Turn-on RNA Mango Beacons for trans-acting fluorogenic nucleic acid detection.

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have trans-acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand. Our first design is bimolecular in nature and uses a DNA inhibition strand to prevent folding of the Mango aptamer core until binding to a target RNA. Our second design is unimolecular in nature, and features hybridization arms flanking the core that inhibit G-quadruplex folding until refolding is triggered by binding to a target RNA strand. Our third design builds upon this structure, and incorporates a self-inhibiting domain into one of the flanking arms that deliberately binds to, and precludes folding of, the aptamer core until a target is bound. This design separates G-quadruplex folding inhibition and RNA target hybridization into separate modules, enabling a more universal unimolecular beacon design. All three Mango Beacons feature high contrasts and low costs when compared to conventional molecular beacons, with excellent potential for in vitro and in vivo applications.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immunostaining.

Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.

A molecular beacon design for a colorimetric loop-mediated isothermal amplification assay.

In this study, a molecular beacon (MB) was designed for colorimetric loop-mediated isothermal amplification (cLAMP). The length of complementary bases on the MB, guanine and cytosine content (GC content), and hybridization sites of complementary bases were investigated as key factors affecting the design of the MB. We designed MBs consisting of 10, 15, and 20 complementary bases located at both ends of the HRPzyme. In the case of the long dumbbell DNA structure amplified from the hlyA gene of Listeria monocytogenes, possessing a flat region (F1c-B1) of 61 base pairs (bp), an MB was designed to intercalate into the flat region between the F1c and B1 regions of the LAMP amplicons. In the case of the short dumbbell DNA structure amplified from the bcfD gene of Salmonella species possessing a flat region (F1c-B1) length of 6 bp, another MB was designed to intercalate into the LoopF or LoopB regions of the LAMP amplicons. The results revealed that the hybridization site of the MB on the LAMP amplicons was not crucial in designing the MB, but the GC content was an important factor. The highest hybridization efficiencies for LAMP amplicons were obtained from hlyA gene-specific and bcfD gene-specific MBs containing 20- and 15-base complementary sequences, respectively, which exhibited the highest GC content. Therefore, designing MBs with a high GC content is an effective solution to overcome the low hybridization efficiency of cLAMP assays. The results obtained can be used as primary data for designing MBs to improve cLAMP accessibility.

Droplet digital molecular beacon-LAMP assay via pico-injection for ultrasensitive detection of pathogens.

A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for the ultrasensitive and quantitative identification of pathogens, leveraging the sequence-specific detection capabilities of MBs. The microfluidic device contained three distinct functional units including droplet generation, pico-injection, and droplet counting. Utilizing a pico-injector, MBs are introduced into each droplet to specifically identify LAMP amplification products, thereby overcoming issues related to temperature incompatibility. Our methodology has been validated through the quantitative detection of Escherichia coli, achieving a detection limit as low as 9 copies/µL in a model plasmid containing the malB gene and 3 CFU/µL in a spiked milk sample. The total analysis time was less than 1.5 h. The sensitivity and robustness of this platform further demonstrated the potential for rapid pathogen detection and diagnosis, particularly when integrated with cutting-edge microfluidic technologies.

OPTILOD: Optimal Beacon Placement for High-Accuracy Indoor Localization of Drones.

For many applications, drones are required to operate entirely or partially autonomously. In order to fly completely or partially on their own, drones need to access location services for navigation commands. While using the Global Positioning System (GPS) is an obvious choice, GPS is not always available, can be spoofed or jammed, and is highly error-prone for indoor and underground environments. The ranging method using beacons is one of the most popular methods for localization, especially for indoor environments. In general, the localization error in this class is due to two factors: the ranging error, and the error induced by the relative geometry between the beacons and the target object to be localized. This paper proposes OPTILOD (Optimal Beacon Placement for High-Accuracy Indoor Localization of Drones), an optimization algorithm for the optimal placement of beacons deployed in three-dimensional indoor environments. OPTILOD leverages advances in evolutionary algorithms to compute the minimum number of beacons and their optimal placement, thereby minimizing the localization error. These problems belong to the Mixed Integer Programming (MIP) class and are both considered NP-hard. Despite this, OPTILOD can provide multiple optimal beacon configurations that minimize the localization error and the number of deployed beacons concurrently and efficiently.

