Puerarin prevents sepsis-associated encephalopathy by regulating the AKT1 pathway in microglia.

BACKGROUND: Previous studies have reported that puerarin possesses cardioprotective, vasodilatory, anti-inflammatory, anti-apoptotic, and hypoglycemic properties. However, the impact of puerarin on sepsis-associated encephalopathy (SAE) remains unexplored. In this study, we explored whether puerarin can modulate microglia-mediated neuroinflammation for the treatment of SAE and delved into the underlying mechanisms. METHODS: We established a murine model of SAE through intraperitoneal injection of lipopolysaccharide (LPS). The puerarin treatment group received pretreatment with puerarin. For in vitro experiments, BV2 cells were pre-incubated with puerarin for 2 h before LPS exposure. We employed network pharmacology, the Morris Water Maze (MWM) test, Novel Object Recognition (NOR) test, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA), Western blotting, and quantitative real-time PCR (qRT-PCR) to elucidate the molecular mechanism of underlying puerarin's effects in SAE treatment. RESULTS: Our findings demonstrate that puerarin significantly reduced the production of inflammatory cytokines (TNF-α and IL-6) in the peripheral blood of LPS-treated mice. Moreover, puerarin treatment markedly ameliorated sepsis-associated cognitive impairment. Puerarin also exhibited inhibitory effects on the release of TNF-α and IL-6 from microglia, thereby preventing hippocampal neuronal cell death. Network pharmacology analysis identified AKT1 as a potential therapeutic target for puerarin in SAE treatment. Subsequently, we validated these results in both in vitro and in vitro experiments. Our study conclusively demonstrated that puerarin reduced LPS-induced phosphorylation of AKT1, with the AKT activator SC79 reversing puerarin's anti-inflammatory effects through the activation of the AKT1 signaling pathway. CONCLUSION: Puerarin exerts an anti-neuroinflammatory effect against SAE by modulating the AKT1 pathway in microglia.

EGFR-T790M Mutation-Derived Interactome Rerouted EGFR Translocation Contributing to Gefitinib Resistance in Non-Small Cell Lung Cancer.

Secondary mutation, T790M, conferring tyrosine kinase inhibitors (TKIs) resistance beyond oncogenic epidermal growth factor receptor (EGFR) mutations presents a challenging unmet need. Although TKI-resistant mechanisms are intensively investigated, the underlying responses of cancer cells adapting drug perturbation are largely unknown. To illuminate the molecular basis linking acquired mutation to TKI resistance, affinity purification coupled mass spectrometry was adopted to dissect EGFR interactome in TKI-sensitive and TKI-resistant non-small cell lung cancer cells. The analysis revealed TKI-resistant EGFR-mutant interactome allocated in diverse subcellular distribution and enriched in endocytic trafficking, in which gefitinib intervention activated autophagy-mediated EGFR degradation and thus autophagy inhibition elevated gefitinib susceptibility. Alternatively, gefitinib prompted TKI-sensitive EGFR translocating toward cell periphery through Rab7 ubiquitination which may favor efficacy to TKIs suppression. This study revealed that T790M mutation rewired EGFR interactome that guided EGFR to autophagy-mediated degradation to escape treatment, suggesting that combination therapy with TKI and autophagy inhibitor may overcome acquired resistance in non-small cell lung cancer.

Choroid plexus-targeted NKCC1 overexpression to treat post-hemorrhagic hydrocephalus.

Post-hemorrhagic hydrocephalus (PHH) refers to a life-threatening accumulation of cerebrospinal fluid (CSF) that occurs following intraventricular hemorrhage (IVH). An incomplete understanding of this variably progressive condition has hampered the development of new therapies beyond serial neurosurgical interventions. Here, we show a key role for the bidirectional Na-K-Cl cotransporter, NKCC1, in the choroid plexus (ChP) to mitigate PHH. Mimicking IVH with intraventricular blood led to increased CSF [K+] and triggered cytosolic calcium activity in ChP epithelial cells, which was followed by NKCC1 activation. ChP-targeted adeno-associated viral (AAV)-NKCC1 prevented blood-induced ventriculomegaly and led to persistently increased CSF clearance capacity. These data demonstrate that intraventricular blood triggered a trans-choroidal, NKCC1-dependent CSF clearance mechanism. Inactive, phosphodeficient AAV-NKCC1-NT51 failed to mitigate ventriculomegaly. Excessive CSF [K+] fluctuations correlated with permanent shunting outcome in humans following hemorrhagic stroke, suggesting targeted gene therapy as a potential treatment to mitigate intracranial fluid accumulation following hemorrhage.

