Development of a Patient-Derived 3D Immuno-Oncology Platform to Potentiate Immunotherapy Responses in Ascites-Derived Circulating Tumor Cells.

High-grade serous ovarian cancer (HGSOC) is responsible for the majority of gynecology cancer-related deaths. Patients in remission often relapse with more aggressive forms of disease within 2 years post-treatment. Alternative immuno-oncology (IO) strategies, such as immune checkpoint blockade (ICB) targeting the PD-(L)1 signaling axis, have proven inefficient so far. Our aim is to utilize epigenetic modulators to maximize the benefit of personalized IO combinations in ex vivo 3D patient-derived platforms and in vivo syngeneic models. Using patient-derived tumor ascites, we optimized an ex vivo 3D screening platform (PDOTS), which employs autologous immune cells and circulating ascites-derived tumor cells, to rapidly test personalized IO combinations. Most importantly, patient responses to platinum chemotherapy and poly-ADP ribose polymerase inhibitors in 3D platforms recapitulate clinical responses. Furthermore, similar to clinical trial results, responses to ICB in PDOTS tend to be low and positively correlated with the frequency of CD3+ immune cells and EPCAM+/PD-L1+ tumor cells. Thus, the greatest response observed with anti-PD-1/anti-PD-L1 immunotherapy alone is seen in patient-derived HGSOC ascites, which present with high levels of systemic CD3+ and PD-L1+ expression in immune and tumor cells, respectively. In addition, priming with epigenetic adjuvants greatly potentiates ICB in ex vivo 3D testing platforms and in vivo tumor models. We further find that epigenetic priming induces increased tumor secretion of several key cytokines known to augment T and NK cell activation and cytotoxicity, including IL-6, IP-10 (CXCL10), KC (CXCL1), and RANTES (CCL5). Moreover, epigenetic priming alone and in combination with ICB immunotherapy in patient-derived PDOTS induces rapid upregulation of CD69, a reliable early activation of immune markers in both CD4+ and CD8+ T cells. Consequently, this functional precision medicine approach could rapidly identify personalized therapeutic combinations able to potentiate ICB, which is a great advantage, especially given the current clinical difficulty of testing a high number of potential combinations in patients.

Advances in MIMO Antenna Design for 5G: A Comprehensive Review.

Multiple-input multiple-output (MIMO) technology has emerged as a highly promising solution for wireless communication, offering an opportunity to overcome the limitations of traffic capacity in high-speed broadband wireless network access. By utilizing multiple antennas at both the transmitting and receiving ends, the MIMO system enhances the efficiency and performance of wireless communication systems. This manuscript specifies a comprehensive review of MIMO antenna design approaches for fifth generation (5G) and beyond. With an introductory glimpse of cellular generation and the frequency spectrum for 5G, profound key enabling technologies for 5G mobile communication are presented. A detailed analysis of MIMO performance parameters in terms of envelope correlation coefficient (ECC), total active reflection coefficient (TARC), mean effective gain (MEG), and isolation is presented along with the advantages of MIMO technology over conventional SISO systems. MIMO is characterized and the performance is compared based on wideband/ultra-wideband, multiband/reconfigurable, circular polarized wideband/circular polarized ultra-wideband/circular polarized multiband, and reconfigurable categories. The design approaches of MIMO antennas for various 5G bands are discussed. It is subsequently enriched with the detailed studies of wideband (WB)/ultra-wideband (UWB), multiband, and circular polarized MIMO antennas with different design techniques. A good MIMO antenna system should be well decoupled among different ports to enhance its performance, and hence isolation among different ports is a crucial factor in designing high-performance MIMO antennas. A summary of design approaches with improved isolation is presented. The manuscript summarizes the various MIMO antenna design aspects for NR FR-1 (new radio frequency range) and NR FR-2, which will benefit researchers in the field of 5G and forthcoming cellular generations.

Inaugurating High-Throughput Profiling of Extracellular Vesicles for Earlier Ovarian Cancer Detection.

