Peste des petits ruminants virus non-structural V and C proteins interact with the NF-κB p65 subunit and modulate pro-inflammatory cytokine gene induction.

Peste des petits ruminants virus (PPRV) is known to induce transient immunosuppression in infected small ruminants by modulating several cellular pathways involved in the antiviral immune response. Our study shows that the PPRV-coded non-structural proteins C and V can interact with the cellular NF-κB p65 subunit. The PPRV-C protein interacts with the transactivation domain (TAD) while PPRV-V interacts with the Rel homology domain (RHD) of the NF-κB p65 subunit. Both viral proteins can suppress the NF-κB transcriptional activity and NF-κB-mediated transcription of cellular genes. PPRV-V protein expression can significantly inhibit the nuclear translocation of NF-κB p65 upon TNF-α stimulation, whereas PPRV-C does not affect it. The NF-κB-mediated pro-inflammatory cytokine gene expression is significantly downregulated in cells expressing PPRV-C or PPRV-V protein. Our study provides evidence suggesting a role of PPRV non-structural proteins V and C in the modulation of NF-κB signalling through interaction with the NF-κB p65 subunit.

Nucleocapsid Protein (N) of Peste des petits ruminants Virus (PPRV) Interacts with Cellular Phosphatidylinositol-3-Kinase (PI3K) Complex-I and Induces Autophagy.

Autophagy is an essential and highly conserved catabolic process in cells, which is important in the battle against intracellular pathogens. Viruses have evolved several ways to alter the host defense mechanisms. PPRV infection is known to modulate the components of a host cell's defense system, resulting in enhanced autophagy. In this study, we demonstrate that the N protein of PPRV interacts with the core components of the class III phosphatidylinositol-3-kinase (PI3K) complex-I and results in the induction of autophagy in the host cell over, thereby expressing this viral protein. Our data shows the interaction between PPRV-N protein and different core components of the autophagy pathway, i.e., VPS34, VPS15, BECN1 and ATG14L. The PPRV-N protein can specifically interact with VPS34 of the PI3K complex-I and colocalize with the proteins of PI3K complex-I in the same sub-cellular compartment, that is, in the cytoplasm. These interactions do not affect the intracellular localization of the different host proteins. The autophagy-related genes were transcriptionally modulated in PPRV-N-expressing cells. The expression of LC3B and SQSTM1/p62 was also modulated in PPRV-N-expressing cells, indicating the induction of autophagic activity. The formation of typical autophagosomes with double membranes was visualized by transmission electron microscopy in PPRV-N-expressing cells. Taken together, our findings provide evidence for the critical role of the N protein of the PPR virus in the induction of autophagy, which is likely to be mediated by PI3K complex-I of the host.

Constitutively Synergistic Multiagent Drug Formulations Targeting MERTK, FLT3, and BCL-2 for Treatment of AML.

PURPOSE: Although high-dose, multiagent chemotherapy has improved leukemia survival rates, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. The development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. METHODS: The dual MERTK/FLT3 inhibitor MRX-2843 and BCL-2 family protein inhibitors were screened in high-throughput against a panel of AML and MLL-rearranged precursor B-cell ALL (infant ALL) cell lines. A neural network model was built to correlate ratiometric drug synergy and target gene expression. Drugs were loaded into liposomal nanocarriers to assess primary AML cell responses. RESULTS: MRX-2843 synergized with venetoclax to reduce AML cell density in vitro. A neural network classifier based on drug exposure and target gene expression predicted drug synergy and growth inhibition in AML with high accuracy. Combination monovalent liposomal drug formulations delivered defined drug ratios intracellularly and recapitulated synergistic drug activity. The magnitude and frequency of synergistic responses were both maintained and improved following drug formulation in a genotypically diverse set of primary AML bone marrow specimens. CONCLUSIONS: We developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and infant ALL cells. We demonstrate ratiometric drug delivery and synergistic cell killing in AML, a result achieved by a systematic, generalizable approach of combination drug screening and nanoscale formulation that may be extended to other drug pairs or diseases in the future.

Development of constitutively synergistic nanoformulations to enhance chemosensitivity in T-cell leukemia.

