A new potential therapeutic approach for ALS: A case report with NGS analysis.

RATIONALE: Amyotrophic lateral sclerosis (ALS) poses a significant clinical challenge due to its rapid progression and limited treatment options, often leading to deadly outcomes. Looking for effective therapeutic interventions is critical to improve patient outcomes in ALS. PATIENT CONCERNS: The patient, a 75-year-old East Asian male, manifested an insidious onset of right-hand weakness advancing with dysarthria. Comprehensive Next-generation sequencing analysis identified variants in specific genes consistent with ALS diagnosis. DIAGNOSES: ALS diagnosis is based on El Escorial diagnostic criteria. INTERVENTIONS: This study introduces a novel therapeutic approach using artificial intelligence phenotypic response surface (AI-PRS) technology to customize personalized drug-dose combinations for ALS. The patient underwent a series of phases of AI-PRS-assisted trials, initially incorporating a 4-drug combination of Ibudilast, Riluzole, Tamoxifen, and Ropinirole. Biomarkers and regular clinical assessments, including nerve conduction velocity, F-wave, H-reflex, electromyography, and motor unit action potential, were monitored to comprehensively evaluate treatment efficacy. OUTCOMES: Neurophysiological assessments supported the ALS diagnosis and revealed the co-presence of diabetic polyneuropathy. Hypotension during the trial necessitated an adaptation to a 2-drug combinational trial (ibudilast and riluzole). Disease progression assessment shifted exclusively to clinical tests of muscle strength, aligning with the patient's well-being. LESSONS: The study raises the significance of personalized therapeutic strategies in ALS by AI-PRS. It also emphasizes the adaptability of interventions based on patient-specific responses. The encountered hypotension incident highlights the importance of attentive monitoring and personalized adjustments in treatment plans. The described therapy using AI-PRS, offering personalized drug-dose combinations technology is a potential approach in treating ALS. The promising outcomes warrant further evaluation in clinical trials for searching a personalized, more effective combinational treatment for ALS patients.

Personalized Tacrolimus Dosing After Liver Transplantation: A Randomized Clinical Trial.

Background: Inter- and intra-individual variability in tacrolimus dose requirements mandates empirical clinician-titrated dosing that frequently results in deviation from a narrow target range. Improved methods to individually dose tacrolimus are needed. Our objective was to determine whether a quantitative, dynamically-customized, phenotypic-outcome-guided dosing method termed Phenotypic Personalized Medicine (PPM) would improve target drug trough maintenance. Methods: In a single-center, randomized, pragmatic clinical trial ( NCT03527238 ), 62 adults were screened, enrolled, and randomized prior to liver transplantation 1:1 to standard-of-care (SOC) clinician-determined or PPM-guided dosing of tacrolimus. The primary outcome measure was percent days with large (>2 ng/mL) deviation from target range from transplant to discharge. Secondary outcomes included percent days outside-of-target-range and mean area-under-the-curve (AUC) outside-of-target-range per day. Safety measures included rejection, graft failure, death, infection, nephrotoxicity, or neurotoxicity. Results: 56 (29 SOC, 27 PPM) patients completed the study. The primary outcome measure was found to be significantly different between the two groups. Patients in the SOC group had a mean of 38.4% of post-transplant days with large deviations from target range; the PPM group had 24.3% of post-transplant days with large deviations; (difference -14.1%, 95% CI: -26.7 to -1.5 %, P=0.029). No significant differences were found in the secondary outcomes. In post-hoc analysis, the SOC group had a 50% longer median length-of-stay than the PPM group [15 days (Q1-Q3: 11-20) versus 10 days (Q1-Q3: 8.5-12); difference 5 days, 95% CI: 2-8 days, P=0.0026]. Conclusions: PPM guided tacrolimus dosing leads to better drug level maintenance than SOC. The PPM approach leads to actionable dosing recommendations on a day-to-day basis. Lay Summary: In a study on 62 adults who underwent liver transplantation, researchers investigated whether a new dosing method called Phenotypic Personalized Medicine (PPM) would improve daily dosing of the immunosuppression drug tacrolimus. They found that PPM guided tacrolimus dosing leads to better drug level maintenance than the standard-of-care clinician-determined dosing. This means that the PPM approach leads to actionable dosing recommendations on a day-to-day basis and can help improve patient outcomes.