Relabeling for Indoor Localization Using Stationary Beacons in Nursing Care Facilities.

In this study, we propose an augmentation method for machine learning based on relabeling data in caregiving and nursing staff indoor localization with Bluetooth Low Energy (BLE) technology. Indoor localization is used to monitor staff-to-patient assistance in caregiving and to gain insights into workload management. However, improving accuracy is challenging when there is a limited amount of data available for training. In this paper, we propose a data augmentation method to reuse the Received Signal Strength (RSS) from different beacons by relabeling to the locations with less samples, resolving data imbalance. Standard deviation and Kullback-Leibler divergence between minority and majority classes are used to measure signal pattern to find matching beacons to relabel. By matching beacons between classes, two variations of relabeling are implemented, specifically full and partial matching. The performance is evaluated using the real-world dataset we collected for five days in a nursing care facility installed with 25 BLE beacons. A Random Forest model is utilized for location recognition, and performance is compared using the weighted F1-score to account for class imbalance. By increasing the beacon data with our proposed relabeling method for data augmentation, we achieve a higher minority class F1-score compared to augmentation with Random Sampling, Synthetic Minority Oversampling Technique (SMOTE) and Adaptive Synthetic Sampling (ADASYN). Our proposed method utilizes collected beacon data by leveraging majority class samples. Full matching demonstrated a 6 to 8% improvement from the original baseline overall weighted F1-score.

Cryologger Ice Tracking Beacon: A Low-Cost, Open-Source Platform for Tracking Icebergs and Ice Islands.

Icebergs and ice islands (large, tabular icebergs) present a significant hazard to marine vessels and infrastructure at a time when demand for access to Arctic waters is increasing. There is a growing demand for in situ iceberg tracking data to monitor their drift trajectories and improve models used for operational forecasting of ice hazards, yet the high cost of commercial tracking devices often prevents monitoring at optimal spatial and temporal resolutions. Here, we provide a detailed description of the Cryologger Ice Tracking Beacon (ITB), a low-cost, robust, and user-friendly data logger and telemeter for tracking icebergs and ice islands based on the Arduino open-source electronics platform. Designed for deployments of at least 2 years with an hourly sampling interval that is remotely modifiable by the end user, the Cryologger ITB provides long-term measurements of position, temperature, pressure, pitch, roll, heading, and battery voltage. Data are transmitted via the Iridium satellite network at user-specified intervals. We present the results of field campaigns in 2018 and 2019, which saw the deployment of 16 ITBs along the coasts of Greenland and Ellesmere and Baffin islands. The overall success of these ITB deployments has demonstrated that inexpensive, open-source hardware and software can provide a reliable and cost-effective method of monitoring icebergs and ice islands in the polar regions.

Enzyme and MicroRNA Dual-Regulated Photodynamic Molecular Beacons for Cell-Selective Amplification of Antitumor Efficacy.

Photodynamic molecular beacons (PMBs) are highly appealing for activatable photodynamic therapy (PDT), but their applications are hindered by limited therapeutic efficacy. Here, by molecular engineering of enzyme-responsive units in the loop region of DNA-based PMBs, we present for the first time the modular design of an enzyme/microRNA dual-regulated PMB (D-PMB) to achieve cancer-cell-selective amplification of PDT efficacy. In the design, the "inert" photosensitizers in D-PMB could be repeatedly activated in the presence of both tumor-specific enzyme and miRNA, leading to amplified generation of cytotoxic singlet oxygen species and therefore enhanced PDT efficacy in vitro and in vivo. By contrast, low photodynamic activity could be observed in healthy cells, as D-PMB activation has been largely avoided by the dual-regulatable design. This work presents a cooperatively activated PDT strategy, which enables enhanced therapeutic efficacy with improved tumor-specificity and thus conceptualizes an approach to expand the repertoire of designing smart tumor treatment modality.

A Multiple Targeting Nanoprobe for Identifying Cancer Metastatic Sites Based on Detection of Various mRNAs in Circulating Tumor Cells.