Advances in multiagent chemotherapy have led to recent improvements in survival for patients with acute lymphoblastic leukemia (ALL); however, a significant fraction do not respond to frontline chemotherapy or later relapse with recurrent disease, after which long-term survival rates remain low. To develop new, effective treatment options for these patients, we conducted a series of high-throughput combination drug screens to identify chemotherapies that synergize in a lineage-specific manner with MRX-2843, a small molecule dual MERTK and FLT3 kinase inhibitor currently in clinical testing for treatment of relapsed/refractory leukemias and solid tumors. Using experimental and computational approaches, we found that MRX-2843 synergized strongly-and in a ratio-dependent manner-with vincristine to inhibit both B-ALL and T-ALL cell line expansion. Based on these findings, we developed multiagent lipid nanoparticle formulations of these drugs that not only delivered defined drug ratios intracellularly in T-ALL, but also improved anti-leukemia activity following drug encapsulation. Synergistic and additive interactions were recapitulated in primary T-ALL patient samples treated with MRX-2843 and vincristine nanoparticle formulations, suggesting their clinical relevance. Moreover, the nanoparticle formulations reduced disease burden and prolonged survival in an orthotopic murine xenograft model of early thymic precursor T-ALL (ETP-ALL), with both agents contributing to therapeutic activity in a dose-dependent manner. In contrast, nanoparticles containing MRX-2843 alone were ineffective in this model. Thus, MRX-2843 increased the sensitivity of ETP-ALL cells to vincristine in vivo. In this context, the additive particles, containing a higher dose of MRX-2843, provided more effective disease control than the synergistic particles. In contrast, particles containing an even higher, antagonistic ratio of MRX-2843 and vincristine were less effective. Thus, both the drug dose and the ratio-dependent interaction between MRX-2843 and vincristine significantly impacted therapeutic activity in vivo. Together, these findings present a systematic approach to high-throughput combination drug screening and multiagent drug delivery that maximizes the therapeutic potential of combined MRX-2843 and vincristine in T-ALL and describe a novel translational agent that could be used to enhance therapeutic responses to vincristine in patients with T-ALL. This broadly generalizable approach could also be applied to develop other constitutively synergistic combination products for the treatment of cancer and other diseases.

Constitutively synergistic multiagent drug formulations targeting MERTK, FLT3, and BCL-2 for treatment of AML.

Although high-dose, multi-agent chemotherapy has improved leukemia survival rates in recent years, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. Development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. To address this challenge, we developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and MLL- rearranged precursor B-cell ALL (infant ALL). In a novel, high-throughput combination drug screen, the MERTK/FLT3 inhibitor MRX-2843 synergized with venetoclax and other BCL-2 family protein inhibitors to reduce AML cell density in vitro . Neural network models based on drug exposure and target gene expression were used to identify a classifier predictive of drug synergy in AML. To maximize the therapeutic potential of these findings, we developed a combination monovalent liposomal drug formulation that maintains ratiometric drug synergy in cell-free assays and following intracellular delivery. The translational potential of these nanoscale drug formulations was confirmed in a genotypically diverse set of primary AML patient samples and both the magnitude and frequency of synergistic responses were not only maintained but were improved following drug formulation. Together, these findings demonstrate a systematic, generalizable approach to combination drug screening, formulation, and development that maximizes therapeutic potential, was effectively applied to develop a novel nanoscale combination therapy for treatment of AML, and could be extended to other drug combinations or diseases in the future.

Therapeutic Targeting of MERTK and BCL-2 in T-Cell and Early T-Precursor Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of childhood ALL. The early T-precursor (ETP-ALL) subset is characterized by an immature T-cell phenotype, chemoresistance, and high rates of induction failure. MERTK receptor tyrosine kinase is ectopically expressed in half of T-ALLs, particularly those with an immature T-cell phenotype, suggesting a role in ETP-ALL. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is essential for ETP-ALL cell survival. Here, we show that MERTK and BCL-2 mRNA and protein are preferentially expressed in ETP-ALL patient samples. The dual MERTK/FLT3 inhibitor MRX-2843 decreased MERTK activation and downstream signaling, inhibited cell expansion, and induced cell death in ETP-ALL cell lines. Further, 54% (21/39) of primary T-ALL patient samples were sensitive to MERTK inhibition. Treatment with MRX-2843 significantly reduced leukemia burden and prolonged survival in cell-line-derived T-ALL and ETP-ALL xenograft models. In a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 markedly reduced peripheral blood leukemia and spleen weight compared to vehicle-treated mice and prolonged survival. MRX-2843 also synergized with venetoclax to provide enhanced anti-leukemia activity in ETP-ALL cell cultures, with a dose ratio of 1:20 MRX-2843:venetoclax providing optimal synergy. These data demonstrate the therapeutic potential of MRX-2843 in patients with T-ALL and provide rationale for clinical development. MRX-2843 monotherapy is currently being tested in patients with relapsed leukemia (NCT04872478). Further, our data indicate that combined MERTK and BCL-2 inhibition may be particularly effective for treatment of ETP-ALL.