Efficacy and safety of an innovative short-course regimen containing clofazimine for treatment of drug-susceptible tuberculosis: a clinical trial.

In preclinical studies, a new antituberculosis drug regimen markedly reduced the time required to achieve relapse-free cure. This study aimed to preliminarily evaluate the efficacy and safety of this four-month regimen, consisting of clofazimine, prothionamide, pyrazinamide and ethambutol, with a standard six-month regimen in patients with drug-susceptible tuberculosis. An open-label pilot randomized clinical trial was conducted among the patients with newly diagnosed bacteriologically-confirmed pulmonary tuberculosis. The primary efficacy end-point was sputum culture negative conversion. Totally, 93 patients were included in the modified intention-to-treat population. The rates of sputum culture conversion were 65.2% (30/46) and 87.2% (41/47) for short-course and standard regimen group, respectively. There was no difference on two-month culture conversion rates, time to culture conversion, nor early bactericidal activity (P > 0.05). However, patients on short-course regimen were observed with lower rates of radiological improvement or recovery and sustained treatment success, which was mainly attributed to higher percent of patients permanently changed assigned regimen (32.1% vs. 12.3%, P = 0.012). The main cause for it was drug-induced hepatitis (16/17). Although lowering the dose of prothionamide was approved, the alternative option of changing assigned regimen was chosen in this study. While in per-protocol population, sputum culture conversion rates were 87.0% (20/23) and 94.4% (34/36) for the respective groups. Overall, the short-course regimen appeared to have inferior efficacy and higher incidence of hepatitis but desired efficacy in per-protocol population. It provides the first proof-of-concept in humans of the capacity of the short-course approach to identify drug regimens that can shorten the treatment time for tuberculosis.

Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication.

Aberrant CpG-island methylation affects ovarian cancer progression. The promotor methylation changes at tumor suppressive genes in ovarian cancer stromal progenitor cells (OCSPCs) and epithelial ovarian cancer (EOC) tissues and their clinical implication remains unexplored. We systemically analyzed the promoter methylation status of 40 tumor suppressor genes (TSGs) associated with cancer in paired epithelial-like and mesenchymal-like OCSPCs and ovarian cancer cells by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The effect of DNA methylation on gene expression was confirmed using qRT-PCR. The differential frequencies of TSGs' promoter methylation among matched epithelial-like or mesenchymal-like OCSPCs from tissues and ascites and ovarian cancer tissues were further validated in cancer tissues and correlated with clinicopathological features and survival outcomes of patients. According to the promoter methylation frequencies of the 40 TSGs, promoters of RASSF1A were the only significantly hypomethylated in epithelial-like OCSPCs from tissues than those from ascites and bulk tumor cells (0% vs 38% vs 45%, P=0.039 by Fisher's exact test). The most frequencies at promotor hypermethylation of TSGs in mesenchymal-like OCSPCs from ascites which processed aggressiveness were CDKN2B (73%) followed by CCND2 (45%) and RASSF1A (45%). Forty-three percent (47/110) of RASSF1A and 45% of CCND2 were validated as a frequently hypermethylated gene in an independent set of 110 EOC tissues in contrast to none (0/60) and 12% (10/60) of benign ovarian cysts (both P<0.001). Functional experiments revealed overexpression of CCND2 or CDKN2B in MSc-OCSPCs decreases EMT, invasion, and spheroid formation in EOC, and abolishes DNMT1 and COL6A3 expression. However, for the expected 5-year overall survival (OS) for patients with methylated RASSF1A, CCND2, and CDKN2B, only RASSF1A was significantly worse than those without methylated RASSF1A (56% vs 80%, p=0.022). Taken together, overexpression of CCND2 and CDKN2B decreased the aggressiveness of mesenchymal-like OCSPCs from ascites which may represent a potential therapeutic target for EOC. Promotor hypomethylation at RASSF1A in OCSPCs from EOC tissues and changes to hypermethylation of EOC and OCSPCs from ascites could predict poor survival outcomes for EOC patients compared to without those changes of CCND2 and CDKN2B.