Identification of cancer metastatic sites is of importance for adjusting therapeutic interventions and treatment choice. However, identifying the location of metastatic lesions with easy accessibility and high safety is challenging. Here we demonstrate that cancer metastatic sites can be accurately detected by a triple targeting nanoprobe. Through coencapsulating molecular beacons probing a cancer biomarker (CXCR4 mRNA), a lung metastatic biomarker (CTSC mRNA), and a bone metastatic biomarker (JAG1 mRNA), the nanoprobe decorated by SYL3C conjugated hyaluronic acid and ICAM-1 specific aptamer conjugated hyaluronic acid can target diverse phenotyped circulating tumor cells (CTCs) during epithelial-mesenchymal and mesenchymal-epithelial transitions in whole blood for sensitive probing. The detection of CTCs from cancer patients shows that the nanoprobe can provide accurate information to distinguish different cancer metastasis statuses including nonmetastasis, lung metastasis, and bone metastasis. This study proposes an efficient screening tool for identifying the location of distant metastatic lesions via facile blood biopsy.

Detecting mir-155-3p through a Molecular Beacon Bead-Based Assay.

Lung cancer (LC) is recognized as one of the most prevalent and lethal cancers worldwide, underscoring an urgent need for innovative diagnostic and therapeutic approaches. MicroRNAs (miRNAs) have emerged as promising biomarkers for several diseases and their progression, such as LC. However, traditional methods for detecting and quantifying miRNAs, such as PCR, are time-consuming and expensive. Herein, we used a molecular beacon (MB) bead-based assay immobilized in a microfluidic device to detect miR-155-3p, which is frequently overexpressed in LC. The assay relies on the fluorescence enhancement of the MB upon binding to the target miRNA via Watson and Crick complementarity, resulting in a conformational change from a stem-loop to a linear structure, thereby bringing apart the fluorophores at each end. This assay was performed on a microfluidic platform enabling rapid and straightforward target detection. We successfully detected miR-155-3p in a saline solution, obtaining a limit of detection (LOD) of 42 nM. Furthermore, we evaluated the method's performance in more complex biological samples, including A549 cells' total RNA and peripheral blood mononuclear cells (PBMCs) spiked with the target miRNA. We achieved satisfactory recovery rates, especially in A549 cells' total RNA.

Tracking real-time proximity in daily life: A new tool to examine social interactions.

Social interactions, spending time together, and relationships are important for individuals' well-being, with people feeling happier when they spend more time with others. So far, most information about the frequency and duration of spending time together is based on self-report questionnaires. Although recent technological innovations have stimulated the development of objective approaches for measuring physical proximity in humans in everyday life, these methods still have substantial limitations. Here we present a novel method, using Bluetooth low-energy beacons and a smartphone application, to measure the frequency and duration of dyads being in close proximity in daily life. This method can also be used to link the frequency and duration of proximity to the quality of interactions, by using proximity-triggered questionnaires. We examined the use of this novel method by exploring proximity patterns of family interactions among 233 participants (77 Dutch families, with 77 adolescents [Mage = 15.9] and 145 parents [Mage = 48.9]) for 14 consecutive days. Overall, proximity-based analyses indicated that adolescents were more often and longer in proximity to mothers than to fathers, with large differences between families in frequency and duration. Proximity-triggered evaluations of the interactions and parenting behavior were generally positive for both fathers and mothers. This innovative method is a promising tool that can be broadly used in other social contexts to yield new and more detailed insights into social proximity in daily life.

Detection of a 7SL RNA-derived small non-coding RNA using Molecular Beacons in vitro and in cells.

Small non-coding RNAs (sncRNA) are involved in many steps of the gene expression cascade and regulate processing and expression of mRNAs by the formation of ribonucleoprotein complexes (RNP) such as the RNA-induced silencing complex (RISC). By analyzing small RNA Seq data sets, we identified a sncRNA annotated as piR-hsa-1254, which is likely derived from the 3'-end of 7SL RNA2 (RN7SL2), herein referred to as snc7SL RNA. The 7SL RNA is an abundant long non-coding RNA polymerase III transcript and serves as structural component of the cytoplasmic signal recognition particle (SRP). To evaluate a potential functional role of snc7SL RNA, we aimed to define its cellular localization by live cell imaging. Therefore, a Molecular Beacon (MB)-based method was established to compare the subcellular localization of snc7SL RNA with its precursor 7SL RNA. We designed and characterized several MBs in vitro and tested those by live cell fluorescence microscopy. Using a multiplex approach, we show that 7SL RNA localizes mainly to the endoplasmic reticulum (ER), as expected for the SRP, whereas snc7SL RNA predominately localizes to the nucleus. This finding suggests a fundamentally different function of 7SL RNA and its derivate snc7SL RNA.