Activated AMPK-mediated glucose uptake and mitochondrial dysfunction is critically involved in the glutamate-induced oxidative injury in HT22 cell.

BACKGROUND: Accumulation of glutamate damages neurons via the reactive oxygen species (ROS) injury, which was involved in the development of neurodegenerative diseases. However, the mechanism of neuronal oxidative stress damage caused by glutamate and the intervention targets still needs to be further studied. This study explored whether 5' adenosine monophosphate-activated protein kinase (AMPK)-induced glucose metabolic and mitochondrial dysfunction were related to glutamate-dependent ROS injury of the neuron. METHODS: Neuronal oxidative stress injury was induced by glutamate treatment in HT-22 cells. Western blotting was used to evaluate the phosphorylation of the AMPK. The XF24 Flux Analyzer was used to measure the effect of glutamate and Compound C (a well-known pharmacological inhibitor of AMPK phosphorylation) on the cellular oxygen consumption rate (OCR) of HT-22 cells. Glucose uptake, intracellular ROS, mitochondrial potential, apoptosis and cell viability were quantified using biochemical assays. RESULTS: Glutamate caused the phosphorylation of AMPK and subsequently promoted the glucose uptake. Furthermore, AMPK-mediated glucose uptake enhanced OCR and increased the intracellular ROS levels in neurons. The pharmacological inhibition of AMPK phosphorylation by Compound C attenuated glutamate-induced toxicity in HT22 cells by regulating the glucose uptake/mitochondrial respiration/ROS pathway. CONCLUSIONS: The AMPK phosphorylation/glucose uptake/mitochondrial respiration/ROS pathway was involved in glutamate-induced excitotoxic injury in HT22 cells. The inhibition of AMPK phosphorylation may be a potential target for the development of therapeutic agents for treating the glutamate-induced neurotoxicity.

Development of a Predictive Model for Optimization of Embryo Transfer Timing Using Blood-Based microRNA Expression Profile.

MicroRNAs (miRNAs) can regulate the expression of genes involved in the establishment of the window of implantation (WOI) in the endometrium. Recent studies indicated that cell-free miRNAs in uterine fluid and blood samples could act as alternative and non-invasive sample types for endometrial receptivity analysis. In this study, we attempt to systematically evaluate whether the expression levels of cell-free microRNAs in blood samples could be used as non-invasive biomarkers for assessing endometrial receptivity status. We profiled the miRNA expression levels of 111 blood samples using next-generation sequencing to establish a predictive model for the assessment of endometrial receptivity status. This model was validated with an independent dataset (n = 73). The overall accuracy is 95.9%. Specifically, we achieved accuracies of 95.9%, 95.9%, and 100.0% for the pre-receptive group, the receptive group, and the post-respective group, respectively. Additionally, we identified a set of differentially expressed miRNAs between different endometrial receptivity statuses using the following criteria: p-value < 0.05 and fold change greater than 1.5 or less than -1.5. In conclusion, the expression levels of cell-free miRNAs in blood samples can be utilized in a non-invasive manner to distinguish different endometrial receptivity statuses.

Improving Infant Hydrocephalus Outcomes in Uganda: A Longitudinal Prospective Study Protocol for Predicting Developmental Outcomes and Identifying Patients at Risk for Early Treatment Failure after ETV/CPC.