Everolimus combined with 5-aza-2-deoxycytidine generated potent anti-tumor effects on ovarian clear cell cancer stem-like/spheroid cells by inhibiting the COL6A3-AKT-mTOR pathway.

Ovarian clear cell cancer stem-like/spheroid cells (OCCCSCs) were associated with recurrence, metastasis, and chemoresistance in ovarian clear cell carcinoma (OCCC). We evaluated the anti-tumor effects of 5-aza-2-deoxycytidine (5-aza-dC) combined with everolimus (RAD001) on human OCCC. We investigated parental OCCCSCs and paclitaxel-resistant cell lines derived from OCCCSCs in vitro and in vivo. A Western blot analysis showed that the 5-aza-dC and RAD001 combination therapy was associated with the COL6A3-AKT-mTOR pathway. The OCCCSCs expressed high levels of stemness markers: CD117, ALDH1, NANOG, OCT4, and CD133. The 5-aza-dC and RAD001 combination inhibited proliferation and survival with up to 100-fold more potency in OCCCSCs compared to OCCC cells. This combination showed significant anti-tumor activity; it preferentially diminished OCCCSC stemness levels and spheroid numbers in vitro. Limiting dilution assays showed that OCCCSCs possessed tumor-initiating capacity. The 5-aza-dC and RAD001 combination significantly enhanced the inhibition of tumor growth compared to the 5-aza-dC or RAD001 alone. OCCCSCs showed higher expression levels of COL6A3, phospho-AKT, phospho-mTOR, and phospho-Rictor compared to OCCCs. Silencing COL6A3 or abolishing the phospho-AKT-mTOR-Rictor pathway with 5-aza-dC and RAD001 treatment further enhanced OCCCSC apoptosis and reduced OCCCSC stemness. In conclusion, 5-aza-dC combined with RAD001 effectively controlled OCCC and OCCCSC growth by inhibiting the COL6A3-AKT-mTOR pathway.

Predicting Long-Term Prognoses and Grading Platinum Sensitivity Using a Novel Progression-Free Interval Criterion in Ovarian Clear Cell Carcinoma: A Multi-Institutional Cohort Study.

This large-scale study aimed to determine the long-term influences of potential prognostic predictors and progression-free interval (PFI) criteria for grading platinum-sensitivity in ovarian clear cell carcinoma (OCCC). We retrospectively reviewed the medical records of OCCC patients presenting at nine tertiary centres (1995−2015), and evaluated patient characteristics, therapeutic factors, clinical outcomes, and hazard ratios for disease progression and death. We enrolled 536 patients (median follow-up, 36.6 months) and developed newly defined distributions of PFIs (seven and 14 months) for grading platinum sensitivity. In the multivariate model, preoperative CA125 levels and chemo-response independently predicted early-stage progression-free survival (PFS) risk. Post-progression cytoreduction correlated with reduced mortality risk. No unfavourable outcomes were observed with respect to coexisting endometriosis, fertility-sparing strategies, or platinum-based regimens. A PFI of <7 months, the strongest predictor of both post-progression mortality and second relapse risks, correlated with chemo-resistance, advanced tumour stage, and shortened post-progression survival. Chemotherapy regimens commonly used in front-line or relapse settings were limited in improving prognoses, especially in the advanced-stage cohort. Clinical trials of novel targeted agents and/or innovative biomarkers for chemoresistance should be comprehensively investigated and offered early to advanced-stage patients or those with OCCC progression occurring within seven months after receiving chemotherapy.

Collagen type VI regulates the CDK4/6-p-Rb signaling pathway and promotes ovarian cancer invasiveness, stemness, and metastasis.