Detecting early cancer through liquid biopsy is challenging due to the lack of specific biomarkers for early lesions and potentially low levels of these markers. The current study systematically develops an extracellular-vesicle (EV)-based test for early detection, specifically focusing on high-grade serous ovarian carcinoma (HGSOC). The marker selection is based on emerging insights into HGSOC pathogenesis, notably that it arises from precursor lesions within the fallopian tube. This work thus establishes murine fallopian tube (mFT) cells with oncogenic mutations and performs proteomic analyses on mFT-derived EVs. The identified markers are then evaluated with an orthotopic HGSOC animal model. In serially-drawn blood of tumor-bearing mice, mFT-EV markers increase with tumor initiation, supporting their potential use in early cancer detection. A pilot clinical study (n = 51) further narrows EV markers to five candidates, EpCAM, CD24, VCAN, HE4, and TNC. The combined expression of these markers distinguishes HGSOC from non-cancer with 89% sensitivity and 93% specificity. The same markers are also effective in classifying three groups (non-cancer, early-stage HGSOC, and late-stage HGSOC). The developed approach, for the first time inaugurated in fallopian tube-derived EVs, could be a minimally invasive tool to monitor women at high risk of ovarian cancer for timely intervention.

Phosphorylation-linked complex profiling identifies assemblies required for Hippo signal integration.

While several computational methods have been developed to predict the functional relevance of phosphorylation sites, experimental analysis of the interdependency between protein phosphorylation and Protein-Protein Interactions (PPIs) remains challenging. Here, we describe an experimental strategy to establish interdependencies between protein phosphorylation and complex formation. This strategy is based on three main steps: (i) systematically charting the phosphorylation landscape of a target protein; (ii) assigning distinct proteoforms of the target protein to different protein complexes by native complex separation (AP-BNPAGE) and protein correlation profiling; and (iii) analyzing proteoforms and complexes in cells lacking regulators of the target protein. We applied this strategy to YAP1, a transcriptional co-activator for the control of organ size and tissue homeostasis that is highly phosphorylated and among the most connected proteins in human cells. We identified multiple YAP1 phosphosites associated with distinct complexes and inferred how both are controlled by Hippo pathway members. We detected a PTPN14/LATS1/YAP1 complex and suggest a model how PTPN14 inhibits YAP1 via augmenting WW domain-dependent complex formation and phosphorylation by LATS1/2.

Profiling extracellular vesicles in circulation enables the early detection of ovarian cancer.

Ovarian cancer is a heterogeneous group of tumors in both cell type and natural history. While outcomes are generally favorable when detected early, the most common subtype, high-grade serous carcinoma (HGSOC), typically presents at an advanced stage and portends less favorable prognoses. Its aggressive nature has thwarted early detection efforts through conventional detection methods such as serum CA125 and ultrasound screening and thus inspired the investigation of novel biomarkers. Here, we report the systematic development of an extracellular-vesicle (EV)-based test to detect early-stage HGSOC. Our study is based on emerging insights into HGSOC biology, notably that it arises from precursor lesions within the fallopian tube before traveling to ovarian and/or peritoneal surfaces. To identify HGSOC marker candidates, we established murine fallopian tube (mFT) cells with oncogenic mutations in Brca1/2, Tp53 , and Pten genes, and performed proteomic analyses on mFT EVs. The identified markers were then evaluated with an orthotopic HGSOC animal model. In serially-drawn blood samples of tumor-bearing mice, mFT-EV markers increased with tumor initiation, supporting their potential use in early cancer detection. A pilot human clinical study ( n = 51) further narrowed EV markers to five candidates, EpCAM, CD24, VCAN, HE4, and TNC. Combined expression of these markers achieved high OvCa diagnostic accuracy (cancer vs. non-cancer) with a sensitivity of 0.89 and specificity of 0.93. The same five markers were also effective in a three-group classification: non-cancer, early-stage (I & II) HGSOC, and late-stage (III & IV) HGSOC. In particular, they differentiated early-stage HGSOC from the rest with a specificity of 0.91. Minimally invasive and repeatable, this EV-based testing could be a versatile and serial tool for informing patient care and monitoring women at high risk for ovarian cancer.

High gain modified Vivaldi vehicular antenna for IoV communications in 5G network.

