A conserved virus-induced cytoplasmic TRAMP-like complex recruits the exosome to target viral RNA for degradation.

RNA degradation is tightly regulated to selectively target aberrant RNAs, including viral RNA, but this regulation is incompletely understood. Through RNAi screening in Drosophila cells, we identified the 3'-to-5' RNA exosome and two components of the exosome cofactor TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex, dMtr4 and dZcchc7, as antiviral against a panel of RNA viruses. We extended our studies to human orthologs and found that the exosome as well as TRAMP components hMTR4 and hZCCHC7 are antiviral. While hMTR4 and hZCCHC7 are normally nuclear, infection by cytoplasmic RNA viruses induces their export, forming a cytoplasmic complex that specifically recognizes and induces degradation of viral mRNAs. Furthermore, the 3' untranslated region (UTR) of bunyaviral mRNA is sufficient to confer virus-induced exosomal degradation. Altogether, our results reveal that signals from viral infection repurpose TRAMP components to a cytoplasmic surveillance role where they selectively engage viral RNAs for degradation to restrict a broad range of viruses.

N,N-Dimethyldithiocarbamate Elicits Pneumococcal Hypersensitivity to Copper and Macrophage-Mediated Clearance.

Streptococcus pneumoniae is a Gram-positive, encapsulated bacterium that is a significant cause of disease burden in pediatric and elderly populations. The rise in unencapsulated disease-causing strains and antimicrobial resistance in S. pneumoniae has increased the need for developing new antimicrobial strategies. Recent work by our laboratory has identified N,N-dimethyldithiocarbamate (DMDC) as a copper-dependent antimicrobial against bacterial, fungal, and parasitic pathogens. As a bactericidal antibiotic against S. pneumoniae, DMDC's ability to work as a copper-dependent antibiotic and its ability to work in vivo warranted further investigation. Here, our group studied the mechanisms of action of DMDC under various medium and excess-metal conditions and investigated DMDC's interactions with the innate immune system in vitro and in vivo. Of note, we found that DMDC plus copper significantly increased the internal copper concentration, hydrogen peroxide stress, nitric oxide stress, and the in vitro macrophage killing efficiency and decreased capsule. Furthermore, we found that in vivo DMDC treatment increased the quantity of innate immune cells in the lung during infection. Taken together, this study provides mechanistic insights regarding DMDC's activity as an antibiotic at the host-pathogen interface.

Four-Year Disease-Free Remission in a Patient With POLE Mutation-Associated Colorectal Cancer Treated Using Anti-PD-1 Therapy.

The stability of the human genome depends upon a delicate balance between replication by high- and low-fidelity DNA polymerases. Aberrant replication by error-prone polymerases or loss of function of high-fidelity polymerases predisposes to genetic instability and, in turn, cancer. DNA polymerase epsilon (Pol ε) is a high-fidelity, processive polymerase that is responsible for the majority of leading strand synthesis, and mutations in Pol ε have been increasingly associated with various human malignancies. The clinical significance of Pol ε mutations, including how and whether they should influence management decisions, remains poorly understood. In this report, we describe a 24-year-old man with an aggressive stage IV high-grade, poorly differentiated colon carcinoma who experienced a dramatic response to single-agent checkpoint inhibitor immunotherapy after rapidly progressing on standard chemotherapy. His response was complete and durable and has been maintained for more than 48 months. Genetic testing revealed a P286R mutation in the endonuclease domain of POLE and an elevated tumor mutational burden of 126 mutations per megabase, both of which have been previously associated with response to immunotherapy. Interestingly, tumor staining for PD-L1 was negative. This case study highlights the importance of genetic profiling of both early and late-stage cancers, the clinical significance of POLE mutations, and how the interplay between genetic instability and immune-checkpoint blockade can impact clinical decision-making.

CC16 Binding to α4ß1 Integrin Protects against Mycoplasma pneumoniae Infection.

Rationale CC16 (club cell secretory protein) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases (asthma and chronic obstructive pulmonary disease). Objectives Although exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating antiinflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Methods Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, leucine-valine-aspartic acid (LVD), a known integrin-binding motif. Recombinant CC16 was generated with and without the putative integrin-binding site. A Mycoplasma pneumoniae mouse model and a fluorescent cellular adhesion assay were used to determine the impact of the LVD site regarding CC16 function during live infection and on cellular adhesion during inflammatory conditions. Measurements and Main Results CC16 bound to integrin α4ß1), also known as the adhesion molecule VLA-4 (very late antigen 4), dependent on the presence of the LVD integrin-binding motif. During infection, recombinant CC16 rescued lung function parameters both when administered to the lung and intravenously but only when the LVD integrin-binding site was intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. Conclusions We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.

