Titania-Graphene Oxide Nanocomposite-Based Philadelphia-Positive Leukemia Therapy.

Philadelphia-positive (Ph+) leukemia is a type of blood cancer also known as acute lymphoblastic leukemia (ALL), affecting 20-30% of adults diagnosed worldwide and having an engraved prognosis as compared to other types of leukemia. The current treatment regimens mainly rely on tyrosine kinase inhibitors (TKIs) and bone marrow transplants. To date, several generations of TKIs have been developed due to associated resistance and frequent relapse, with cardiovascular system anomalies being the most devastating complication. Nanotechnology has the potential to address these limitations by the targeted drug delivery and controlled release of TKIs. This study focused on the titanium dioxide (TiO2) and graphene oxide (GO) nanocomposite employment to load nilotinib and ponatinib TKIs for therapy of Ph+ leukemia cell line (K562) and Ba/F3 cells engineered to express BCR-ABL oncogene. Meanwhile, after treatment, the oncogene expressing fibroblast cells (Rat-1 P185) were evaluated for their colony formation ability under 3D conditions. To validate the nanocomposite formation, the TiO2-GO nanocomposites were characterized by scanning electron microscope, DLS, XRD, FTIR, zeta potential, EDX, and element mapping. The TKI-loaded TiO2-GO was not inferior to the free drugs after evaluating their effects by a cell viability assay (XTT), apoptosis induction, and colony formation inhibition. The cell signaling pathways of the mammalian target of rapamycin (mTOR), signal transducers and activators of transcription 5 (STAT5), and extracellular signal-regulated kinase (Erk1/2) were also investigated by Western blot. These signaling pathways were significantly downregulated in the TKI-loaded TiO2-GO-treated groups. Based on the findings above, we can conclude that TiO2-GO exhibited excellent drug delivery potential that can be used for Ph+ leukemia therapy in the future, subject to further investigations.

Lyophilization Based Isolation of Exosomes.

Exosomes are nanoscale extracellular vesicles which regulate intercellular communication. They have great potential for application in nanomedicine. However, techniques for their isolation are limited by requirements for advanced instruments and costly reagents. In this study, we developed a lyophilization-based method for isolating exosomes from cultured cells. The isolated exosomes were characterized for protein content using Bradford assay, and for size distribution and shape using scanning electron microscopy (SEM) and nanoparticles tracking analysis (NTA). In addition, CD63, CD9, CD81, HSP70 and TSG101 were evaluated as essential exosomal surface markers using Western blot. Drug loading and release studies were performed to confirm their drug delivery properties using an in vitro model. Exosomes were also loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. All these characterizations confirmed successful exosome isolation with measurements of less than 150 nm, having a typical shape, and by expressing the known exosome surface protein markers. Finally, tyrosine kinase inhibitors (dasatinib and ponatinib) were loaded on the exosomes to evaluate their anticancer effects on leukemia cells (K562 and engineered Ba/F3-BCR-ABL) using MTT and Annexin-PI assays. The expression of MUC1 protein on the exosomes isolated from MCF-7 cells also indicated that their potential diagnostic properties were intact. In conclusion, we developed a new method for exosome isolation from cultured cells. These exosomes met all the essential requirements in terms of characterization, drug loading and release ability, and inhibition of proliferation and apoptosis induction in Ph+ leukemia cells. Based on these results, we are confident in presenting the lyophilization-based exosome isolation method as an alternative to traditional techniques for exosome isolation from cultured cells.

Diastereoselective [3 + 2] Cycloaddition of Quinoxalin-2(1H)-ones with Donor-Acceptor Cyclopropanes: Efficient Synthesis of Tetrahydro pyrrolo[1,2-a]quinoxalin-4(5H)-ones.

A ytterbium triflate-catalyzed diastereoselective [3 + 2] cycloaddition of quinoxalinones with donor-acceptor cyclopropanes and cyclobutanes is described. A series of tetrahydropyrrolo-quinoxalinone derivatives were obtained in high yields (up to 96%) with excellent diastereoselectivities (up to 46:1). Other medicinally important heterocycles like benzoxazinone, isoquinoxalinone, and dibenzoxazepine derivatives were also suitable for the desired annulation reaction. The current method is applicable for the scale-up reaction. Further, the utility of this annulation reaction is demonstrated by the synthesis of densely functionalized proline derivatives.

Cu(II)-Catalyzed C-N, C-O, C-Cl, C-S, and C-Se Bond Formation via C(sp2)-H Activation Using 7-Azaindole as an Intrinsic Directing Group.

A general protocol has been developed for the construction of carbon-heteroatom (C-N, C-Cl, C-O, C-S, and C-Se) bonds using the bench stable, earth-abundant, and environmentally benign copper catalyst. Only oxygen is sufficient to regenerate the copper catalyst. Control experiments suggested that the proto-demetalation step is reversible. Depending on the coupling partner, the reaction follows either disproportionation or radical pathways to complete the catalytic cycle. The synthetic utility of the developed protocol has been demonstrated via various functional group transformations.