The expression of collagen VI in primary ovarian tumors may correlate with tumor grade and response to chemotherapy. We have sought to elucidate the role of collagen VI in promoting ovarian cancer tumor growth and metastasis. Here we examined the effects of collagen VI on ovarian carcinoma stromal progenitor cells (OCSPCs). Epithelial-like OCSPCs (epi-OCSPCs) and mesenchymal-like OCSPCs (msc-OCSPCs) were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Differentially expressed genes were integrated with survival-related genes using The Cancer Genome Atlas (TCGA) data and confirmed in our samples. The roles of candidate genes and signaling pathways were further explored. We found that SKOV3/msc-OCSPCs possessed greater migration, invasion, and spheroid formation than SKOV3/epi-OCSPCs (P < 0.001). Expression of collagen alpha-3 (VI; COL6A3), which encodes collagen VI, was 90-fold higher in msc-OCSPCs than in epi-OCSPCs. Analysis of TCGA data and our samples indicated that high expression of COL6A3 was correlated with advanced-stage carcinoma (P < 0.01) and shorter overall survival (P < 0.01). In vitro, adding collagen VI, msc-OCSPCs, or knockdown collagen VI in msc-OCSPCs to epithelial ovarian carcinoma (EOC) cells augmented or decreased invasion and spheroid formation. Tumor dissemination to the peritoneal cavity and lung in mice following intraperitoneal coinjection with msc-OCSPCs and SKOV3-Luc cells and intravenous injection with COL6A3 and ES2 cells derived spheroids was significantly greater compare to coinjection with SKOV3-Luc cells alone or in combination with msc-OCSPCs/shCOL6A3 cells and msc-OCSPCs and ES2 derived spheroids. Knockdown of COL6A3 abolished the expression of DNMT1, CDK4, CDK6, and p-Rb in msc-OCSPCs and EOC spheroids. In contrast, overexpression of COL6A3 enhanced the expression of CDK4, CDK6, and p-Rb in SKOV3 cells. EOC spheroid formation, invasion, tumor growth, and metastasis were inhibited when COL6A3 downstream signaling pathway was blocked using CDK4/6 inhibitor LEE011. Our results suggested that collagen VI regulates the CDK4/6-p-Rb signaling pathway and promotes EOC invasiveness, stemness, and metastasis.

Validation of a universal and highly sensitive two-dimensional liquid chromatography-tandem mass spectrometry methodology for the quantification of pyrazinamide, ethambutol, protionamide, and clofazimine in different biological matrices.

A novel and potent anti-tuberculosis drug combination pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO), and clofazimine (CFZ) that rapidly kills Mycobacterium tuberculosis (Mtb) in the lungs has been identified using the artificial-intelligence-enabled parabolic response surface approach. A universal and highly sensitive two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method for the simultaneous determination of PZA, EMB, PTO, and CFZ in various biological samples in different states (liquid samples: plasma, bile, and urine; solid samples: tissue and feces) using simple pretreatment was established and validated. For the first dimension of this column-switching arrangement, the automated purification and enrichment of the drugs were achieved on a Polar-RP column. The subsequent analytical separation was performed on an Agilent Zorbax SB-Aq column, and the total loop time was 7.5 min. The positive-ionization mode with multiple reaction monitoring was used for detection. The sensitivity was good with no carry-over detected, and the lower limit of quantification ranged from 100 to 500 pg/mL. This quantification method was fully validated and proved to be robust in accordance with US Food and Drug Administration guidelines. High recoveries (85.3-111.4%) and accuracies (92.1-109.3%), together with high precision values (0.5-13.8%), were verified in all matrices. All standard curves showed favorable linearities with r2 > 0.995. This validated method was applied to study plasma pharmacokinetics, tissue distribution, and excretion in Sprague-Dawley rats after oral administration of the drug combination.

Enabling Technologies for Personalized and Precision Medicine.

Individualizing patient treatment is a core objective of the medical field. Reaching this objective has been elusive owing to the complex set of factors contributing to both disease and health; many factors, from genes to proteins, remain unknown in their role in human physiology. Accurately diagnosing, monitoring, and treating disorders requires advances in biomarker discovery, the subsequent development of accurate signatures that correspond with dynamic disease states, as well as therapeutic interventions that can be continuously optimized and modulated for dose and drug selection. This work highlights key breakthroughs in the development of enabling technologies that further the goal of personalized and precision medicine, and remaining challenges that, when addressed, may forge unprecedented capabilities in realizing truly individualized patient care.