Infant hydrocephalus poses a severe global health burden; 80% of cases occur in the developing world where patients have limited access to neurosurgical care. Surgical treatment combining endoscopic third ventriculostomy and choroid plexus cauterization (ETV/CPC), first practiced at CURE Children's Hospital of Uganda (CCHU), is as effective as standard ventriculoperitoneal shunt (VPS) placement while requiring fewer resources and less post-operative care. Although treatment focuses on controlling ventricle size, this has little association with treatment failure or long-term outcome. This study aims to monitor the progression of hydrocephalus and treatment response, and investigate the association between cerebral physiology, brain growth, and neurodevelopmental outcomes following surgery. We will enroll 300 infants admitted to CCHU for treatment. All patients will receive pre/post-operative measurements of cerebral tissue oxygenation (SO2), cerebral blood flow (CBF), and cerebral metabolic rate of oxygen consumption (CMRO2) using frequency-domain near-infrared combined with diffuse correlation spectroscopies (FDNIRS-DCS). Infants will also receive brain imaging, to monitor tissue/ventricle volume, and neurodevelopmental assessments until two years of age. This study will provide a foundation for implementing cerebral physiological monitoring to establish evidence-based guidelines for hydrocephalus treatment. This paper outlines the protocol, clinical workflow, data management, and analysis plan of this international, multi-center trial.

Comparing visual inspection and performance observation for evaluation of hospital cleanliness.

BACKGROUND: Environmental cleaning is an effective measure to prevent infections. However, performance observation has been rarely delineated. This study aimed to compare correlations among visual inspection, performance observation, and effectiveness by using adenosine triphosphate bioluminescence (ATP bioluminescence) as a comparator to find out which method is better to assess hospital cleanliness. METHODS: This prospective study was conducted at a medical center from April 2019 to October 2020. Seven high-touch surfaces were evaluated during and after terminal cleaning by performance observation, visual inspection, and ATP bioluminescence. RESULTS: The scores by performance observation, visual inspection, and ATP were 55.4%, 87.5%, and 26.6% after cleaning. The correlations between performance observation and visual inspection and between performance observation and ATP interpretation were weak positive (φ = 0.300, 0.324, P < .001). No correlation was between the visual inspection and ATP interpretation (φ=0.137). The median of ATP readings was lower in "compliant" group by performance observation and "clean" group by visual inspection than "not compliant" group and "not clean" group (P < .001). CONCLUSIONS: Performance observation combined with ATP would be preferred to include to audit cleanliness on high-risk surfaces. Visual inspection would be a rapid and time-saving assessment tool on low-risk surfaces.

A data-independent acquisition-based global phosphoproteomics system enables deep profiling.

Phosphoproteomics can provide insights into cellular signaling dynamics. To achieve deep and robust quantitative phosphoproteomics profiling for minute amounts of sample, we here develop a global phosphoproteomics strategy based on data-independent acquisition (DIA) mass spectrometry and hybrid spectral libraries derived from data-dependent acquisition (DDA) and DIA data. Benchmarking the method using 166 synthetic phosphopeptides shows high sensitivity (<0.1 ng), accurate site localization and reproducible quantification (~5% median coefficient of variation). As a proof-of-concept, we use lung cancer cell lines and patient-derived tissue to construct a hybrid phosphoproteome spectral library covering 159,524 phosphopeptides (88,107 phosphosites). Based on this library, our single-shot streamlined DIA workflow quantifies 36,350 phosphosites (19,755 class 1) in cell line samples within two hours. Application to drug-resistant cells and patient-derived lung cancer tissues delineates site-specific phosphorylation events associated with resistance and tumor progression, showing that our workflow enables the characterization of phosphorylation signaling with deep coverage, high sensitivity and low between-run missing values.

Integrating site-specific peptide reporters and targeted mass spectrometry enables rapid substrate-specific kinase assay at the nanogram cell level.

