First-in-class MKK4 inhibitors enhance liver regeneration and prevent liver failure.

Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i).

The Angiopoietin Signaling Pathway Is Involved in Inflammatory Processes in Hospitalized COVID-19 Patients.

The viral agent SARS-CoV-2 clearly affects several organ systems, including the cardiovascular system. Angiopoietins are involved in vascular integrity and angiogenesis. Angiopoietin-1 (Ang1) promotes vessel stabilization, while angiopoietin-2 (Ang2), which is usually expressed at low levels, is significantly elevated in inflammatory and angiogenic conditions.

A Rapid, Isothermal, and Point-of-Care System for COVID-19 Diagnostics.

The COVID-19 pandemic has had a profound, detrimental effect on economies and societies worldwide. Where the pandemic has been controlled, extremely high rates of diagnostic testing for the SARS-CoV-2 virus have proven critical, enabling isolation of cases and contact tracing. Recently, diagnostic testing has been supplemented with wastewater measures to evaluate the degree to which communities have infections.

ACKR4 in Tumor Cells Regulates Dendritic Cell Migration to Tumor-Draining Lymph Nodes and T-Cell Priming.

Colorectal cancer (CRC) is one of the most common malignancies in both morbidity and mortality. Immune checkpoint blockade (ICB) treatments have been successful in a portion of mismatch repair-deficient (dMMR) CRC patients but have failed in mismatch repair-proficient (pMMR) CRC patients. Atypical Chemokine Receptor 4 (ACKR4) is implicated in regulating dendritic cell (DC) migration.

Implementation of a pooled surveillance testing program for asymptomatic SARS-CoV-2 infections in K-12 schools and universities.

Background: The negative impact of continued school closures during the height of the COVID-19 pandemic warrants the establishment of cost-effective strategies for surveillance and screening to safely reopen and monitor for potential in-school transmission. Here, we present a novel approach to increase the availability of repetitive and routine COVID-19 testing that may ultimately reduce the overall viral burden in the community.

Methods: We implemented a testing program using the SalivaClear࣪ pooled surveillance method that included students, faculty and staff from K-12 schools (student age range 5-18 years) and universities (student age range >18 years) across the country (Mirimus Clinical Labs, Brooklyn, NY).

Ephrin-A1 and the sheddase ADAM12 are upregulated in COVID-19.

More than 3.5 million people have died globally from COVID-19, yet an effective therapy is not available. It is, therefore, important to understand the signaling pathways that mediate disease progression in order to identify new molecular targets for therapeutic development.

Administration of high titer convalescent anti-SARS-CoV-2 plasma: From donor selection to monitoring recipient outcomes.

Early in the SARS-CoV-2 pandemic, convalescent plasma (CP) therapy was proposed as a treatment for severely ill patients. We conducted a CP treatment protocol under the Mayo Clinic Extended Access Program at University Hospital Brooklyn (UHB). Potential donors were screened with a lateral flow assay (LFA) for IgM and IgG antibodies against the SARS-CoV-2 S1 receptor-binding domain (RBD).

Prediction of potent shRNAs with a sequential classification algorithm.

We present SplashRNA, a sequential classifier to predict potent microRNA-based short hairpin RNAs (shRNAs). Trained on published and novel data sets, SplashRNA outperforms previous algorithms and reliably predicts the most efficient shRNAs for a given gene. Combined with an optimized miR-E backbone, >90% of high-scoring SplashRNA predictions trigger >85% protein knockdown when expressed from a single genomic integration.

Cohesin loss alters adult hematopoietic stem cell homeostasis, leading to myeloproliferative neoplasms.

The cohesin complex (consisting of Rad21, Smc1a, Smc3, and Stag2 proteins) is critically important for proper sister chromatid separation during mitosis. Mutations in the cohesin complex were recently identified in a variety of human malignancies including acute myeloid leukemia (AML). To address the potential tumor-suppressive function of cohesin in vivo, we generated a series of shRNA mouse models in which endogenous cohesin can be silenced inducibly.