While targeting the sixth generation (6G) wireless communication which incorporates mainly the Internet of Things (IoT), the Internet of Vehicles (IoV) becomes a vital component to be implemented. A vehicular antenna is designed by using the modified design of the Vivaldi antenna that can cover all the frequency bands for the long-term evolution (LTE) and the mid-band fifth-generation (5G) wireless systems (ranging from 3.3 GHz to 6.5 GHz). The proposed design is optimized by analyzing the effect of the variation in the dimensions of the antenna, changing the position of the taper, the rectangular slot dimensions and the diameter of the circular slot etc. The presented automotive antenna operates at four frequency bands ranging from 3.62 GHz - 4.03 GHz (410 MHz), 4.17 GHz-5.38 GHz (1210 MHz), 5.57 GHz-6.16 GHz (590 MHz) and from 6.23 GHz - 6.64 GHz (410 MHz) with the minimum reflection coefficient of -39 dB, -39.06 dB, -50.42 dB and -12.45 dB, respectively. The proposed antenna offers the high gain of 7.6 dB, 6.7 dB, 9.3 dB and 7.4 dB for the four bands of the operating spectrum, respectively. The analysis of the reflection and radiation characteristics of the antenna when placed on the vehicle confirms the suitability of an antenna for the smart vehicle to vehicle communications.

Cells under pressure: how yeast cells respond to mechanical forces.

In their natural habitats, unicellular fungal microbes are exposed to a myriad of mechanical cues such as shear forces from fluid flow, osmotic changes, and contact forces arising from microbial expansion in confined niches. While the rigidity of the cell wall is critical to withstand such external forces and balance high internal turgor pressure, it poses mechanical challenges during physiological processes such as cell growth, division, and mating that require cell wall remodeling. Thus, even organisms as simple as yeast have evolved complex signaling networks to sense and respond to intrinsic and extrinsic mechanical forces. In this review, we summarize the type and origin of mechanical forces experienced by unicellular yeast and discuss how these forces reorganize cell polarity and how pathogenic fungi exploit polarized assemblies to track weak spots in host tissues for successful penetration. We then describe mechanisms of force-sensing by conserved sets of mechanosensors. Finally, we elaborate downstream mechanotransduction mechanisms that orchestrate appropriate cellular responses, leading to improved mechanical fitness.

Wideband miniaturized patch radiator for Sub-6 GHz 5G devices.

In this article, the compact wideband elliptically slotted semi-circular patch radiator with the defected ground structure for sub-6 GHz applications is designed and developed. The proposed miniaturized patch radiator offers flexibility in adjusting the band of operation by varying the slot dimensions. The effective size reduction is achieved by comparing different iterations in the process of designing and the size of the regular circular-shaped radiator is reduced into the semi-circular radiating patch. The impact of the variation of effective radius of the semi-circular patch, major-axis radius of the elliptical slot, ground plane length, and feed line width is investigated. The size of the proposed radiator is 23.885 × 23.885 × 1.405 mm3. This compact structure manifests the wide bandwidth of 2140 MHz (3.2 GHz-5.34 GHz) with 50% of fractional bandwidth (FBW). The measured results show good agreement with the simulated results. The various parameters validate the utility of the radiator in the C band of super-high frequency (SHF) spectrum and n77 (3.3 GHz-4.2 GHz), n78 (3.3 GHz-3.8 GHz), and n79 (4.4 GHz-5 GHz) bands of the frequency range 1 (FR1) of the sub-6 GHz 5G spectrum.

Crosstalk and spatiotemporal regulation between stress-induced MAP kinase pathways and pheromone signaling in budding yeast.

Budding yeast, Saccharomyces cerevisiae, has been widely used as a model system to study cellular signaling in response to internal and external cues. Yeast was among the first organisms in which the architecture, feedback mechanisms and physiological responses of various MAP kinase signaling cascades were studied in detail. Although these MAP kinase pathways are activated by different signals and elicit diverse cellular responses, such as adaptation to stress and mating, they function as an interconnected signaling network, as they influence each other and, in some cases, even share components. Indeed, various stress signaling pathways interfere with pheromone signaling that triggers a distinct cellular differentiation program. However, the molecular mechanisms responsible for this crosstalk are still poorly understood. Here, we review the general topology of the yeast MAP kinase signaling network and highlight recent and new data revealing how conflicting intrinsic and extrinsic signals are interpreted to orchestrate appropriate cellular responses.

Quantitative analysis of yeast MAPK signaling networks and crosstalk using a microfluidic device.