Dysregulation of phosphorylation-mediated signaling drives the initiation and progression of many diseases. A substrate-specific kinase assay capable of quantifying the altered site-specific phosphorylation of its phenotype-dependent substrates provides better specificity to monitor a disease state. We report a sensitive and rapid substrate-specific kinase assay by integrating site-specific peptide reporter and multiple reaction monitoring (MRM)-MS platform for relative and absolute quantification of substrate-specific kinase activity at the sensitivity of nanomolar kinase and nanogram cell lysate. Using non-small cell lung cancer as a proof-of-concept, three substrate peptides selected from constitutive phosphorylation in tumors (HDGF-S165, RALY-S135, and NRD1-S94) were designed to demonstrate the feasibility. The assay showed good accuracy (<15% nominal deviation) and reproducibility (<15% CV). In PC9 cells, the measured activity for HDGF-S165 was 3.2 ± 0.2 fmol µg-1 min-1, while RALY-S135 and NRD1-S94 showed 4- and 20-fold higher activity at the sensitivity of 25 ng and 5 ng lysate, respectively, suggesting different endogenous kinases for each substrate peptide. Without the conventional shotgun phosphoproteomics workflow, the overall pipeline from cell lysate to MS data acquisition only takes 3 h. The multiplexed analysis revealed differences in the phenotype-dependent substrate phosphorylation profiles across six NSCLC cell lines and suggested a potential association of HDGF-S165 and NRD1-S94 with TKI resistance. With the ease of design, sensitivity, accuracy, and reproducibility, this approach may offer rapid and sensitive assays for targeted quantification of the multiplexed substrate-specific kinase activity of small amounts of sample.

Choroid plexus NKCC1 mediates cerebrospinal fluid clearance during mouse early postnatal development.

Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation, such as hydrocephalus, can negatively affect perinatal neurodevelopment. The mechanisms regulating CSF clearance during the postnatal critical period are unclear. Here, we show that CSF K+, accompanied by water, is cleared through the choroid plexus (ChP) during mouse early postnatal development. We report that, at this developmental stage, the ChP showed increased ATP production and increased expression of ATP-dependent K+ transporters, particularly the Na+, K+, Cl-, and water cotransporter NKCC1. Overexpression of NKCC1 in the ChP resulted in increased CSF K+ clearance, increased cerebral compliance, and reduced circulating CSF in the brain without changes in intracranial pressure in mice. Moreover, ChP-specific NKCC1 overexpression in an obstructive hydrocephalus mouse model resulted in reduced ventriculomegaly. Collectively, our results implicate NKCC1 in regulating CSF K+ clearance through the ChP in the critical period during postnatal neurodevelopment in mice.

Proteogenomics of Non-smoking Lung Cancer in East Asia Delineates Molecular Signatures of Pathogenesis and Progression.

Lung cancer in East Asia is characterized by a high percentage of never-smokers, early onset and predominant EGFR mutations. To illuminate the molecular phenotype of this demographically distinct disease, we performed a deep comprehensive proteogenomic study on a prospectively collected cohort in Taiwan, representing early stage, predominantly female, non-smoking lung adenocarcinoma. Integrated genomic, proteomic, and phosphoproteomic analysis delineated the demographically distinct molecular attributes and hallmarks of tumor progression. Mutational signature analysis revealed age- and gender-related mutagenesis mechanisms, characterized by high prevalence of APOBEC mutational signature in younger females and over-representation of environmental carcinogen-like mutational signatures in older females. A proteomics-informed classification distinguished the clinical characteristics of early stage patients with EGFR mutations. Furthermore, integrated protein network analysis revealed the cellular remodeling underpinning clinical trajectories and nominated candidate biomarkers for patient stratification and therapeutic intervention. This multi-omic molecular architecture may help develop strategies for management of early stage never-smoker lung adenocarcinoma.

Effects of food supplementation on cognitive function, cerebral blood flow, and nutritional status in young children at risk of undernutrition: randomized controlled trial.