NEXUS trial: a multicenter phase II clinical study evaluating the efficacy and safety of the perioperative use of encorafenib, binimetinib, and cetuximab in patients with previously untreated surgically resectable BRAF V600E mutant colorectal oligometastases.

BACKGROUND: The optimal treatment strategy for resectable BRAF V600E mutant colorectal oligometastases (CRM) has not been established due to the rarity and rapid progression of the disease. Since the unresectable recurrence rate is high, development of novel perioperative therapies are warranted. On December 2020, the BEACON CRC triplet regimen of encorafenib, binimetinib, and cetuximab was approved for unresectable metastatic colorectal cancer in Japan. METHODS: The NEXUS trial is a multicenter phase II clinical study evaluating the efficacy and safety of the perioperative use of encorafenib, binimetinib, and cetuximab in patients with previously untreated surgically resectable BRAF V600E mutant CRM. The key inclusion criteria are as follows: histologically diagnosed with colorectal adeno/adenosquamous carcinoma; RAS wild-type and BRAF V600E mutation by tissue or blood; and previously untreated resectable distant metastases. The triplet regimen (encorafenib: 300 mg daily; binimetinib: 45 mg twice daily; cetuximab: 400 mg/m2, then 250 mg/m2 weekly, 28 days/cycle) is administered for 3 cycles each before and after curative resection. The primary endpoint of the study is the 1-year progression-free survival (PFS) rate and the secondary end points are the PFS, disease-free survival, overall survival, and objective response rate. The sample size is 32 patients. Endpoints in the NEXUS trial as well as integrated analysis with the nationwide registry data will be considered for seeking regulatory approval for the perioperative use of the triplet regimen. DISCUSSION: The use of the triplet regimen in the perioperative period is expected to be safe and effective in patients with resectable BRAF V600E mutant CRM. TRIAL REGISTRATION: jRCT2031220025, April. 16, 2022.

Advancements in Rapid and Affordable Diagnostic Testing for Respiratory Infectious Diseases: Evaluation of Aptamer Beacon Technology for Rapid and Sensitive Detection of SAR-CoV-2 in Breath Condensate.

The demand for rapid and efficient diagnostic point-of-care tests for respiratory infectious diseases has become increasingly critical in the current landscape. The emphasis on accessibility has been underscored over the past year, making it crucial to have biological components that exhibit fast and accurate kinetics. The foundation for precise, swift, and effective testing relies on the availability of highly responsive biological agents. Two published aptamer DNA sequences designated Song and MSA52 and their truncated internal stem-loop structures were studied for their potential to serve as aptamer beacons for rapid COVID detection. The candidate beacons were covalently labeled with Atto 633 dye attached to their 5' ends and Iowa Black quencher attached to their 3' ends. The whole aptamer structures exhibited the greatest fluorescence signal intensities and higher fluorescence background than their truncated internal stem-loop beacon structures suggesting that the distance between fluorophores and quenchers was greater for the whole aptamer beacon candidates versus the isolated stem-loop structures. Beacon candidates were tested against two heat- or gamma radiation-killed SARS-CoV-2 Washington 1/2020 virus samples and three different COVID spike (S) proteins to test their effectiveness. Despite the higher background fluorescence, the whole aptamer beacons showed better signal-to-noise ratios and were selected for further investigation. Limit of detection (LOD) studies revealed that both the whole Song and whole MSA52 aptamer beacon candidates had a LOD of 9.61 × 103 genome equivalents in phosphate-buffered saline using the red channel of a Promega Quantus™ fluorometer which correlated well with confirmatory spectrofluorometry. Cross-reactivity studies using numerous COVID variants, related coronaviruses, and other common respiratory pathogens suggested greater COVID selectivity for the whole MSA52 versus the whole Song aptamer beacon candidate, indicating promise for specific COVID detection. Importantly, both whole aptamer beacon candidates exhibited very rapid "bind and detect" fluorescence increases within the first 1-2 min of mixing the beacons with killed SARS-CoV-2 viruses in 100 µl samples. Overall, this work illustrates the strong potential for aptamer beacons for rapid, on-site detection and presumptive diagnosis of COVID in breath condensates or other small liquid samples. This research highlights the strong potential of aptamer beacons for addressing the need for fast and convenient diagnostic tools in global health contexts, especially in resource-limited settings.