Virus-induced translational arrest through 4EBP1/2-dependent decay of 5'-TOP mRNAs restricts viral infection.

The mosquito-transmitted bunyavirus, Rift Valley fever virus (RVFV), is a highly successful pathogen for which there are no vaccines or therapeutics. Translational arrest is a common antiviral strategy used by hosts. In response, RVFV inhibits two well-known antiviral pathways that attenuate translation during infection, PKR and type I IFN signaling. Despite this, translational arrest occurs during RVFV infection by unknown mechanisms. Here, we find that RVFV infection triggers the decay of core translation machinery mRNAs that possess a 5'-terminal oligopyrimidine (5'-TOP) motif in their 5'-UTR, including mRNAs encoding ribosomal proteins, which leads to a decrease in overall ribosomal protein levels. We find that the RNA decapping enzyme NUDT16 selectively degrades 5'-TOP mRNAs during RVFV infection and this decay is triggered in response to mTOR attenuation via the translational repressor 4EBP1/2 axis. Translational arrest of 5'-TOPs via 4EBP1/2 restricts RVFV replication, and this increased RNA decay results in the loss of visible RNA granules, including P bodies and stress granules. Because RVFV cap-snatches in RNA granules, the increased level of 5'-TOP mRNAs in this compartment leads to snatching of these targets, which are translationally suppressed during infection. Therefore, translation of RVFV mRNAs is compromised by multiple mechanisms during infection. Together, these data present a previously unknown mechanism for translational shutdown in response to viral infection and identify mTOR attenuation as a potential therapeutic avenue against bunyaviral infection.

Non-shunt Management of Communicating Hydrocephalus in an Immunocompetent Host With Disseminated Central Nervous System Coccidioidomycosis.

Disseminated coccidioidomycosis is associated with significant morbidity and mortality. Involvement of the meninges is often fatal if untreated, typically requiring lifelong antifungal therapy and neurosurgical intervention. We present the case of a young male without any known immunocompromising conditions who opted exclusively for medical management of newly diagnosed coccidioidomycosis meningitis with communicating hydrocephalus and discuss the controversy associated with this approach. This case highlights the importance of shared decision-making between patient and clinician, even if the plan diverges from available guidelines. Furthermore, we discuss clinical considerations in approaching the close outpatient monitoring of patients with central nervous system coccidioidomycosis with hydrocephalus.

Peripheral T-Cell Lymphoma in a Patient Previously Diagnosed With Sarcoidosis.

Sarcoidosis is a multisystem disorder characterized by granulomatous inflammation on histopathological evaluation. Diagnosis of sarcoidosis requires thorough elimination of malignancy and alternative causes of noncaseating granulomatous inflammation. Sarcoidosis and several subtypes of lymphoma have similar clinical presentations and can potentially have similar histopathological findings. Patients with a histopathology-confirmed diagnosis of sarcoidosis are at higher risk of developing malignancies. In this report, we present a case of a 64-year-old male diagnosed with sarcoidosis 2 years before presenting to the emergency department with a 4-month history of generalized weakness, cough, and very high fever. After a thorough workup involving cervical lymph node biopsy and bone marrow biopsy, he was diagnosed with peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). Due to the patient's current lymphoma diagnosis and features noted on pathology, a retrospective review of the prior biopsy specimen was performed, finding similar hematopathological features on both initial lymph node biopsy diagnosing sarcoidosis and current biopsies diagnosing lymphoma. Given these findings, our patient likely had early manifestation of PTCL misdiagnosed as sarcoidosis. In summary, lymphoma should be considered in all patients with suspected sarcoidosis, especially those who do not respond to treatment or who present with persistent hematological abnormalities.

Evaluation of Host Constitutive and Ex Vivo Coccidioidal Antigen-Stimulated Immune Response in Dogs with Naturally Acquired Coccidioidomycosis.