Cellular Signaling Analysis shows antiviral, ribavirin-mediated ribosomal signaling modulation.

As antiviral drug resistance develops and new viruses emerge there is a pressing need to develop strategies to rapidly develop antiviral therapeutics. Here we use phospho-specific flow cytometry to assess perturbations of many different cellular signaling pathways during treatment with drug combinations that are highly effective in blocking Herpes simplex virus type 1 (HSV-1) infection. We discovered two antiviral drug combinations act on distinct signaling pathways, either STAT1 or S6 phosphorylation, to block HSV-1 infection. We focused on upregulation of S6 phosphorylation by HSV-1 infection, and our subsequent finding that ribavirin antagonizes this upregulation of S6 phosphorylation. We go on to show that the S6 kinase inhibitor SL0101 blocks HSV-1 replication in vitro and in an in vivo animal model of HSV-1 infection. Overall, we have used an unbiased analysis of cellular signaling pathways during treatment by antiviral drug combinations to discover a novel antiviral drug target against HSV-1 infection. The outcomes of the approach we present highlight the importance of analyzing how antiviral drugs modulate cellular and pathogen-induced signaling as a method to discover new drug therapy targets.

Artificial intelligence enabled parabolic response surface platform identifies ultra-rapid near-universal TB drug treatment regimens comprising approved drugs.

BACKGROUND: Shorter, more effective treatments for tuberculosis (TB) are urgently needed. While many TB drugs are available, identification of the best regimens is challenging because of the large number of possible drug-dose combinations. We have found consistently that responses of cells or whole animals to drug-dose stimulations fit a parabolic response surface (PRS), allowing us to identify and optimize the best drug combinations by testing only a small fraction of the total search space. Previously, we used PRS methodology to identify three regimens (PRS Regimens I-III) that in murine models are much more effective than the standard regimen used to treat TB. However, PRS Regimens I and II are unsuitable for treating drug-resistant TB and PRS Regimen III includes an experimental drug. Here, we use PRS methodology to identify from an expanded pool of drugs new highly effective near-universal drug regimens comprising only approved drugs. METHODS AND FINDINGS: We evaluated combinations of 15 different drugs in a human macrophage TB model and identified the most promising 4-drug combinations. We then tested 14 of these combinations in Mycobacterium tuberculosis-infected BALB/c mice and chose for PRS dose optimization and further study the two most potent regimens, designated PRS Regimens IV and V, consisting of clofazimine (CFZ), bedaquiline (BDQ), pyrazinamide (PZA), and either amoxicillin/clavulanate (AC) or delamanid (DLM), respectively. We then evaluated the efficacy in mice of optimized PRS Regimens IV and V, as well as a 3-drug regimen, PRS Regimen VI (CFZ, BDQ, and PZA), and compared their efficacy to PRS Regimen III (CFZ, BDQ, PZA, and SQ109), previously shown to reduce the time to achieve relapse-free cure in mice by 80% compared with the Standard Regimen (isoniazid, rifampicin, PZA, and ethambutol). Efficacy measurements included early bactericidal activity, time to lung sterilization, and time to relapse-free cure. PRS Regimens III-VI all rapidly sterilized the lungs and achieved relapse-free cure in 3 weeks (PRS Regimens III, V, and VI) or 5 weeks (PRS Regimen IV). In contrast, mice treated with the Standard Regimen still had high numbers of bacteria in their lungs after 6-weeks treatment and none achieved relapse-free cure in this time-period. CONCLUSIONS: We have identified three new regimens that rapidly sterilize the lungs of mice and dramatically shorten the time required to achieve relapse-free cure. All mouse drug doses in these regimens extrapolate to doses that are readily achievable in humans. Because PRS Regimens IV and V contain only one first line drug (PZA) and exclude fluoroquinolones and aminoglycosides, they should be effective against most TB cases that are multidrug resistant (MDR-TB) and many that are extensively drug-resistant (XDR-TB). Hence, these regimens have potential to shorten dramatically the time required for treatment of both drug-sensitive and drug-resistant TB. If clinical trials confirm that these regimens dramatically shorten the time required to achieve relapse-free cure in humans, then this radically shortened treatment has the potential to improve treatment compliance, decrease the emergence of drug resistance, and decrease the healthcare burden of treating both drug-sensitive and drug-resistant TB.