A novel aptamer beacon for rapid screening of recombinant cells and in vivo monitoring of recombinant proteins.

Recombinant protein drugs, which are typically produced by mammalian host cells, have been approved for the treatment of a range of diseases. Accordingly, systems for selecting recombinant cell lines with efficient protein expression and for testing the content of recombinant proteins in vivo are crucial to the large-scale production and application of protein-based therapeutic drugs. In this study, we designed three aptamer beacons to detect His-tag, a common label of recombinant proteins. We found that all three beacons could specifically and quantitatively measure the His-tagged recombinant proteins with a short reaction time. Among these three beacons, the 6H5-MU beacon had the highest sensitivity for His polypeptides with a detection limit of 250 ng/mL and the shortest detection time within 1 min. Furthermore, we established a rapid and highly effective recombinant cell line construction system, which could obtain monoclonal cell lines with high yields of target proteins within 21 days, by combining 6H5-MU with pSB, a novel plasmid composed of a Sleeping Beauty transposase and a transposon. Finally, 6H5-MU also discriminately tested the serum concentration of His-tagged recombinant proteins in vivo, with consistent results compared to enzyme-linked immunosorbent assay (ELISA). We thus established a rapid and high-throughput method for generating recombinant cell lines and in vivo monitoring of recombinant protein levels, thereby providing a new platform for the development and preparation of recombinant protein drugs. KEY POINTS: • The 6H5-MU aptamer beacon rapidly and accurately binds to His-tagged recombinant proteins. • A system for rapid and high-throughput generation of recombinant cell lines is established using 6H5-MU and pSB. • 6H5-MU allows in vivo monitoring of recombinant protein levels.

Moral beacons: Understanding moral character and moral influence.

OBJECTIVE: We introduce the concept of moral beacons-individuals who are higher in moral character than their peers and prominent within their social environment-and examine the degree to which moral beacons increase the moral awareness of their peers. BACKGROUND: Using data from cohorts of students in graduate business education across two universities, we applied theory and methods from organizational behavior, personality psychology, and social networks analysis to test two research questions about moral beacons. METHOD: We used latent profile analysis of data from personality questionnaires and social network surveys completed by graduate business students at two universities (N = 502) to identify individuals classified as moral beacons. We used peer nominations and an in-class business case discussion exercise to assess moral influence. RESULTS: Latent profile analysis identified a latent class of moral beacons in our sample. These individuals received more nominations from their peers in end-of-class surveys as guides for moral thought and action and positively impacted the moral awareness of their peers in a discussion of a difficult business case about possible lead poisoning of employees, but did not significantly change their counterparts' moral awareness in a different case. CONCLUSIONS: These results provide promising initial evidence that moral beacons can be distinguished from their peers by both moral character and social prominence and can act as guides for others, at times encouraging greater consideration of the moral aspects of situations and decisions. As these results are the first of their kind, we encourage further replication and investigations of moral beacons and moral influence in other settings.

Determination of adenosine by CRISPR-Cas12a system based on duplexed aptamer and molecular beacon reporter linked to gold nanoparticles.

Adenosine as a potential tumor marker is of great value for clinical disease diagnosis. Since the CRISPR-cas12a system is only capable of recognizing nucleic acid targets we expanded the CRISPR-cas12a system to determine small molecules by designing a duplexed aptamer (DA) converting g-RNA recognition of adenosine to recognition of aptamer complementary DNA strands (ACD). To further improve the sensitivity of determination, we designed a molecule beacon (MB)/gold nanoparticle (AuNP)-based reporter, which has higher sensitivity than traditional ssDNA reporter. In addition, the AuNP-based reporter enables more efficient and fast determination. The determination of adenosine under 488-nm excitation can be realized within 7 min, which is more than 4 times faster than traditional ssDNA reporter. The linear determination range of the assay to adenosine was 0.5-100 µM with the determination limit of 15.67 nM. The  assay was applied to recovery determination of adenosine in serum samples with satisfactory results. The recoveries were between 91 and 106% and the RSD values of different concertation were below  4.8%. This sensitive, highly selective, and stable sensing system is expected to play a role in the clinical determination of adenosine and other biomolecules.