Evaluation for Bleeding Disorders in Suspected Child Abuse.

Bruising or bleeding in a child can raise the concern for child abuse. Assessing whether the findings are the result of trauma and/or whether the child has a bleeding disorder is critical. Many bleeding disorders are rare, and not every child with bruising/bleeding that may raise a concern for abuse requires an evaluation for bleeding disorders. However, in some instances, bleeding disorders can present in a manner similar to child abuse. Bleeding disorders cannot be ruled out solely on the basis of patient and family history, no matter how extensive. The history and clinical evaluation can be used to determine the necessity of an evaluation for a possible bleeding disorder, and prevalence and known clinical presentations of individual bleeding disorders can be used to guide the extent of laboratory testing. This clinical report provides guidance to pediatricians and other clinicians regarding the evaluation for bleeding disorders when child abuse is suspected.

Creating a Collaborative Trauma-Informed Interdisciplinary Citywide Victim Services Model Focused on Health Care for Survivors of Human Trafficking.

Although human trafficking is recognized as a public health issue, research on the health effects of human trafficking and best intervention practices is limited. We describe 2 citywide collaborative victim services models, the THRIVE (Trafficking, Healthcare, Resources, and Interdisciplinary Victim Services and Education) Clinic at the University of Miami and Jackson Health System in Miami, Florida, and the Greater Houston Area Pathways for Advocacy-based, Trauma-Informed Healthcare (PATH) Collaborative at Baylor College of Medicine, CommonSpirit Health, and San Jose Clinic in Houston, Texas, funded in part by the Office for Victims of Crime, which focus on trauma-informed health care delivery for victims of human trafficking. From June 2015 through September 2021, the THRIVE Clinic served 214 patients with an average age of 28.7 years at the time of their first visit. From October 2017 through September 2021, the PATH Collaborative received 560 suspected trafficking referrals, 400 of which screened positive for labor or sex trafficking. These models serve as a framework for replication of interdisciplinary practices to provide health care for this unique population and preliminary information about the strategies put in place to assist victims during their recovery. Key lessons include the importance of a citywide needs assessment, patient navigators, interdisciplinary care, and building community partnerships to ensure safe housing, transportation, identification, health insurance, vocation services, input from survivors, peer-to-peer mentorship, and medical-legal services. Further research is needed to understand the detrimental health effects of trafficking and the health care needs of victims. In addition, a need exists to develop optimal models of care for recovery and reintegration for this patient population and to address public health, legal, and medical policies to ensure access to and sustainability of comprehensive, trauma-informed, interdisciplinary victim services.

Predicting defects in imbalanced data using resampling methods: an empirical investigation.

The development of correct and effective software defect prediction (SDP) models is one of the utmost needs of the software industry. Statistics of many defect-related open-source data sets depict the class imbalance problem in object-oriented projects. Models trained on imbalanced data leads to inaccurate future predictions owing to biased learning and ineffective defect prediction. In addition to this large number of software metrics degrades the model performance. This study aims at (1) identification of useful metrics in the software using correlation feature selection, (2) extensive comparative analysis of 10 resampling methods to generate effective machine learning models for imbalanced data, (3) inclusion of stable performance evaluators-AUC, GMean, and Balance and (4) integration of statistical validation of results. The impact of 10 resampling methods is analyzed on selected features of 12 object-oriented Apache datasets using 15 machine learning techniques. The performances of developed models are analyzed using AUC, GMean, Balance, and sensitivity. Statistical results advocate the use of resampling methods to improve SDP. Random oversampling portrays the best predictive capability of developed defect prediction models. The study provides a guideline for identifying metrics that are influential for SDP. The performances of oversampling methods are superior to undersampling methods.

Engineered Cytokines for Cancer and Autoimmune Disease Immunotherapy.