OBJECTIVE: To assess the effects of food supplementation on improving working memory and additional measures including cerebral blood flow in children at risk of undernutrition. DESIGN: Randomized controlled trial. SETTING: 10 villages in Guinea-Bissau. PARTICIPANTS: 1059 children aged 15 months to 7 years; children younger than 4 were the primary population. INTERVENTIONS: Supervised isocaloric servings (≈1300 kJ, five mornings each week, 23 weeks) of a new food supplement (NEWSUP, high in plant polyphenols and omega 3 fatty acids, within a wide variety and high fortification of micronutrients, and a high protein content), or a fortified blended food (FBF) used in nutrition programs, or a control meal (traditional rice breakfast). MAIN OUTCOME MEASUREMENTS: The primary outcome was working memory, a core executive function predicting long term academic achievement. Additional outcomes were hemoglobin concentration, growth, body composition, and index of cerebral blood flow (CBFi). In addition to an intention-to-treat analysis, a predefined per protocol analysis was conducted in children who consumed at least 75% of the supplement (820/925, 89%). The primary outcome was assessed by a multivariable Poisson model; other outcomes were assessed by multivariable linear mixed models. RESULTS: Among children younger than 4, randomization to NEWSUP increased working memory compared with the control meal (rate ratio 1.20, 95% confidence interval 1.02 to 1.41, P=0.03), with a larger effect in the per protocol population (1.25, 1.06 to 1.47, P=0.009). NEWSUP also increased hemoglobin concentration among children with anemia (adjusted mean difference 0.65 g/dL, 95% confidence interval 0.23 to 1.07, P=0.003) compared with the control meal, decreased body mass index z score gain (-0.23, -0.43 to -0.02, P=0.03), and increased lean tissue accretion (2.98 cm2, 0.04 to 5.92, P=0.046) with less fat (-5.82 cm2, -11.28 to -0.36, P=0.04) compared with FBF. Additionally, NEWSUP increased CBFi compared with the control meal and FBF in both age groups combined (1.14 mm2/s×10-8, 0.10 to 2.23, P=0.04 for both comparisons). Among children aged 4 and older, NEWSUP had no significant effect on working memory or anemia, but increased lean tissue compared with FBF (4.31 cm2, 0.34 to 8.28, P=0.03). CONCLUSIONS: Childhood undernutrition is associated with long term impairment in cognition. Contrary to current understanding, supplementary feeding for 23 weeks could improve executive function, brain health, and nutritional status in vulnerable young children living in low income countries. Further research is needed to optimize nutritional prescriptions for regenerative improvements in cognitive function, and to test effectiveness in other vulnerable groups. TRIAL REGISTRATION: ClinicalTrials.gov NCT03017209.

Silencing of circFoxO3 Protects HT22 Cells from Glutamate-Induced Oxidative Injury via Regulating the Mitochondrial Apoptosis Pathway.

Recent studies demonstrated that FoxO3 circular RNA (circFoxO3) plays an important regulatory role in tumourigenesis and cardiomyopathy. However, the role of circFoxO3 in neurodegenerative diseases remains unknown. The aim of this study was to examine the possible role of circFoxO3 in neurodegenerative diseases and the underlying mechanisms. To model human neurodegenerative conditions, hippocampus-derived neurons were treated with glutamate. Using molecular and cellular biology approaches, we found that circFoxO3 expression was significantly higher in the glutamate treatment group than that in the control group. Furthermore, silencing of circFoxO3 protected HT22 cells from glutamate-induced oxidative injury through the inhibition of the mitochondrial apoptotic pathway. Collectively, our study demonstrates that endogenous circFoxO3 plays a key role in inducing apoptosis and neuronal cell death and may act as a novel therapeutic target for neurodegenerative diseases.

Dysregulation of Dual-Specificity Phosphatases by Epstein-Barr Virus LMP1 and Its Impact on Lymphoblastoid Cell Line Survival.