Simultaneous determination of the potent anti-tuberculosis regimen-Pyrazinamide, ethambutol, protionamide, clofazimine in beagle dog plasma using LC-MS/MS method coupled with 96-well format plate.

Tuberculosis is one of the top concerns in the world and acutely threatens human health. A new potent candidate regimen containing pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO) and clofazimine (CFZ) was proposed by Parabolic Response Surface/Feedback System Control (FSC/PRS) system and showed excellent outcomes in vitro and vivo studies. Here, a convenient liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneously determination of four compounds in beagle dog plasma. The plasma samples, 50 µL for each, were pretreated by methanol on 96-well format plates and a further dilution step was designed to reduce predictable matrix effect and lessen the burden of subsequent analysis. The chromatographic separation was achieved on an Agilent SB-Aq column (4.6 mm × 150 mm, 5 µm) at 30 °C by a gradient elution within 6 min. The mobile phase was a mixture of 0.2% formic acid-5 mM ammonium acetate aqueous solution (phase A) and 0.2% formic acid methanol (phase B) with a total flow rate of 1 mL/min. The 30% of post-column eluant was injected into mass spectrometer, equipped with electrospray ionization (ESI) source under positive mode and multiple-reaction monitoring (MRM). This quantification method was proved to be satisfied in selectivity, accuracy, precision, linearity (r2 > 0.998), recovery, matrix effect and stability. Under the specialized conditions, the calibration curves ranged from 20 to 5000 ng/mL for PZA, 1 to 500 ng/mL for EMB, 1 to 500 ng/mL for PTO, and 1 to 200 ng/mL for CFZ. The quantitative accuracy was further assessed under different degrees of hemolyses in detail. This method was proved to be robust and efficient, and successfully applied to the pharmacokinetic study of the new regimen in Beagle dogs.

Ultra-rapid near universal TB drug regimen identified via parabolic response surface platform cures mice of both conventional and high susceptibility.

As current treatment of tuberculosis is burdensomely long, provoking non-adherence and drug resistance, effective short-course treatments are needed. Using the output-driven parabolic response surface (PRS) platform, we have identified drug regimens that treat tuberculosis more rapidly in mice than the current Standard Regimen used in humans. We show that PRS Regimen III, comprising clofazimine, SQ109, bedaquiline and pyrazinamide, rapidly sterilizes the lung both in conventionally studied BALB/c mice and in C3HeB/FeJ mice, highly susceptible mice that develop massive necrotic granulomatous lung lesions akin to those in humans, achieving relapse-free cure in only 4 weeks (p<0.0001 versus Standard Regimen). In contrast, the Standard Regimen required 16 weeks to attain lung culture negative status and 20 weeks to achieve relapse-free cure. Thus, PRS Regimen III dramatically cuts by ~80% the time to relapse-free cure in mouse tuberculosis models. PRS Regimen III, with three nonstandard drugs, can potentially treat both drug-sensitive and most drug-resistant tuberculosis.

Optimizing drug combinations against multiple myeloma using a quadratic phenotypic optimization platform (QPOP).

Multiple myeloma is an incurable hematological malignancy that relies on drug combinations for first and secondary lines of treatment. The inclusion of proteasome inhibitors, such as bortezomib, into these combination regimens has improved median survival. Resistance to bortezomib, however, is a common occurrence that ultimately contributes to treatment failure, and there remains a need to identify improved drug combinations. We developed the quadratic phenotypic optimization platform (QPOP) to optimize treatment combinations selected from a candidate pool of 114 approved drugs. QPOP uses quadratic surfaces to model the biological effects of drug combinations to identify effective drug combinations without reference to molecular mechanisms or predetermined drug synergy data. Applying QPOP to bortezomib-resistant multiple myeloma cell lines determined the drug combinations that collectively optimized treatment efficacy. We found that these combinations acted by reversing the DNA methylation and tumor suppressor silencing that often occur after acquired bortezomib resistance in multiple myeloma. Successive application of QPOP on a xenograft mouse model further optimized the dosages of each drug within a given combination while minimizing overall toxicity in vivo, and application of QPOP to ex vivo multiple myeloma patient samples optimized drug combinations in patient-specific contexts.