Oncogene-independent resistance in Philadelphia chromosome - positive (Ph+) acute lymphoblastic leukemia (ALL) is mediated by activation of AKT/mTOR pathway.

Tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, dasatinib, and ponatinib have significantly improved the life expectancy of Philadelphia chromosome-positive (Ph+) acute lymphocytic leukemia (ALL) patients; however, resistance to TKIs remains a major clinical challenge. Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL1 have emerged as the predominant cause of acquired resistance. In approximately 30% of patients, the mechanism of resistance to TKIs remains elusive. This study aimed to investigate mechanisms of nonmutational resistance in Ph+ ALL. Here we report the development of a nonmutational resistance cell line SupB15-RT; conferring resistance to approved ABL kinase inhibitors (AKIs) and allosteric inhibitors GNF-2, ABL001, and crizotinib, except for dasatinib (IC90 50nM), a multitarget kinase inhibitor. We found that the AKT/mTOR pathway is activated in these cells and their proliferation inhibited by Torin-1 with an IC50 of 24.7 nM. These observations were confirmed using 3 different ALL patient-derived long term cultures (PDLTCs): (1) HP (BCR-ABL1 negative), (2) PH (BCR-ABL1 positive and responsive to TKIs) and (3) BV (BCR-ABL1 positive and nonmutational resistant to TKIs). Furthermore, Torin-1 and NVP-BEZ235 induced apoptosis in PH and BV cells but not in HP cells. Our experiments provide evidence of the involvement of AKT/mTOR pathway in the evolution of nonmutational resistance in Ph+ ALL which will assist in developing novel targeted therapy for Ph+ ALL patients with BCR-ABL1 independent nonmutational resistance.

AXL receptor tyrosine kinase: a possible therapeutic target in acute promyelocytic leukemia.

BACKGROUND: Acute promyelocytic leukemia (APL) is a subset of acute myeloid leukemia (AML) which is characterized by the fusion of promyelocytic leukemia PML and retinoic acid receptor- alpha (RAR-alpha) genes. All-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO) have resulted in durable cytogenetic and molecular remissions in most APL patients and have altered the natural history of the disease. Most APL patients treated with ATRA and/or ATO are now anticipated to have a nearly normal life expectancy. Unfortunately, relapse and resistance to the current treatment occur in APL patients and the outcome remains dismal in these refractory patients. AXL receptor tyrosine kinase (AXL-RTK) has been shown to increase tumour burden, provide resistance to therapy and is critical to maintain cancer stem cells (CSCs) in chronic myeloid leukemia (CML) by stabilizing ß-catenin in the Wnt/ß-catenin signalling pathway. However, the role of AXL-RTK has not been explored in PML/RARα-positive APL. This study aimed to explore the role of AXL-RTK receptor in PML/RARα-positive APL. METHODS AND RESULTS: By using biochemical and pharmacological approaches, here we report that targeting of AXL-RTK is related to the down-regulation of ß-catenin target genes including c-myc (p < 0.001), AXIN2 (p < 0.001), and HIF1α (p < 0.01) and induction of apoptosis in PML/RARα-positive APL cell line. Resistance to all-trans retinoic acid (ATRA) was also overcomed by targeting AXL-RTK with R428 in APL (p < 0.05). CONCLUSION: Our results provide clear evidence of the involvement of AXL-RTK in leukemogenic potential of PML/RARα-positive APL and suggest targeting of AXL-RTK in the treatment of therapy resistant APL patients.

Crizotinib acts as ABL1 inhibitor combining ATP-binding with allosteric inhibition and is active against native BCR-ABL1 and its resistance and compound mutants BCR-ABL1T315I and BCR-ABL1T315I-E255K.

Resistance remains the major clinical challenge for the therapy of Philadelphia chromosome-positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common "gatekeeper" mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph- cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia-like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.

COVID-19 transmission in a theme-park

BackgroundAs COVID-19 vaccination coverage increases, public health and industries are contemplating re-opening measures of public spaces, including theme-parks. To re-open, theme-parks must provide public health mitigation plans. Questions on implementation of public health mitigation strategies such as park cleaning, COVID-19 testing, and enforcement of social distancing and the wearing of personal protective equipment (PPE) in the park remain. MethodsWe have developed a mathematical model of COVID-19 transmission in a theme-park that considers direct human-human and indirect environment-human transmission of the virus. The model thus tracks the changing infection/disease landscape of all visitors, workers, and environmental reservoirs in a theme park setting. FindingsModels results show that theme-park public health mitigation must include mechanisms that reduce virus contamination of the environment to ensure that workers and visitors are protected from COVID-19 transmission in the park. Thus, cleaning rates and mitigation of human-environment contact increases in importance. ConclusionOur findings have important practical implications in terms of public health as policy- and decision-makers are equipped with a mathematical tool that can guide theme-parks in developing public health mitigation strategies for a safe re-opening.