The strongest evidence of the oncogenicity of Epstein-Barr virus (EBV) in vitro is its ability to immortalize human primary B lymphocytes into lymphoblastoid cell lines (LCLs). Yet the underlying mechanisms explaining how the virus tempers the growth program of the host cells have not been fully elucidated. The mitogen-activated protein kinases (MAPKs) are implicated in many cellular processes and are constitutively activated in LCLs. We questioned the expression and regulation of the dual-specificity phosphatases (DUSPs), the main negative regulator of MAPKs, during EBV infection and immortalization. Thirteen DUSPs, including 10 typical and 3 atypical types of DUSPs, were tested. Most of them were downregulated after EBV infection. Here, a role of viral oncogene latent membrane protein 1 (LMP1) in limiting DUSP6 and DUSP8 expression was identified. Using MAPK inhibitors, we found that LMP1 activates extracellular signal-regulated kinase (ERK) or p38 to repress the expression of DUSP6 and DUSP8, with corresponding substrate specificity. Morphologically, overexpression of DUSP6 and DUSP8 attenuates the ability of EBV-immortalized LCL cells to clump together. Mechanistically, apoptosis induced by restoring DUSP6 and DUSP8 in LCLs indicated a novel mechanism for LMP1 to provide a survival signal during EBV immortalization. Collectively, this report provides the first description of the interplay between EBV genes and DUSPs and contributes considerably to the interpretation of MAPK regulation in EBV immortalization.IMPORTANCE Infections by the ubiquitous Epstein-Barr virus (EBV) are associated with a wide spectrum of lymphomas and carcinomas. It has been well documented that activation levels of MAPKs are found in cancer cells to translate various external or intrinsic stimuli into cellular responses. Physiologically, the dual-specificity phosphates (DUSPs) exhibit great ability in regulating MAPK activities with respect to their capability of dephosphorylating MAPKs. In this study, we found that DUSPs were generally downregulated after EBV infection. EBV oncogenic latent membrane protein 1 (LMP1) suppressed DUSP6 and DUSP8 expression via MAPK pathway. In this way, LMP1-mediated MAPK activation was a continuous process. Furthermore, DUSP downregulation was found to contribute greatly to prevent apoptosis of EBV-infected cells. To sum up, this report sheds light on a novel molecular mechanism explaining how EBV maintains the unlimited proliferation status of the immortalized cells and provides a new link to understand EBV-induced B cell survival.

A novel 3D-printed computer-assisted piezocision guide for surgically facilitated orthodontics.

Surgical interventions on the alveolar ridges aimed at facilitating orthodontic tooth movement have been extensively reported. However, unexpected events or complications still occur in daily practice. The purpose of this report was to present a novel 3-dimensional (3D) computer-assisted piezocision guide (CAPG) designed to be translucent for increased visibility, rigid for enhanced support during guidance, and porous for profuse irrigation during procedure. Such a design can function to minimize the risk of surgical complications. In this case, we present a novel 3D-printed CAPG to facilitate a minimally invasive periodontal accelerated osteogenic orthodontics (PAOO) procedure with a guide that provides accuracy, adequate visibility, and greater access for the coolant to reach the surgery site. By navigating the cone-beam computed tomography data, we precisely know the cortical bone thickness, root direction, and interrelations between anatomic structures in an individual situation, which allows us to design our cutting slot for the required length and depth according to the operator's knowledge. Finally, 3D printing was applied, transferring our surgical plan to fabricate the CAPG. Moreover, the well designed pores on the CAPG allow effective irrigation during the piezocision procedure. This minimally invasive procedure was uneventful, and no devitalized tooth or alveolar bone was found.

Neuroprotective Effects of HSF1 in Retinal Ischemia-Reperfusion Injury.

Purpose: Retinal ischemia, a common cause of several vision-threatening diseases, contributes to the death of retinal neurons, particularly retinal ganglion cells (RGCs). Heat shock transcription factor 1 (HSF1), a stress-responsive protein, has been shown to be important in response to cellular stress stimuli, including ischemia. This study is to investigate whether HSF1 has a role in retinal neuronal injury in a mouse model of retinal ischemia-reperfusion (IR). Methods: IR was induced by inserting an infusion needle into the anterior chamber of the right eye and elevating a saline reservoir connected to the needle to raise the intraocular pressure to 110 mm Hg for 45 minutes. HSF1, Hsp70, molecules in the endoplasmic reticulum (ER) stress branches, tau phosphorylation, inflammatory molecules, and RGC injury were determined by immunohistochemistry, Western blot, or quantitative PCR. Results: HSF1 expression was significantly increased in the retina 6 hours after IR. Using our novel transgenic mice carrying full-length human HSF gene, we demonstrated that IR-induced retinal neuronal apoptosis and necroptosis were abrogated 12 hours after IR. RGCs and their function were preserved in the HSF1 transgenic mice 7 days after IR. Mechanistically, the beneficial effects of HSF1 may be mediated by its induction of chaperone protein Hsp70 and alleviation of ER stress, leading to decreased tau phosphorylation and attenuated inflammatory response 12 to 24 hours after IR. Conclusions: These data provide compelling evidence that HSF1 is neuroprotective against retinal IR injury, and boosting HSF1 expression may be a beneficial strategy to limit neuronal degeneration in retinal diseases.

