%) and photon-photon mix (88.88%). The lowest value of hotspot was seen in the partially wide tangent technique (112.69% ± 1.92). Hotspot is unacceptably high in both photon-electron mix and 30/70 photon-electron mix (> 120%). Left lung mean dose for all techniques on a pair-wise comparison showed no statistical difference. Left lung V20 values for partially wide tangent was 37.56% ± 8.17 and for photon-photon mix it was 40.49% ± 3.36. The mean heart dose with partially wide tangent was 9.43 ± 3.15 Gy and with photon-photon mix it was 10.10 ± 2.70 Gy. The mean heart dose for photon-electron mix was 7.56 ± 1.95 Gy and for 30/70 photon-electron mix it was 7.98 ± 2.16 Gy. CONCLUSION: No single technique satisfies all the criteria. The decision should be made on a case-by-case basis, considering the anatomy of the patient, availability of electron facilities and setup accuracy and reproducibility.

Ror ß expression in activated macrophages and dental pulp stem cells.

AIM: To assess the expression of Retinoic acid-related orphan receptor beta (Ror ß) in human inflamed dental pulp stem cells (hI-DPSCs) and during macrophage phenotypic conversion. METHODOLOGY: Commercially procured THP-1 monocytes conversion to macrophages was judged by their morphology, the percentage of adherent cells and the expression of CD-14 surface marker. THP-1 macrophage cell viability following LPS, IFN-γ/IL-4, IL-13 stimulus was evaluated at 24 and 48h. The phenotypic conversion of macrophages to M1 and M2 was confirmed by flow cytometry and Western blot analysis. Cytokine release following polarization was estimated by the BD cytokine flex kit. The expression of Ror ß in THP-1 macrophages and hI-DPSCs following LPS, IFN-γ/IL-4, IL-13 stimulus was assessed by Western blot analysis. Statistical significance was analysed using one-way Anova followed by Tukey's Post hoc test. RESULTS: THP-1 monocytes pretreated with PMA (100 ng mL-1 ) for 48 h followed by culturing in PMA-free media for another 48 h yielded cells with morphological characteristics similar to macrophages with a high percentage of adherence capability and CD-14 expression. Macrophages treated with LPS 100 ng mL-1 and IFN-γ 20 ng mL-1 or IL-4 20 ng mL-1 had high expression of the respective M1 and M2 CD markers in flow cytometry and Western blot analysis. Cytokine release studies demonstrated the expression of IL-1ß, TNF-α and IL-10 in the M1-polarized macrophages (P < 0.01), whilst TGF- ß levels were seen in the M1 and M2-polarized macrophages. Ror ß expression was upregulated when macrophages and hI-DPSCs were treated with anti-inflammatory cytokines. CONCLUSION: Ror ß was expressed in THP-1 macrophages and hI-DPSCs during their resting stage. Upregulated expression of Ror ß occurred following an anti-inflammatory stimulus.

Design, Synthesis, and Biological Evaluation of a Series of Oxazolone Carboxamides as a Novel Class of Acid Ceramidase Inhibitors.

Acid ceramidase (AC) is a cysteine hydrolase that plays a crucial role in the metabolism of lysosomal ceramides, important members of the sphingolipid family, a diversified class of bioactive molecules that mediate many biological processes ranging from cell structural integrity, signaling, and cell proliferation to cell death. In the effort to expand the structural diversity of the existing collection of AC inhibitors, a novel class of substituted oxazol-2-one-3-carboxamides were designed and synthesized. Herein, we present the chemical optimization of our initial hits, 2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a, which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent AC inhibitor with optimal physicochemical and metabolic properties, showing target engagement in human neuroblastoma SH-SY5Y cells and a desirable pharmacokinetic profile in mice, following intravenous and oral administration. 32b enriches the arsenal of promising lead compounds that may therefore act as useful pharmacological tools for investigating the potential therapeutic effects of AC inhibition in relevant sphingolipid-mediated disorders.

The mechanism of cancer drug addiction in ALK-positive T-Cell lymphoma.