Seismic Beacon-A New Instrument for Detection of Changes in Rock Massif.

The seismic beacon is a new instrument that allows for the measurement of changes in a rock massif with high sensitivity. It is based on effects, which affect the propagation of harmonic seismic waves generated continuously with stable and precise frequency and amplitude. These seismic waves are registered by a system of seismic stations. The amplitude of the seismic signal is very small, and it is normally hidden in a seismic noise. Special techniques are applied to increase the signal-to-noise ratio. In 2020, the first prototype of the seismic beacon was constructed in a laboratory, and field tests were performed in 2022 and 2023. During the tests, the changes in spectral amplitude and phase of seismic waves were detected, which is interpreted as the changes in material properties. These measurements testified the basic functionality of the device. The seismic beacon has been developed primarily for the detection of critical stress before an earthquake, which is manifested by non-linear effects such as higher harmonics generation. In addition, it could be used, for example, in the detection of magma movements, groundwater level changes, changes in hydrocarbon saturation in rocks during the extraction of oil and natural gas, or the penetration of gases and liquids into the earth's crust.

A Minimalist Self-Localization Approach for Swarm Robots Based on Active Beacon in Indoor Environments.

When performing indoor tasks, miniature swarm robots are suffered from their small size, poor on-board computing power, and electromagnetic shielding of buildings, which means that some traditional localization methods, such as global positioning system (GPS), simultaneous localization and mapping (SLAM), and ultra-wideband (UWB), cannot be employed. In this paper, a minimalist indoor self-localization approach for swarm robots is proposed based on active optical beacons. A robotic navigator is introduced into a swarm of robots to provide locally localization services by actively projecting a customized optical beacon on the indoor ceiling, which contains the origin and the reference direction of localization coordinates. The swarm robots observe the optical beacon on the ceiling via a bottom-up-view monocular camera, and extract the beacon information on-board to localize their positions and headings. The uniqueness of this strategy is that it uses the flat, smooth, and well-reflective ceiling in the indoor environment as a ubiquitous plane for displaying the optical beacon; meanwhile, the bottom-up view of swarm robots is not easily blocked. Real robotic experiments are conducted to validate and analyze the localization performance of the proposed minimalist self-localization approach. The results show that our approach is feasible and effective, and can meet the needs of swarm robots to coordinate their motion. Specifically, for the stationary robots, the average position error and heading error are 2.41 cm and 1.44°; when the robots are moving, the average position error and heading error are less than 2.40 cm and 2.66°.

Beacon Success Rate versus Gateway Density in Sub-GHz Sensor Networks.

Multiple Gateways (GWs) provide network connectivity to Internet of Things (IoT) sensors in a Wide Area Network (WAN). The End Nodes (ENs) can connect to any GW by discovering and acquiring its periodic beacons. This provides GW diversity, improving coverage area. However, simultaneous periodic beacon transmissions among nearby GWs lead to interference and collisions. In this study, the impact of such intra-network interference is analyzed to determine the maximum number of GWs that can coexist. The paper presents a new collision model that considers the combined effects of the Medium Access Control (MAC) and Physical (PHY) layers. The model takes into account the partial overlap durations and relative power of all colliding events. It also illustrates the relationship between the collisions and the resulting packet loss rates. A performance evaluation is presented using a combination of analytical and simulation methods, with the former validating the simulation results. The system models are developed from experimental data obtained from field measurements. Numerical results are provided with Gaussian Frequency Shift Keying (GFSK) modulation. This paper provides guidance on selecting GFSK modulation parameters for low bit-rate and narrow-bandwidth IoT applications. The analysis and simulation results show that larger beacon intervals and frequency hopping help in reducing beacon loss rates, at the cost of larger beacon acquisition latency. On the flip side, the gateway discovery latency reduces with increasing GW density, thanks to an abundance of beacons.