Pd-Catalyzed C-H Halogenation of Indolines and Tetrahydroquinolines with Removable Directing Group.

Pd-catalyzed directing-group-assisted regioselective halogenations to C7 of indolines and C8 of tetrahydroquinolines were achieved in good to excellent yields. The practicality and utility of the developed method have been illustrated by various functional group transformations such as arylation, alkenylation, cyanation, and silylation utilizing the installed synthetic handle. The concise synthesis of primaquine, an antimalarial drug, and formal syntheses of two bioactive natural products, hippadine and pratosine, have also been demonstrated.

Cu(II)-Catalyzed Ortho C(sp2)-H Diarylamination of Arylamines To Synthesize Triarylamines.

A copper-catalyzed, directed ortho C-H diarylamination of indoles, indolines, anilines, and N-aryl-7-azaindoles has been established. Only copper salt as the catalyst and oxygen as the terminal oxidant are used to synthesize triarylamines using various diarylamines including carbazole and phenothiazine. Mechanistic interrogation reveals that copper plays a dual role.

Cu(II)-Mediated Cross-Dehydrogenative Coupling of Indolines with Sulfonamides, Carboxamides, and Amines.

A facile and efficient Cu-mediated protocol for the cross-dehydrogenative coupling of indoline with sulfonamides, carboxamides, and anilines is reported. The reaction takes place through Cu-mediated C7-H activation via a 6-membered metallacycle to afford the amide and amine derivatives in good yields with a wide range of functional group tolerance. The importance of the protocol has been demonstrated by synthesizing the antiproliferative agent, ER-67836.

CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells.

Adoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-ß is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αß and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR-modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer-reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αß TCR resulted in more efficient redirection of CD4+ and CD8+ T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies.

Early Versus Delayed Laparoscopic Cholecystectomy For Acute Cholecystitis.

BACKGROUND: Laparoscopic cholecystectomy is considered the gold standard for the management of acute cholecystitis but controversy surrounds the timings of the surgery. Studies are available favouring both early and delayed laparoscopic cholecystectomy. The objective of this study was to compare early versus delayed laparoscopic cholecystectomy for acute cholecystitis. METHODS: This quasi-experimental study included 180 patients irrespective of their age and sex presented at department of Surgery, Lahore General Hospital between January to December 2014 with a diagnosis of acute cholecystitis were assigned randomly to early laparoscopic cholecystectomy within 24 h of admission or to initial conservative treatment followed by delayed laparoscopic cholecystectomy, 6-12 weeks later. RESULTS: The mean operating time was 64.32 min vs. 58.24 min in the delayed group, conversion rate (early 15.5% vs. delayed 14.4%). The mean postoperative hospital stay was 1.67 days in the earlier group and 4.38 days in the delayed group. Overall mortality was zero. CONCLUSIONS: Early laparoscopic cholecystectomy for acute cholecystitis is safe, offering economic benefit of much shorter hospital stay and quick recovery.

BCR: a new target in resistance mediated by BCR/ABL-315I?

Targeting BCR/ABL with Tyrosine kinase inhibitors (TKIs) is a proven concept for the treatment of Philadelphia chromosome-positive (Ph+) leukemias but the "gatekeeper" mutation T315I confers resistance against all approved TKIs, with the only exception of ponatinib, a multi-targeted kinase inhibitor. Besides resistance to TKIs, T315I also confers additional features to the leukemogenic potential of BCR/ABL, involving endogenous BCR. Therefore we studied the role of BCR on BCR/ABL mutants lacking functional domains indispensable for the oncogenic activity of BCR/ABL. We used the factor independent growth of murine myeloid progenitor 32D cells and the transformation of Rat-1 fibroblasts both mediated by BCR/ABL. Here we report that T315I restores the capacity to mediate factor-independent growth and transformation potential of loss-of-function mutants of BCR/ABL. Targeting endogenous Bcr abrogated the capacity of oligomerization deficient mutant of BCR/ABL-T315I to mediate factor independent growth of 32D cells and strongly reduced their transformation potential in Rat-1 cells, as well as led to the up-regulation of mitogen activated protein kinase (MAPK) pathway. Our data show that the T315I restores the capacity of loss-of-function mutants to transform cells which is dependent on the transphosphorylation of endogenous Bcr, which becomes a putative therapeutic target to overcome resistance by T315I.

INTESTINAL OBSTRUCTION DUE TO RARE INTERNAL SUPRAVESICAL HERNIA.