Everolimus following 5-aza-2-deoxycytidine is a promising therapy in paclitaxel-resistant clear cell carcinoma of the ovary.

Our previous study showed that 5-aza-2-deoxycytidine (5-aza-dC) could inhibit tumor growth by enhancing the susceptibility of ovarian clear cell carcinoma (OCCC) to paclitaxel through decreasing AKT/mTOR expressions. The objective of the study is to evaluate the antitumor efficacy of everolimus (RAD001) and 5-aza-2-deoxycytidine (5-aza-dC) by targeting AKT/mTOR and EZH2 in OCCC. Paclitaxel-sensitive and resistant OCCC cell lines were established. In vitro proliferative and apoptotic assays and flow cytometry were performed. The expressions of EZH2, PIK3IP1, phospho-AKT, phospho-mTOR and phospho-Rictor in the OCCC cell lines were evaluated by Western blotting. In vivo animal experiments with RAD001 and 5-aza-dC were performed. RAD001 alone showed significant in vitro antitumor activity and inhibited in vivo tumor growth in paclitaxel-sensitive and resistant OCCC cells. In addition, 5-aza-dC enhanced the antitumor effects when combined with paclitaxel or RAD001 in both paclitaxel-sensitive and resistant tumors. Activation of phospho-AKT ser473 and PIK3IP1 was observed in RAD001-treated paclitaxel-sensitive and resistant OCCC cells. In contrast, inhibition of phospho-AKT ser473 and EZH2 was observed with RAD001 following 5-aza-dC treatment of paclitaxel-sensitive and resistant OCCC cells. Furthermore, RAD001 following 5-aza-dC enhanced apoptosis of paclitaxel-sensitive and resistant OCCC cells. RAD001 following 5-aza-dC may be a promising treatment strategy for the treatment of both chemo-sensitive and resistant OCCC. Further clinical studies are warranted.

Effective drug combination for Caenorhabditis elegans nematodes discovered by output-driven feedback system control technique.

Infections from parasitic nematodes (or roundworms) contribute to a significant disease burden and productivity losses for humans and livestock. The limited number of anthelmintics (or antinematode drugs) available today to treat these infections are rapidly losing their efficacy as multidrug resistance in parasites becomes a global health challenge. We propose an engineering approach to discover an anthelmintic drug combination that is more potent at killing wild-type Caenorhabditis elegans worms than four individual drugs. In the experiment, freely swimming single worms are enclosed in microfluidic drug environments to assess the centroid velocity and track curvature of worm movements. After analyzing the behavioral data in every iteration, the feedback system control (FSC) scheme is used to predict new drug combinations to test. Through a differential evolutionary search, the winning drug combination is reached that produces minimal centroid velocity and high track curvature, while requiring each drug in less than their EC50 concentrations. The FSC approach is model-less and does not need any information on the drug pharmacology, signaling pathways, or animal biology. Toward combating multidrug resistance, the method presented here is applicable to the discovery of new potent combinations of available anthelmintics on C. elegans, parasitic nematodes, and other small model organisms.

Continuous Adaptive Population Reduction (CAPR) for Differential Evolution Optimization.

Differential evolution (DE) has been applied extensively in drug combination optimization studies in the past decade. It allows for identification of desired drug combinations with minimal experimental effort. This article proposes an adaptive population-sizing method for the DE algorithm. Our new method presents improvements in terms of efficiency and convergence over the original DE algorithm and constant stepwise population reduction-based DE algorithm, which would lead to a reduced number of cells and animals required to identify an optimal drug combination. The method continuously adjusts the reduction of the population size in accordance with the stage of the optimization process. Our adaptive scheme limits the population reduction to occur only at the exploitation stage. We believe that continuously adjusting for a more effective population size during the evolutionary process is the major reason for the significant improvement in the convergence speed of the DE algorithm. The performance of the method is evaluated through a set of unimodal and multimodal benchmark functions. In combining with self-adaptive schemes for mutation and crossover constants, this adaptive population reduction method can help shed light on the future direction of a completely parameter tune-free self-adaptive DE algorithm.