Cytokine signaling is critical to a range of biological processes including cell development, tissue repair, aging, and immunity. In addition to acting as key signal mediators of the immune system, cytokines can also serve as potent immunotherapies with more than 20 recombinant products currently Food and Drug Administration (FDA)-approved to treat conditions including hepatitis, multiple sclerosis, arthritis, and various cancers. Yet despite their biological importance and clinical utility, cytokine immunotherapies suffer from intrinsic challenges that limit their therapeutic potential including poor circulation, systemic toxicity, and low tissue- or cell-specificity. In the past decade in particular, methods have been devised to engineer cytokines in order to overcome such challenges and here, the myriad strategies are reviewed that may be employed in order to improve the therapeutic potential of cytokine and chemokine immunotherapies with applications in cancer and autoimmune disease therapy, as well as tissue engineering and regenerative medicine. For clarity, these strategies are collected and presented as they vary across size scales, ranging from single amino acid substitutions, to larger protein-polymer conjugates, nano/micrometer-scale particles, and macroscale implants. Together, this work aims to provide readers with a timely view of the field of cytokine engineering with an emphasis on early-stage therapeutic approaches.

Progress Update in Pediatric Renal Tumors.

PURPOSE OF REVIEW: Pediatric renal tumors account for 7% of new cancer diagnoses in children. Here, we will review results from recently completed clinical trials informing the current standard of care and discuss targeted and immune therapies being explored for the treatment of high risk or relapsed/refractory pediatric renal malignancies. RECENT FINDINGS: Cooperative group trials have continued to make improvements in the care of children with pediatric tumors. In particular, trials that standardize treatment of rare cancers (e.g., bilateral Wilms tumor) have improved outcomes significantly. We have seen improvements in event free and overall survival in recently completed clinical trials for many pediatric renal tumors. Still, there are subsets of rarer cancers where outcomes remain poor and new therapeutic strategies are needed. Future trials aim to balance treatment toxicity with treatment efficacy for those with excellent outcomes while identifying novel therapeutics for those with poor outcomes.

Use of pegylated interferon in young patients with polycythemia vera and essential thrombocythemia.

Myeloproliferative neoplasms (MPN) are rare disorders in young patients, and because of this, standardized treatment recommendations are not available. Pediatric patients are more frequently treated with hydroxyurea than interferon, yet there are no data suggesting this is the best practice. Current treatment guidelines for adults suggest using interferon as upfront therapy in young patients. We reviewed the cases of 13 young patients with polycythemia vera or essential thrombocythemia, who were treated with interferon. Extreme thrombocytosis was well controlled and the medication was tolerated by many. Our work shows the need for prospective studies evaluating interferon in our youngest patients with MPN.

The molecular biology of intracellular events during Coronavirus infection cycle.

CoV-2 which is the causative agent of COVID-19 belongs to genus betacoronaviruses. The sequence analysis of S protein of CoV-2 has shown that it has acquired a 'polybasic cleavage site' consisting of 12 aminoacids that has been predicted to enable its cleavage by other cellular proteases possibly increasing its transmissibility. The aminoacids present in receptor binding domain of S protein of SARS CoV which are critical for its binding to cellular receptor are different in CoV-2. The presence of heptanucleotide slippery sequence in ORF1 resulting in ribosomal frameshifting, and presence of transcription regulatory sequences between ORFs resulting in discontinuous transcription, are peculiar features of Coronavirus infection cycle. The exonuclease activity of nsp14 provides possible proofreading ability to RNA polymerase makes coronaviruses different from other RNA viruses allowing coronaviruses to maintain their relatively large genome size. This mini-review summarizes the peculiar features of Coronaviruses genome and the critical events during the infection cycle with focus on CoV-2.

Anemia and chronic kidney disease: making sense of the recent trials.

Anemia is a very common complication of chronic kidney disease (CKD). Anemia confers significant risk of cardiovascular disease and contributes to decreased quality of life. Anemia in CKD patients can be multi-factorial, including but not invariably due to the underlying renal insufficiency. Identifying the type of anemia is important in this group of patients and can often be challenging. Diagnosing anemia of renal disease due to erythropoietin (EPO) deficiency is a diagnosis of exclusion. Erythropoiesis stimulating agents (ESA) are the mainstay for the treatment of anemia secondary to CKD. However, over the last four years the use of ESA in the treatment of anemia in CKD patients has undergone a severe interrogation as several trials have reported adverse outcomes with targeting higher hemoglobin (Hb) levels with these agents. Thereby, this review describes the pathophysiology of anemia in CKD patients, diagnosis and the current role of ESA's as it relates to anemia of CKD as well as safety and efficacy of ESA's.