Eukaryotic cells developed complex mitogen-activated protein kinase (MAPK) signaling networks to sense their intra- and extracellular environment and respond to various stress conditions. For example, S. cerevisiae uses five distinct MAP kinase pathways to orchestrate meiosis or respond to mating pheromones, osmolarity changes and cell wall stress. Although each MAPK module has been studied individually, the mechanisms underlying crosstalk between signaling pathways remain poorly understood, in part because suitable experimental systems to monitor cellular outputs when applying different signals are lacking. Here, we investigate the yeast MAPK signaling pathways and their crosstalk, taking advantage of a new microfluidic device coupled to quantitative microscopy. We designed specific micropads to trap yeast cells in a single focal plane, and modulate the magnitude of a given stress signal by microfluidic serial dilution while keeping other signaling inputs constant. This approach enabled us to quantify in single cells nuclear relocation of effectors responding to MAPK activation, like Yap1 for oxidative stress, and expression of stress-specific reporter expression, like pSTL1-qV and pFIG1-qV for high-osmolarity or mating pheromone signaling, respectively. Using this quantitative single-cell analysis, we confirmed bimodal behavior of gene expression in response to Hog1 activation, and quantified crosstalk between the pheromone- and cell wall integrity (CWI) signaling pathways. Importantly, we further observed that oxidative stress inhibits pheromone signaling. Mechanistically, this crosstalk is mediated by Pkc1-dependent phosphorylation of the scaffold protein Ste5 on serine 185, which prevents Ste5 recruitment to the plasma membrane.

Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5.

Cells continuously adapt cellular processes by integrating external and internal signals. In yeast, multiple stress signals regulate pheromone signaling to prevent mating under unfavorable conditions. However, the underlying crosstalk mechanisms remain poorly understood. Here, we show that mechanical stress activates Pkc1, which prevents lysis of pheromone-treated cells by inhibiting polarized growth. In vitro Pkc1 phosphorylates conserved residues within the RING-H2 domains of the scaffold proteins Far1 and Ste5, which are also phosphorylated in vivo. Interestingly, Pkc1 triggers dispersal of Ste5 from mating projections upon mechanically induced stress and during cell-cell fusion, leading to inhibition of the MAPK Fus3. Indeed, RING phosphorylation interferes with Ste5 membrane association by preventing binding to the receptor-linked Gßγ protein. Cells expressing nonphosphorylatable Ste5 undergo increased lysis upon mechanical stress and exhibit defects in cell-cell fusion during mating, which is exacerbated by simultaneous expression of nonphosphorylatable Far1. These results uncover a mechanical stress-triggered crosstalk mechanism modulating pheromone signaling, polarized growth, and cell-cell fusion during mating.

Protein kinase C and calcineurin cooperatively mediate cell survival under compressive mechanical stress.

Cells experience compressive stress while growing in limited space or migrating through narrow constrictions. To survive such stress, cells reprogram their intracellular organization to acquire appropriate mechanical properties. However, the mechanosensors and downstream signaling networks mediating these changes remain largely unknown. Here, we have established a microfluidic platform to specifically trigger compressive stress, and to quantitatively monitor single-cell responses of budding yeast in situ. We found that yeast senses compressive stress via the cell surface protein Mid2 and the calcium channel proteins Mid1 and Cch1, which then activate the Pkc1/Mpk1 MAP kinase pathway and calcium signaling, respectively. Genetic analysis revealed that these pathways work in parallel to mediate cell survival. Mid2 contains a short intracellular tail and a serine-threonine-rich extracellular domain with spring-like properties, and both domains are required for mechanosignaling. Mid2-dependent spatial activation of the Pkc1/Mpk1 pathway depolarizes the actin cytoskeleton in budding or shmooing cells, thereby antagonizing polarized growth to protect cells under compressive stress conditions. Together, these results identify a conserved signaling network responding to compressive mechanical stress, which, in higher eukaryotes, may ensure cell survival in confined environments.

Room-temperature isopiestic distillation of in situ generated arsenious chloride and its application for the determination of trace level impurities in arsenious oxide.

The room-temperature vapor pressures of HCl and AsCl3 in the presence of hydrochloric acid have been utilized in situ for the removal of arsenious oxide matrix as AsCl3 by isopiestic distillation. Reagent purification, sample dissolution, and matrix separation have been carried out in one step with a clean environment for both analyte and analyst. Quantitative recoveries have been achieved for most of the elements including volatile elements. Blank levels were found to be in nanograms per gram of As2O3 despite use of common reagents and simple reaction vessel. The utility of the present method for the analysis of As2O3 samples has been demonstrated.