Supravesical hernias are very rarely seen and reported as a possible cause of small bowel obstruction. The proper diagnosis of which is usually made intra-operatively as the preliminary diagnosis despite the availability of advanced radiological investigations which are not very helpful. There are two anatomical variants of supravesical hernias, of which internal one usually presents as small bowel obstruction. We present a case of 62 year old man, presented to us in emergency with three day history of absolute constipation, abdominal distention, and vomiting. On exploratory laparotomy, the terminal ileum loops were retrieved from the supravesical hernial defect by gentle traction and the defect was closed. Post-operative period remained uneventful. Supravesical hernias though very rare still can be a possible cause of small bowel obstruction. The diagnosis is usually made per operatively where the bowel loops are seen to be strangulated in the hernia defect.

AUDIT OF SURGICAL EMERGENCY AT LAHORE GENERAL HOSPITAL.

BACKGROUND: Audit is a quality improvement process that seeks to improve patient care and outcomes through systematic review of care against explicit criteria and the review of change. Objective of this study was to report the patterns of admissions in our surgical emergency and the comparison of results with the available data. METHODS: All the patients presented in the surgical emergency of Unit III from April to December 2014. Detail of all surgical patients admitted during the period was recorded from the emergency entry register maintained by the staff nurse. Demographic data, mode of admission, diagnosis and outcomes were recorded on a pro forma. RESULTS: Total number of patients were 11140, out of which 5998 (53.8%) were males and 5142 (46%) were females, mostly were between 18-56 years of age. Emergency surgeries were performed in 650 of our cases whereas the rest of the patients were managed conservatively, treated at minor operation theatre (MOT), referred to their concerned emergencies or discharged. The most common presentation was road traffic accidents followed by trauma, urological emergencies and intestinal obstruction. Overall mortality was estimated as 1.5%. CONCLUSIONS: Surgical audit should be made a regular practice to serve as an important and effective tool of accountibilty on clinical outcomes and self evaluation and in improving the quality of our health care system.

The functional interplay between the t(9;22)-associated fusion proteins BCR/ABL and ABL/BCR in Philadelphia chromosome-positive acute lymphatic leukemia.

The hallmark of Philadelphia chromosome positive (Ph(+)) leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph(+) leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid leukemia (CML) and 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96(ABL/BCR) for the pathogenesis of Ph(+) ALL. The co-expression of p96(ABL/BCR) enhanced the kinase activity and as a consequence, the transformation potential of p185(BCR/ABL). Targeting p96(ABL/BCR) by RNAi inhibited growth of Ph(+) ALL cell lines and Ph(+) ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96(ABL/BCR) and p185(BCR/AB)L in hematopoietic stem cells. Co-expression of p96(ABL/BCR) abolished the capacity of p185(BCR/ABL) to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96(ABL/BCR) for the pathogenesis of Ph(+) ALL.

Intention and willingness in understanding Ritalin misuse among Iranian medical college students: a cross-sectional study.

Ritalin misuse can create powerful stimulant effects and serious health risks. The main aim of present study was compared that two cognitive construct (behavioral intention or behavioral willingness) for predicting Ritalin misuse. This cross-sectional study was conducted among 264 Iranian medical college students; participants selected in random sampling, and data were collected by using self-report questionnaire. Data were analyzed by SPSS version 21 at 95% significant level. Our findings showed, the three predictor variables of (1) attitude, (2) subjective norms, and (3) prototype accounted for 29% of the variation in intention and 25% of the variation in willingness to Ritalin misuse. In addition, behavioral intention was a stronger prediction factor compared to willingness for Ritalin misuse, with odds ratio estimate of 1.607 [95% CI: 1.167, 2.213]. There is some support to use the prototype willingness model to design interventions to improve individuals' beliefs that academic goals are achievable without the misuse of Ritalin.

Using the PRECEDE model in understanding determinants of quality of life among Iranian male addicts.

Quality of Life (QOL) in opiate-addicted patients who are receiving methadone maintenance therapy is one of the important issues to be considered in the treatment of addiction. To determine a needs assessment using the PRECEDE model to find out factors related to QOL among Iranian male opiate addicts. This cross-sectional study was conducted in Kermanshah, Iran in 2013. A total of 762 male opiate addicts, who were referred to addiction treatment centers for receiving methadone maintenance treatment, were randomly selected to participate voluntarily in the study. SF-12, predisposing factors, enabling factors, reinforcement factors, and methadone maintenance treatment intention were used to find the related factors. Data were analyzed by the SPSS software (ver. 21.0) using the t-test, one-way analysis of variance (ANOVA), bivariate correlations, and linear regression at 95% significant level. Linear regression analysis showed the determinant variable accounted for 17% of the variation in QOL. Our findings suggest, providing social support for addicts could be beneficial results for the increasing quality of life among them.