Constitutively active MyD88/CD40 costimulation enhances expansion and efficacy of chimeric antigen receptor T cells targeting hematological malignancies.

Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting "low" cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.

Targeted Sequencing of Circulating Tumor DNA to Monitor Genetic Variants and Therapeutic Response in Metastatic Colorectal Cancer.

Substantial improvements have been made in the management of metastatic colorectal cancer (mCRC) in the last two decades, but disease monitoring remains underdeveloped. Circulating tumor DNA (ctDNA) is a promising prognostic and predictive biomarker; however, ctDNA as a marker for mCRC patients is not well established, and there is still no consensus about how to utilize it most cost-effectively. In this study, we aim to investigate plasma ctDNA levels as a biomarker for therapeutic response of mCRC patients. We performed next-generation sequencing (NGS) by using a 12-gene panel to identify genetic variants in 136 tumor tissue and ctDNA samples from 32 mCRC patients. Genetic variants were detected in approximately 70% of samples, and there was a high concordance (85%) between tumor tissue and plasma ctDNA. We observed ctDNA changes in 18 follow-up patients, including the emergence of new variants. Changes in ctDNA levels significantly correlated with tumor shrinkage (P = 0.041), and patients with a ctDNA decrease >80% after treatment had a longer progression-free survival compared with patients with a ctDNA decrease of <80% (HR, 0.22; P = 0.015). The objective response rate among patients with a ctDNA decrease of >80% was better than those with a ctDNA decrease <80% (OR, 0.026; P = 0.007). In conclusion, this study demonstrates that monitoring of genetic ctDNA variants can serve as a valuable biomarker for therapeutic efficacy in mCRC patients, and that using a moderate-sized 12-gene NGS panel may be suitable for such clinical monitoring. Mol Cancer Ther; 17(10); 2238-47. ©2018 AACR.

Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments.

T cells compete with malignant cells for limited nutrients within the solid tumor microenvironment. We found that effector memory CD4 T cells respond distinctly from other T cell subsets to limiting glucose and can maintain high levels of interferon-γ (IFN-γ) production in a nutrient-poor environment. Unlike naive (TN) or central memory T (TCM) cells, effector memory T (TEM) cells fail to upregulate fatty acid synthesis, oxidative phosphorylation, and reductive glutaminolysis in limiting glucose. Interference of fatty acid synthesis in naive T cells dramatically upregulates IFN-γ, while increasing exogenous lipids in media inhibits production of IFN-γ by all subsets, suggesting that relative ratio of fatty acid metabolism to glycolysis is a direct predictor of T cell effector activity. Together, these data suggest that effector memory T cells are programmed to have limited ability to synthesize and metabolize fatty acids, which allows them to maintain T cell function in nutrient-depleted microenvironments.

A study to evaluate the potential of an in silico approach for predicting dipeptidyl peptidase-IV inhibitory activity in vitro of protein hydrolysates.

A total of 294 edible protein sequences and 5 commercial proteases listed in the BIOPEP database were analyzed in silico. The frequency (A), a parameter in silico described previously, was examined further to calculating the ratio of truncated peptides with Xaa-proline and/or Xaa-alanine to all peptide fragments in a protein hydrolyzed with a protease, using the BIOPEP database. Then the in vitro DPP-IV inhibitory activity was determined using the same 15 protein and protease combinations to evaluate their relationship. The result shows that A values considering the number of Xaa-proline+Xaa-alanine exhibited a strong correlation with in vitro DPP-IV inhibition rates by Pearson's correlation analysis (r=0.6993; P<0.05). Therefore, the in silico approach is effective to predict DPP-IV inhibitory activities in vitro of protein hydrolysates.