Rational new strategies are needed to treat tumors resistant to kinase inhibitors. Mechanistic studies of resistance provide fertile ground for development of new approaches. Cancer drug addiction is a paradoxical resistance phenomenon, well-described in MEK-ERK-driven solid tumors, in which drug-target overexpression promotes resistance but a toxic overdose of signaling if the inhibitor is withdrawn. This can permit prolonged control of tumors through intermittent dosing. We and others showed previously that cancer drug addiction arises also in the hematologic malignancy ALK-positive anaplastic large-cell lymphoma (ALCL) resistant to ALK-specific tyrosine kinase inhibitors (TKIs). This is driven by the overexpression of the fusion kinase NPM1-ALK, but the mechanism by which ALK overactivity drives toxicity upon TKI withdrawal remained obscure. Here we reveal the mechanism of ALK-TKI addiction in ALCL. We interrogated the well-described mechanism of MEK/ERK pathway inhibitor addiction in solid tumors and found it does not apply to ALCL. Instead, phosphoproteomics and confirmatory functional studies revealed that the STAT1 overactivation is the key mechanism of ALK-TKI addiction in ALCL. The withdrawal of TKI from addicted tumors in vitro and in vivo leads to overwhelming phospho-STAT1 activation, turning on its tumor-suppressive gene-expression program and turning off STAT3's oncogenic program. Moreover, a novel NPM1-ALK-positive ALCL PDX model showed a significant survival benefit from intermittent compared with continuous TKI dosing. In sum, we reveal for the first time the mechanism of cancer drug addiction in ALK-positive ALCL and the benefit of scheduled intermittent dosing in high-risk patient-derived tumors in vivo.

Objective Assessment of Microsurgery Competency-In Search of a Validated Tool.

Microsurgical skill acquisition is an integral component of training in plastic surgery. Current microsurgical training is based on the subjective Halstedian model. An ideal microsurgery assessment tool should be able to deconstruct all the subskills of microsurgery and assess them objectively and reliably. For our study, to analyze the feasibility, reliability, and validity of microsurgery skill assessment, a video-based objective structured assessment of technical skill tool was chosen. Two blinded experts evaluated 40 videos of six residents performing microsurgical anastomosis for arteriovenous fistula surgery. The generic Reznick's global rating score (GRS) and University of Western Ontario microsurgical skills acquisition/assessment (UWOMSA) instrument were used as checklists. Correlation coefficients of 0.75 to 0.80 (UWOMSA) and 0.71 to 0.77 (GRS) for interrater and intrarater reliability showed that the assessment tools were reliable. Convergent validity of the UWOMSA tool with the prevalidated GRS tool showed good agreement. The mean improvement of scores with years of residency was measured with analysis of variance. Both UWOMSA ( p -value: 0.034) and GRS ( p -value: 0.037) demonstrated significant improvement in scores from postgraduate year 1 (PGY1) to PGY2 and a less marked improvement from PGY2 to PGY3. We conclude that objective assessment of microsurgical skills in an actual clinical setting is feasible. Tools like UWOMSA are valid and reliable for microsurgery assessment and provide feedback to chart progression of learning. Acceptance and validation of such objective assessments will help to improve training and bring uniformity to microsurgery education.

Halitosis and Pain Post Electrocautery Adenoidectomy.

Background and Objectives: Electrocautery adenoidectomy (ECA) is a common procedure performed in paediatric otolaryngology. ECA has been preferred over curettage adenoidectomy due to its lower intraoperative bleeding rates, decreased procedure time, and higher subjective success. However, post-ECA symptoms of pain and halitosis have never been studied. The objective of our study was to identify the pattern of post-ECA halitosis and pain in the paediatric population. Materials and Methods: This is a single centre, prospective observational study that uses visual analogue scales (VAS) by parent proxy to assess post-ECA pain and halitosis in paediatric patients (age < 18) in South Australia. A total of 19 patients were enrolled in the study and followed for seven days. Results: Postoperative pain and halitosis reaches a peak 3 days post-ECA (median = 2 for pain; median = 6 for halitosis) but resolves 7 days post-ECA (median = 0 for both). Conclusions: Our study demonstrates that halitosis and pain occur over a seven-day period in patients undergoing ECA and will resolve post-operatively with simple analgesia and without antibiotics.