The early innate immune response to coccidioidomycosis has proven to be pivotal in directing the adaptive immune response and disease outcome in mice and humans but is unexplored in dogs. The objectives of this study were to evaluate the innate immune profile of dogs with coccidioidomycosis and determine if differences exist based on the extent of infection (i.e., pulmonary or disseminated). A total of 28 dogs with coccidioidomycosis (pulmonary, n = 16; disseminated, n = 12) and 10 seronegative healthy controls were enrolled. Immunologic testing was performed immediately, without ex vivo incubation (i.e., constitutive), and after coccidioidal antigen stimulation of whole blood cultures. Whole blood cultures were incubated with a phosphate-buffered solution (PBS) (negative control) or a coccidioidal antigen (rCTS1 (105-310); 10 µg/mL) for 24 h. A validated canine-specific multiplex bead-based assay was used to measure 12 cytokines in plasma and cell culture supernatant. Serum C-reactive protein (CRP) was measured with an ELISA assay. Leukocyte expression of toll-like receptors (TLRs)2 and TLR4 was measured using flow cytometry. Dogs with coccidioidomycosis had higher constitutive plasma keratinocyte chemotactic (KC)-like concentrations (p = 0.02) and serum CRP concentrations compared to controls (p < 0.001). Moreover, dogs with pulmonary coccidioidomycosis had higher serum CRP concentrations than those with dissemination (p = 0.001). Peripheral blood leukocytes from dogs with coccidioidomycosis produced higher concentrations of tumor necrosis factor (TNF)-α (p = 0.0003), interleukin (IL)-6 (p = 0.04), interferon (IFN)-γ (p = 0.03), monocyte chemoattractant protein (MCP)-1 (p = 0.02), IL-10 (p = 0.02), and lower IL-8 (p = 0.003) in supernatants following coccidioidal antigen stimulation when compared to those from control dogs. There was no detectable difference between dogs with pulmonary and disseminated disease. No differences in constitutive or stimulated leukocyte TLR2 and TLR4 expression were found. These results provide information about the constitutive and coccidioidal antigen-specific stimulated immune profile in dogs with naturally acquired coccidioidomycosis.

Novel dithiocarbamate derivatives are effective copper-dependent antimicrobials against Streptococcal species.

Despite the availability of several vaccines against multiple disease-causing strains of Streptococcus pneumoniae, the rise of antimicrobial resistance and pneumococcal disease caused by strains not covered by the vaccine creates a need for developing novel antimicrobial strategies. N,N-dimethyldithiocarbamate (DMDC) was found to be a potent copper-dependent antimicrobial against several pathogens, including S. pneumoniae. Here, DMDCs efficacy against Streptococcal pathogens Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus anginosus was tested using bactericidal and inductively coupled plasma - optical emission spectrometry. After confirming DMDC as broad-spectrum streptococcal antimicrobial, DMDC was derivatized into five compounds. The derivatives' effectiveness as copper chelators using DsRed2 and as copper-dependent antimicrobials against S. pneumoniae TIGR4 and tested in bactericidal and animal models. Two compounds, sodium N-benzyl-N-methyldithiocarbamate and sodium N-allyl-N-methyldithiocarbamate (herein "Compound 3" and "Compound 4"), were effective against TIGR4 and further, D39 and ATCC® 6303™ _(a type 3 capsular strain). Both Compound 3 and 4 increased the pneumococcal internal concentrations of copper to the same previously reported levels as with DMDC and copper treatment. However, in an in vivo murine pneumonia model, Compound 3, but not Compound 4, was effective in significantly decreasing the bacterial burden in the blood and lungs of S. pneumoniae-infected mice. These derivatives also had detrimental effects on the other streptococcal species. Collectively, derivatizing DMDC holds promise as potent bactericidal antibiotics against relevant streptococcal pathogens.

A quantitative enzyme-linked immunoassay (ELISA) to approximate complement-fixing antibody titers in serum from patients with coccidioidomycosis.

Coccidioidomycosis is most frequently diagnosed serologically, and the quantitative test for complement-fixing antibodies is considered prognostically useful. Because complement-fixing antibody testing is complex, labor-intensive, and poorly standardized, an enzyme-linked immunoassay (ELISA) alternative would be attractive. In this report, we restrict the complement-fixing, antibody-binding domain to a 200-amino-acid recombinant peptide of the known antigen. Over-lapping truncations of this peptide do not bind complement-fixing antibodies, suggesting that the responsible epitope(s) are conformational. Further, anchoring the antigenic peptide to the ELISA plate by means of a C-terminal biotin-mimic peptide tag instead of allowing the peptide to randomly adhere to the plastic plate improves sensitivity of antibody detection by 1-2 logs in different sera. The newly developed ELISA shows a significant quantitative correlation with complement-fixing antibody titers. This ELISA shows potential as the basis for a new quantitative assay for coccidioidal antibodies.