Nephrin Preserves Podocyte Viability and Glomerular Structure and Function in Adult Kidneys.

Nephrin is required during kidney development for the maturation of podocytes and formation of the slit diaphragm junctional complex. Because nephrin expression is downregulated in acquired glomerular diseases, nephrin deficiency is considered a pathologic feature of glomerular injury. However, whether nephrin deficiency exacerbates glomerular injury in glomerular diseases has not been experimentally confirmed.

In vivo RNA interference models of inducible and reversible Sirt1 knockdown in kidney cells.

The silent mating type information regulation 2 homolog 1 gene (Sirt1) encodes an NAD-dependent deacetylase that modifies the activity of well-known transcriptional regulators affected in kidney diseases. Sirt1 is expressed in the kidney podocyte, but its function in the podocyte is not clear. Genetically engineered mice with inducible and reversible Sirt1 knockdown in widespread, podocyte-specific, or tubular-specific patterns were generated.

PTEN action in leukaemia dictated by the tissue microenvironment.

PTEN encodes a lipid phosphatase that is underexpressed in many cancers owing to deletions, mutations or gene silencing. PTEN dephosphorylates phosphatidylinositol (3,4,5)-triphosphate, thereby opposing the activity of class I phosphatidylinositol 3-kinases that mediate growth- and survival-factor signalling through phosphatidylinositol 3-kinase effectors such as AKT and mTOR. To determine whether continued PTEN inactivation is required to maintain malignancy, here we generate an RNA interference-based transgenic mouse model that allows tetracycline-dependent regulation of PTEN in a time- and tissue-specific manner.

Creating transgenic shRNA mice by recombinase-mediated cassette exchange.

RNA interference (RNAi) enables sequence-specific, experimentally induced silencing of virtually any gene by tapping into innate regulatory mechanisms that are conserved among most eukaryotes. The principles that enable transgenic RNAi in cell lines can also be used to create transgenic animals, which express short-hairpin RNAs (shRNAs) in a regulated or tissue-specific fashion. However, RNAi in transgenic animals is somewhat more challenging than RNAi in cultured cells.

Targeting synthetic lethal interactions between Myc and the eIF4F complex impedes tumorigenesis.

The energetically demanding process of translation is linked to multiple signaling events through mTOR-mediated regulation of eukaryotic initiation factor (eIF)4F complex assembly. Disrupting mTOR constraints on eIF4F activity can be oncogenic and alter chemotherapy response, making eIF4F an attractive antineoplastic target. Here, we combine a newly developed inducible RNAi platform and pharmacological targeting of eIF4F activity to define a critical role for endogenous eIF4F in Myc-dependent tumor initiation.

G Protein-regulated inducer of neurite outgrowth (GRIN) modulates Sprouty protein repression of mitogen-activated protein kinase (MAPK) activation by growth factor stimulation.

Gα(o/i) interacts directly with GRIN (G protein-regulated inducer of neurite outgrowth). Using the yeast two-hybrid system, we identified Sprouty2 as an interacting partner of GRIN. Gα(o) and Sprouty2 bind to overlapping regions of GRIN, thus competing for GRIN binding.

A pipeline for the generation of shRNA transgenic mice.

RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches-inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use.

Control of the senescence-associated secretory phenotype by NF-κB promotes senescence and enhances chemosensitivity.

Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells.

Reversible suppression of an essential gene in adult mice using transgenic RNA interference.

RNAi has revolutionized loss-of-function genetics by enabling sequence-specific suppression of virtually any gene. Furthermore, tetracycline response elements (TRE) can drive expression of short hairpin RNAs (shRNAs) for inducible and reversible target gene suppression. Here, we demonstrate the feasibility of transgenic inducible RNAi for suppression of essential genes.

A rapid and scalable system for studying gene function in mice using conditional RNA interference.

RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16(INK4a), p19(ARF) and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo.

Tissue-specific and reversible RNA interference in transgenic mice.

Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice.