TNF plays a crucial role in inflammation by signaling via T cell TNFR2.

TNF, produced largely by T and innate immune cells, is potently proinflammatory, as are cytokines such as IFN-γ and IL-17 produced by Th1 and Th17 cells, respectively. Here, we asked if TNF is upstream of Th skewing toward inflammatory phenotypes. Exposure of mouse CD4+ T cells to TNF and TGF-ß generated Th17 cells that express low levels of IL-17 (ROR-γt+IL-17lo) and high levels of inflammatory markers independently of IL-6 and STAT3. This was mediated by the nondeath TNF receptor TNFR2, which also contributed to the generation of inflammatory Th1 cells. Single-cell RNA sequencing of central nervous system-infiltrating CD4+ T cells in mouse experimental autoimmune encephalomyelitis (EAE) found an inflammatory gene expression profile similar to cerebrospinal fluid-infiltrating CD4+ T cells from patients with multiple sclerosis. Notably, TNFR2-deficient CD4+ T cells produced fewer inflammatory mediators and were less pathogenic in EAE and colitis. IL-1ß, a Th17-skewing cytokine, induced TNF and proinflammatory granulocyte-macrophage colony-stimulating factor (GM-CSF) in T cells, which was inhibited by disruption of TNFR2 signaling, demonstrating IL-1ß can function indirectly via the production of TNF. Thus, TNF is not just an effector but also an initiator of inflammatory Th differentiation.

Optineurin Deficiency and Insufficiency Lead to Higher Microglial TDP-43 Protein Levels.

Mutations in optineurin, a ubiquitin-binding adaptor protein, cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease of motor neurons linked to chronic inflammation and protein aggregation. The majority of ALS patients, including those carrying the optineurin mutations, exhibit cytoplasmic mislocalization, ubiquitination, and aggregation of nuclear TAR DNA-binding protein 43 kDa (TDP-43). To address the crosstalk between optineurin and TDP-43, we generated optineurin knockout (KO) neuronal and microglial cell lines using the CRISPR/Cas9 approach. Interestingly, we observed that loss of optineurin resulted in elevated TDP-43 protein expression in microglial BV2 but not neuronal Neuro 2a and NSC-34 cell lines. No changes were observed at the mRNA level, suggesting that this increase was post-translationally regulated. To confirm this observation in primary cells, we then used microglia and macrophages from an optineurin loss-of-function mouse model that lacks the C-terminal ubiquitin-binding region (Optn470T), mimicking optineurin truncations in ALS patients. As observed in the BV2 cells, we also found elevated basal levels of TDP-43 protein in Optn470T microglia and bone marrow-derived macrophages. To test if inflammation could further enhance TDP-43 accumulation in cells lacking functional optineurin, we stimulated them with lipopolysaccharide (LPS), and we observed a significant increase in TDP-43 expression following LPS treatment of WT cells. However, this was absent in both BV2 Optn KO and primary Optn470T microglia, which exhibited the same elevated TDP-43 levels as in basal conditions. Furthermore, we did not observe nuclear TDP-43 depletion or cytoplasmic aggregate formation in either Optn470T microglia or LPS-treated WT or Optn470T microglia. Taken together, our results show that optineurin deficiency and insufficiency post-translationally upregulate microglial TDP-43 protein levels and that elevated TDP-43 levels in cells lacking functional optineurin could not be further increased by an inflammatory stimulus, suggesting the presence of a plateau.

Dynamics of Collisions and Adsorption in the Stochastic Electrochemistry of Emulsion Microdroplets.

Current-time recordings of emulsified toluene microdroplets containing 20 mM Ferrocene (Fc), show electrochemical oxidation peaks from individual adsorption events on disk microelectrodes (5 µm diameter). The average droplet diameter (∼0.7 µm) determined from peak area integration was close to Dynamic Light Scattering measurements (∼1 µm). Random walk simulations were performed deriving equations for droplet electrolysis using the diffusion and thermal velocity expressions from Einstein. The simulations show that multiple droplet-electrode collisions, lasting ∼0.11 µs each, occur before a droplet wanders away. Updating the Fc-concentration at every collision shows that a droplet only oxidizes ∼0.58% of its content in one collisional journey. In fact, it would take ∼5.45 × 106 collisions and ∼1.26 h to electrolyze the Fc in one droplet with the collision frequency derived from the thermal velocity (∼0.52 cm/s) of a 1 µm-droplet. To simulate adsorption, the droplet was immobilized at first contact with the electrode while the electrolysis current was computed. This approach along with modeling of instrumental filtering, produced the best match of experimental peaks, which were attributed to electrolysis from single adsorption events instead of multiple consecutive collisions. These results point to a heightened sensitivity and speed when relying on adsorption instead of collisions. The electrochemical current for the former is limited by the probability of adsorption per collision, whereas for the latter, the current depends on the collision frequency and the probability of electron transfer per collision (J. Am. Chem. Soc. 2017, 139, 16923-16931).

Unique properties of TCR-activated p38 are necessary for NFAT-dependent T-cell activation.

Nuclear factor of activated T cells (NFAT) transcription factors are required for induction of T-cell cytokine production and effector function. Although it is known that activation via the T-cell antigen receptor (TCR) results in 2 critical steps, calcineurin-mediated NFAT1 dephosphorylation and NFAT2 up-regulation, the molecular mechanisms underlying each are poorly understood. Here we find that T cell p38, which is activated by an alternative pathway independent of the mitogen-activated protein (MAP) kinase cascade and with different substrate specificities, directly controls these events. First, alternatively (but not classically) activated p38 was required to induce the expression of the AP-1 component c-Fos, which was necessary for NFAT2 expression and cytokine production. Second, alternatively (but not classically) activated p38 phosphorylated NFAT1 on a heretofore unidentified site, S79, and in its absence NFAT1 was unable to interact with calcineurin or migrate to the nucleus. These results demonstrate that the acquisition of unique specificities by TCR-activated p38 orchestrates NFAT-dependent T-cell functions.

A High-Order EMSIW MIMO Antenna for Space-Constrained 5G Smartphone.

This paper proposes a high-order MIMO antenna operating at 3.5 GHz for a 5G new radio. Using an eighth-mode substrate integrated waveguide (EMSIW) cavity and considering a typical smartphone scenario, a two-element MIMO antenna is developed and extended to a twelve-element MIMO. These MIMO elements are closely spaced, and by employing multiple diversity techniques, high isolation is achieved without using a decoupling network. The asymmetric EMSIW structures resulted in radiation pattern diversity, and their orthogonal placement provides polarization diversity. The radiation characteristics and diversity performance are parametrically optimized for a two-element MIMO antenna. The experimental results exhibited 6.0 dB and 10.0 dB bandwidths of 250 and 100 MHz, respectively. The measured and simulated radiation patterns are closely matched with a peak gain of 3.4 dBi and isolation ≥36 dB. Encouraged with these results, higher-order MIMO, namely, four- and twelve-element MIMO are investigated, and isolation ≥35 and ≥22 dB are achieved, respectively. The channel capacity is found equal to 56.37 bps/Hz for twelve-element MIMO, which is nearly 6.25 times higher than the two-element counterpart. The hand and head proximity analysis reveal that the proposed antenna performances are within the acceptable limit. A detailed comparison with the previous works demonstrates that the proposed antenna offers a simple, low-cost, and compact MIMO antenna design solution with a high diversity performance.

Calcineurin is an adaptor required for assembly of the TCR signaling complex.

The serine/threonine phosphatase calcineurin is a component of the T cell receptor (TCR) signalosome, where it promotes T cell activation by dephosphorylating LckS59. Using small interfering RNA (siRNA)-mediated knockdown and CRISPR-Cas9-targeted genetic disruption of the calcineurin A chain α and ß isoforms, we find that calcineurin also functions as an adaptor in TCR-signaled human T cells. Unlike inhibition of its phosphatase activity, in the absence of calcineurin A, TCR signaling results in attenuated actin rearrangement, markedly reduced TCR-Lck microcluster formation and recruitment of the adaptor RhoH, and diminished phosphorylation of critical targets downstream of Lck such as TCRζ and ZAP-70. Reconstitution of deficient T cells with either calcineurin Aα or Aß restores TCR microcluster formation and signaling, as does reconstitution with a phosphatase-inactive Aα chain. These results assign a non-enzymatic adaptor function to calcineurin in the formation and stabilization of a functional TCR signaling complex.

Centrosome amplification and aneuploidy driven by the HIV-1-induced Vpr•VprBP•Plk4 complex in CD4+ T cells.

HIV-1 infection elevates the risk of developing various cancers, including T-cell lymphoma. Whether HIV-1-encoded proteins directly contribute to oncogenesis remains unknown. We observe that approximately 1-5% of CD4+ T cells from the blood of people living with HIV-1 exhibit over-duplicated centrioles, suggesting that centrosome amplification underlies the development of HIV-1-associated cancers by driving aneuploidy. Through affinity purification, biochemical, and cellular analyses, we discover that Vpr, an accessory protein of HIV-1, hijacks the centriole duplication machinery and induces centrosome amplification and aneuploidy. Mechanistically, Vpr forms a cooperative ternary complex with an E3 ligase subunit, VprBP, and polo-like kinase 4 (Plk4). Unexpectedly, however, the complex enhances Plk4's functionality by promoting its relocalization to the procentriole assembly and induces centrosome amplification. Loss of either Vpr's C-terminal 17 residues or VprBP acidic region, the two elements required for binding to Plk4 cryptic polo-box, abrogates Vpr's capacity to induce these events. Furthermore, HIV-1 WT, but not its Vpr mutant, induces multiple centrosomes and aneuploidy in human primary CD4+ T cells. We propose that the Vpr•VprBP•Plk4 complex serves as a molecular link that connects HIV-1 infection to oncogenesis and that inhibiting the Vpr C-terminal motif may reduce the occurrence of HIV-1-associated cancers.

Sonographic Characteristics and Pathology Correlation of Breast Imaging Reporting and Data System (BI-RADS) Category 4 Lesions.

INTRODUCTION: The Breast Imaging-Reporting and Database System (BI-RADS) category 4 is designated for breast lumps that do not display the typical features of malignancy but still raise enough suspicion to warrant a recommendation for a biopsy, as malignancy cannot be ruled out through imaging alone. The main objective of this study was to investigate the sonographic characteristics and pathology correlation of BI-RADS 4 breast lesions and determine the positive predictive rate of BI-RADS 4 lesions in diagnosing breast cancer, using histopathology as the gold standard. METHODS: This was a cross-sectional study conducted at the Department of Radiology, Aga Khan University Hospital in Karachi, spanning from May 2021 to August 2022, with a duration of 15 months. The study focused on female patients over the age of 18 who presented with suspicious breast lesions on ultrasound. Both mammography and ultrasound-guided core needle biopsy were performed on these patients, followed by a detailed histopathological evaluation of the biopsy specimens. To calculate the positive predictive value (PPV), true positive cases were identified through both histopathology and ultrasonography. RESULTS: A total of 227 cases were categorized as BI-RADS 4 lesions, with the patients' mean age being 47.8 ± 14.3 years (range: 17 - 88). Among the biopsied lesions, 101 cases were confirmed to be true positive for breast malignancies, resulting in a PPV for malignancy of 44.9%. Conversely, there were 124 false positive cases out of the 227 BI-RADS 4 category lesions (54.63%). The primary indication for presentation was a breast lump, and out of the 101 confirmed malignant cases, 70 (69.3%) were associated with malignancy. CONCLUSION: BI-RADS 4 can be utilized to assess suspicious breast lumps; however, for more reliable results and to avoid false negatives, histopathological confirmation should complement the imaging findings.

Centrosome amplification and aneuploidy driven by the HIV-1-induced Vpr•VprBP•Plk4 complex in CD4+ T cells.

HIV-1 infection elevates the risk of developing various cancers, including T-cell lymphoma. Whether HIV-1-encoded proteins directly contribute to oncogenesis remains unknown. We observed that approximately 1-5% of CD4+ T cells from the blood of people living with HIV-1 exhibit over-duplicated centrioles, suggesting that centrosome amplification underlies the development of HIV-1-associated cancers by driving aneuploidy. Through affinity purification, biochemical, and cell biology analyses, we discovered that Vpr, an accessory protein of HIV-1, hijacks the centriole duplication machinery and induces centrosome amplification and aneuploidy. Mechanistically, Vpr formed a cooperative ternary complex with an E3 ligase subunit, VprBP, and polo-like kinase 4 (Plk4). Unexpectedly, however, the complex enhanced Plk4's functionality by promoting its relocalization to the procentriole assembly and induced centrosome amplification. Loss of either Vpr's C-terminal 17 residues or VprBP acidic region, the two elements required for binding to Plk4 cryptic polo-box, abrogated Vpr's capacity to induce all these events. Furthermore, HIV-1 WT, but not its Vpr mutant, induced multiple centrosomes and aneuploidy in primary CD4+ T cells. We propose that the Vpr•VprBP•Plk4 complex serves as a molecular link that connects HIV-1 infection to oncogenesis and that inhibiting the Vpr C-terminal motif may reduce the occurrence of HIV-1-associated cancers.

Proximity Ligation Assay (PLA).

Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (<40 nm distance) at endogenous protein levels. It exploits specific antibodies identifying (either directly or indirectly) the two proteins of interest and takes advantage of specific DNA primers covalently linked to the antibodies. A hybridization step followed by a PCR amplification with fluorescent probes then permits visualization of spots of proximity by fluorescence microscopy.

Phytochemistry, Traditional Uses and Pharmacological Properties of Enhydra fluctuans Lour: a Comprehensive Review.

BACKGROUND: Enhydra fluctuans Lour, a tropical herb, commonly known as helencha or harkuch, belongs to the family Asteraceae. It is an edible semi-aquatic herbaceous vegetable plant with serrate leaves and grows commonly in different parts of the world. Enhydra fluctuans possesses potential pharmacological role against inflammation, cancer, diarrhea, microbial infection, diabetes, etc. Aim: This review aims to provide the most current information on the botanical characterization, distribution, traditional uses, chemical constituents, as well as the pharmacological activities of Enhydra fluctuans Lour. MATERIALS AND METHODS: The recently updated information on Enhydra fluctuans was gathered from scientific journals, books, and worldwide accepted scientific databases via a library and electronic search PubMed, Elsevier, Google Scholar, Springer, Scopus, Web of Science, Wiley online library. All of the full-text articles and abstracts related to Enhydra were screened. The most important and relevant articles were carefully chosen for study in this review. RESULTS: Crude extracts and isolated compounds of Enhydra fluctuans Lour have been reported to be pharmacologically active against cytoprotective, analgesic and anti-inflammatory, antimicrobial, anticancer, antidiarrheal, antihelmintic, CNS depressant, hepatoprotective, thrombolytic, antidiabetic, antioxidant, phagocytic and cytotoxic, and neuroprotective potential activities. DISCUSSION: Phytochemical analysis from different studies has reported Germacranolide, Sesquiterpene lactone, Flavonoid, Essential oil, Steroid, Diterpenoid, Melampolide, Sesquiterpene lactone, and Isoflavone glycoside as major compounds of Enhydra fluctuans Lour. CONCLUSION: However, more research is needed to explore the mode of action of bioactive components of the plant and its therapeutic capabilities.

Novel mannopyranoside esters as sterol 14α-demethylase inhibitors: Synthesis, PASS predication, molecular docking, and pharmacokinetic studies.

Direct unimolar one-step valeroylation of methyl α-d-mannopyranoside (MDM) furnished mainly 6-O-valeroate. However, similar reaction catalyzed by DMAP resulted 3,6-di-O-valeroate (21%) and 6-O-valeroate (47%) indicating reactivity sequence as 6-OH>3-OH>2-OH,4-OH. To get potential antimicrobial agents, 6-O-valeroate was converted into four 2,3,4-di-O-acyl esters, and 3,6-di-O-valeroate was converted into 2,4-di-O-acetate. Direct tetra-O-valeroylation of MDM gave a mixture of 2,3,4,6-tetra-O-valeroate and 2,3,6-tri-O-valeroate indicating that the C2-OH is more reactive than the equatorial C4-OH. The activity spectra analysis along with in vitro antimicrobial evaluation clearly indicated that these novel MDM esters had better antifungal activities over antibacterial agents. In this connection, molecular docking indicated that these MDM esters acted as competitive inhibitors of sterol 14α-demethylase (CYP51), an essential enzyme for clinical target to cure several infectious diseases. Furthermore, pharmacokinetic studies revealed that these MDM esters may be worth considering as potent candidates for oral and topical administration. Structure activity relationship (SAR) affirmed that saturated valeric chain (C5) in combination with caprylic (C8) chains was more promising CYP51 inhibitor over conventional antifungal antibiotics.

Synthesis, PASS predication, in vitro antimicrobial evaluation and pharmacokinetic study of novel n-octyl glucopyranoside esters.

Octyl ß-d-glucopyranoside (OBG), prepared from d-glucose and octan-1-ol employing MW method, was subjected to direct dimolar valeroylation in pyridine at room temperature (25 °C) with valeroyl chloride. This mainly furnished the corresponding 3,6-di-O-valeroate in 57% yield indicating the regioselectivity at C-6 and C-3 positions. For structural elucidation and to get newer glucopyranosides of potential antimicrobial 3,6-di-O-valeroate was further converted into four novel 2,4-di-O-acyl esters reasonably in good yields. Per-O-acetate and per-O-benzoate of OBG were also prepared for SAR study. PASS predication and in vitro antimicrobial studies established them as better antifungal agent than that of antibacterial. SAR study along with AdmetSAR and SwissADME suggested that incorporation of alkanoyl and aromatic ester groups on octyl glucopyranoside core increase antimicrobial potentiality in very low concentration (10 µgmL-1). Molecular docking revealed that novel 2,4-di-O-tosyl ester and 2,3,4,6-tetra-O-benzoyl ester may act as competitive inhibitors of lanosterol 14-alpha demethylase.

Proximity Ligation Assay (PLA).

Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances <40 nm) at endogenous protein levels. It exploits specific antibodies identifying (either directly or indirectly) the two proteins of interest and utilizes specific DNA primers covalently linked to the antibodies. A hybridization step followed by DNA amplification with fluorescent probes permit visualization of spots of proximity by fluorescence microscopy. Since the development of PLA in 2002, it has been increasingly used to detect the interaction between two proteins with high sensitivity and specificity. It is a simple and sensitive technique to study protein-protein interaction in cells. © 2018 by John Wiley & Sons, Inc.

Discovery and Characterization of a Biologically Active Non-ATP-Competitive p38 MAP Kinase Inhibitor.

Mitogen-activated protein kinase (MAPK) p38 is part of a broad and ubiquitously expressed family of MAPKs whose activity is responsible for mediating an intracellular response to extracellular stimuli through a phosphorylation cascade. p38 is central to this signaling node and is activated by upstream kinases while being responsible for activating downstream kinases and transcription factors via phosphorylation. Dysregulated p38 activity is associated with numerous autoimmune disorders and has been implicated in the progression of several types of cancer. A number of p38 inhibitors have been tested in clinical trials, with none receiving regulatory approval. One characteristic shared by all of the compounds that failed clinical trials is that they are all adenosine triphosphate (ATP)-competitive p38 inhibitors. Seeing this lack of mechanistic diversity as an opportunity, we screened ~32,000 substances in search of novel p38 inhibitors. Among the inhibitors discovered is a compound that is both non-ATP competitive and biologically active in cell-based models for p38 activity. This is the first reported discovery of a non-ATP-competitive p38 inhibitor that is active in cells and, as such, may enable new pharmacophore designs for both therapeutic and basic research to better understand and exploit non-ATP-competitive inhibitors of p38 activity.

Selective inhibition of the p38 alternative activation pathway in infiltrating T cells inhibits pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive neoplasm characterized by a marked fibro-inflammatory microenvironment, the presence of which can promote both cancer induction and growth. Therefore, selective manipulation of local cytokines is an attractive, although unrealized, therapeutic approach. T cells possess a unique mechanism of p38 mitogen-activated protein kinase (MAPK) activation downstream of T cell receptor (TCR) engagement through the phosphorylation of Tyr323 (pY323). This alternative p38 activation pathway is required for pro-inflammatory cytokine production. Here we show in human PDAC that a high percentage of infiltrating pY323(+) T cells was associated with large numbers of tumor necrosis factor (TNF)-α- and interleukin (IL)-17-producing CD4(+) tumor-infiltrating lymphocytes (TILs) and aggressive disease. The growth of mouse pancreatic tumors was inhibited by genetic ablation of the alternative p38 pathway, and transfer of wild-type CD4(+) T cells, but not those lacking the alternative pathway, enhanced tumor growth in T cell-deficient mice. Notably, a plasma membrane-permeable peptide derived from GADD45-α, the naturally occurring inhibitor of p38 pY323(+) (ref. 7), reduced CD4(+) TIL production of TNF-α, IL-17A, IL-10 and secondary cytokines, halted growth of implanted tumors and inhibited progression of spontaneous KRAS-driven adenocarcinoma in mice. Thus, TCR-mediated activation of CD4(+) TILs results in alternative p38 activation and production of protumorigenic factors and can be targeted for therapeutic benefit.

Counter-regulation of T cell effector function by differentially activated p38.

Unlike the MAP kinase (MAPK) cascade that phosphorylates p38 on the activation loop, T cell receptor (TCR) signaling results in phosphorylation on Tyr-323 (pY323, alternative pathway). Using mice expressing p38α and p38ß with Y323F substitutions, we show that alternatively but not MAPK cascade-activated p38 up-regulates the transcription factors NFATc1 and IRF4, which are required for proliferation and cytokine production. Conversely, activation of p38 with UV or osmotic shock mitigated TCR-mediated activation by phosphorylation and cytoplasmic retention of NFATc1. Notably, UVB treatment of human psoriatic lesions reduced skin-infiltrating p38 pY323(+) T cell IRF4 and IL-17 production. Thus, distinct mechanisms of p38 activation converge on NFATc1 with opposing effects on T cell immunity, which may underlie the beneficial effect of phototherapy on psoriasis.