Demonstration of N,N-Dimethyldithiocarbamate as a Copper-Dependent Antibiotic against Multiple Upper Respiratory Tract Pathogens.

Transition metals are necessary cofactors and structural elements in living systems. Exposure to high concentrations of biologically important transition metals, such as zinc and copper, results in cell toxicity. At the infection site, the immune system deploys metal sorbent proteins (e.g., lactoferrin and calprotectin) to starve pathogens of necessary metals (such as iron), while phagocytes expose engulfed pathogens to high levels of other metals, such as copper and zinc. The opportunistic pathogen Streptococcus pneumoniae (the pneumococcus) encounters macrophages during initial and protracted infections. The pneumococcus employs a copper export pathway, which improves colonization and persistent infection of the nasopharynx and the upper respiratory tract. Because copper is tightly regulated in the host, we instead sought to leverage the localized power of nutritional immunity by identifying small molecules with copper-dependent toxicity (CDT) through a targeted screen of compounds for antibiotic efficacy. We chose to include dithiocarbamates, based on the copper synergy observed in other organisms with 1-(diethylthiocarbamoyldisulfanyl)-N,N-diethyl-methanethioamide (tetraethylthiuram disulfide, disulfiram). We observed CDT of some dithiocarbamates in S. pneumoniae. Only N,N-dimethyldithiocarbamate (DMDC) was consistently toxic across a range of concentrations with copper both in vitro and in vivo against the pneumococcus. We also observed various degrees of CDT in vitro using DMDC in Staphylococcus aureus, Coccidioides posadasii, and Schistosoma mansoni. Collectively, we demonstrate that the compound DMDC is a potent bactericidal compound against S. pneumoniae with antimicrobial efficacy against bacterial and fungal pathogens. IMPORTANCE With the rise of antibiotic resistance, approaches that add new antimicrobials to the current repertoire are vital. Here, we investigate putative and known copper ionophores in an attempt to intoxicate bacteria and use ionophore/copper synergy, and we ultimately find success with N,N-dimethyldithiocarbamate (DMDC). We show that DMDC has in vitro efficacy in a copper-dependent manner and kills pathogens across three different kingdoms, Streptococcus pneumoniae, Coccidioides posadasii, and Schistosoma mansoni, and in vivo efficacy against S. pneumoniae. As such, dithiocarbamates represent a new potential class of antimicrobials and thus warrant further mechanistic investigation.

Gastric Cardia Adenocarcinoma with Metastasis to the Scalp: A Case Report.

Cutaneous metastasis is a rare manifestation of advanced gastrointestinal (GI) cancers. Gastric adenocarcinoma rarely presents with cutaneous metastasis, as cutaneous manifestations occur in less than 1% of upper GI tract malignancies. Here, we present the case of a patient with advanced gastric cardia adenocarcinoma with metastasis to the right occipital region of the scalp. Following shave biopsy, the immunohistochemistry (IHC) and molecular profile of the scalp lesion were analyzed, both of which confirmed metastasis and guided the treatment approach. The lesion demonstrated programmed death ligand-1 (PD-L1), an immune checkpoint protein, positivity by IHC, which led to the recommendation for treatment with immunotherapy as per the National Comprehensive Cancer Network (NCCN) guidelines. Clinicians should conduct dermatologic examinations in patients with a history of gastric cancer or who are currently undergoing chemotherapy for gastric cancer in order to monitor for disease progression or metastatic lesions. The aim of this report is to increase awareness of scalp metastasis as an indicator of advanced internal visceral carcinoma for earlier diagnosis and improved management of the condition.

Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors.

Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the cop box. Using this motif, we sought to determine whether genes outside the cop operon are also regulated by the CopY repressor, which was previously shown in Lactococcus lactis We found that S. pneumoniae CopY did not bind to cop operators upstream of these candidate genes in vitro During this process, we found that the cop box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the S. pneumoniaecop operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant (Kd ) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs.IMPORTANCE Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the cop operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.