Directing three-dimensional multicellular morphogenesis by self-organization of vascular mesenchymal cells in hyaluronic acid hydrogels.

BACKGROUND: Physical scaffolds are useful for supporting cells to form three-dimensional (3D) tissue. However, it is non-trivial to develop a scheme that can robustly guide cells to self-organize into a tissue with the desired 3D spatial structures. To achieve this goal, the rational regulation of cellular self-organization in 3D extracellular matrix (ECM) such as hydrogel is needed. RESULTS: In this study, we integrated the Turing reaction-diffusion mechanism with the self-organization process of cells and produced multicellular 3D structures with the desired configurations in a rational manner. By optimizing the components of the hydrogel and applying exogenous morphogens, a variety of multicellular 3D architectures composed of multipotent vascular mesenchymal cells (VMCs) were formed inside hyaluronic acid (HA) hydrogels. These 3D architectures could mimic the features of trabecular bones and multicellular nodules. Based on the Turing reaction-diffusion instability of morphogens and cells, a theoretical model was proposed to predict the variations observed in 3D multicellular structures in response to exogenous factors. It enabled the feasibility to obtain diverse types of 3D multicellular structures by addition of Noggin and/or BMP2. CONCLUSIONS: The morphological consistency between the simulation prediction and experimental results probably revealed a Turing-type mechanism underlying the 3D self-organization of VMCs in HA hydrogels. Our study has provided new ways to create a variety of self-organized 3D multicellular architectures for regenerating biomaterial and tissues in a Turing mechanism-based approach.

Detection of lower levels of SNAP25 using multiple microarray systems and its functional significance in medulloblastoma.

Medulloblastoma (MB) is the most common pediatric malignant brain tumor and patients with high-risk or recurrent MB respond poorly to current therapies, and have a higher related mortality. For this reason, potential molecules related to MB need be identified in order to develop targets for the development of novel therapeutics. In the present study, we compared MB microarray data obtained using different microarray systems and significant targets were selected by gene annotation and enrichment analysis. Genes for soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) annotated with the function 'vesicle' were identified and one of these proteins, synaptosomal-associated protein 25 (SNAP25), was found to have significantly lower expression levels in MB. In addition, SNAP25 was detected in a very low number of MB cells as shown by western blot analysis and immunohistochemical analyses of archived and formalin-fixed/paraffin-embedded human MB specimens. We found that SNAP25 altered the morphology and the chemotherapeutic effects of arabinofuranosyl cytidine (Ara-C) on SNAP25-expressing MB cells. On the whole, our data indicate that the expression of SNAP25 is crucial for dendrite formation and is associated with the effects of targeted chemotherapy. The detection of SNAP25 expression in MB cells may thus be essential for the chemotherapeutic application of Ara-C.

Drug regimens identified and optimized by output-driven platform markedly reduce tuberculosis treatment time.

The current drug regimens for treating tuberculosis are lengthy and onerous, and hence complicated by poor adherence leading to drug resistance and disease relapse. Previously, using an output-driven optimization platform and an in vitro macrophage model of Mycobacterium tuberculosis infection, we identified several experimental drug regimens among billions of possible drug-dose combinations that outperform the current standard regimen. Here we use this platform to optimize the in vivo drug doses of two of these regimens in a mouse model of pulmonary tuberculosis. The experimental regimens kill M. tuberculosis much more rapidly than the standard regimen and reduce treatment time to relapse-free cure by 75%. Thus, these regimens have the potential to provide a markedly shorter course of treatment for tuberculosis in humans. As these regimens omit isoniazid, rifampicin, fluoroquinolones and injectable aminoglycosides, they would be suitable for treating many cases of multidrug and extensively drug-resistant tuberculosis.