Persistent symptoms of SARS‐CoV‐2 are prevalent weeks to months following the infection. To date, it is difficult to disentangle the direct from the indirect effects of SARS‐CoV‐2, including lockdown, social, and economic factors.The study aims to characterize the prevalence of symptoms, functional capacity, and quality of life at 12 months in outpatient symptomatic individuals tested positive for SARS‐CoV‐2 compared to individuals tested negative.From 23 April to 27 July 2021, outpatient symptomatic individuals tested for SARS‐CoV‐2 at the Geneva University Hospitals were followed up 12 months after their test date.At 12 months, out of the 1447 participants , 33.4% reported residual mild to moderate symptoms following SARS‐CoV‐2 infection compared to 6.5% in the control group. Symptoms included fatigue (16% vs. 3.1%), dyspnea (8.9% vs. 1.1%), headache (9.8% vs. 1.7%), insomnia (8.9% vs. 2.7%), and difficulty concentrating (7.4% vs. 2.5%). When compared to the control group, 30.5% of SARS‐CoV‐2 positive individuals reported functional impairment at 12 months versus 6.6%. SARS‐CoV‐2 infection was associated with the persistence of symptoms  and functional impairment  overall, and in subgroups of women, men, individuals younger than 40 years, those between 40–59 years, and in individuals with no past medical or psychiatric history.SARS‐CoV‐2 infection leads to persistent symptoms over several months, including in young healthy individuals, in addition to the pandemic effects, and potentially more than other common respiratory infections. Symptoms impact functional capacity up to 12 months post infection.  Coxiella burnetii [Post‐acute sequelae of SARS‐CoV‐2 infection (PASC), long COVID, or post‐COVID syndrome , 2, 3 haburnetii . To dateburnetii , 14, 15.burnetii , 16 but Additionally, only a few studies using patient records have compared post SARS‐CoV‐2 symptoms to the general population of adults tested negative for the infection , 18, 19,In this study, we describe the prevalence of symptoms, functional capacity, productivity, and quality of life at 12 months in individuals tested positive for SARS‐CoV‐2 compared to individuals tested negative during the same time period (defined as the control group). The study hypothesis postulates that SARS‐CoV‐2 positive individuals suffer more from persistent symptoms leading to functional impairment on top of the pandemic effects when compared to SARS‐CoV‐2 negative individuals. We also assess the association between SARS‐CoV‐2 infection and the long‐term persistence of symptoms.From 23 April to 27 July 2021, an online questionnaire was sent to all adults, 12 months after being tested for SARS‐CoV‐2 infection (RT‐PCR test between 15 March 2020 and 30 July 2020) at the outpatient testing center of the Geneva University Hospitals (Switzerland). Overall, 3515 adults had a valid mobile phone number or email address, and were contacted for follow‐up. Individuals tested positive for SARS‐CoV‐2 were considered as the group of interest for SARS‐CoV‐2 infection and those tested negative for SARS‐CoV‐2 were considered as the control group. All individuals gave consent and the study was approved by the Cantonal Research Ethics Commission of Geneva, Switzerland (protocol number 2021‐00389). Only individuals with a laboratory‐confirmed test date were included in this study.The questionnaire was distributed via REDCap, and a personal link was sent to each participant via email or SMS in case an email address was not available. The follow‐up questionnaire included questions about baseline characteristics, comorbidities, self‐rated health, symptoms at the time of testing, evolution of symptoms since testing, current symptoms over the past 2 weeks, symptom intensity and frequency when present, treatment, number of contacts with the healthcare system , functional capacity, productivity, and quality of life.Age categories were defined as “below 40,” “40–59 years,” and “60 years and above” on the basis of previous studies suggesting that middle age may be a predictor of persistent symptoms . Symptomp‐value <0.05 was considered significant. Prevalence estimates of each symptom were adjusted for the time from infection, age, sex, education, profession, working in healthcare setting, smoking, physical activity, COVID‐19 vaccination status, symptoms at presentation, hospitalization, and the following comorbidities when present prior to testing: overweight or obese, hypertension, respiratory disease, cardiovascular disease, diabetes, immunosuppression, hypothyroidism, anemia, migraine, tension headache, sleeping disorder, anxiety, depression, any psychiatric condition, irritable bowel syndrome, chronic pain syndrome, and chronic fatigue.Data were collected using REDCap v11.0.3 and analyzed using the statistical software Stata, version 16.0 (StataCorp). Descriptive analyses included percentages with comparisons using chi‐square tests or Fisher's exact test when appropriate. A Logistic regression models were used to evaluate associations between SARS‐CoV‐2 infection and persistent symptoms at 12 months, as well as SARS‐CoV‐2 infection and functional impairment at 12 months. Only participants reporting onset of new symptoms at testing were included in the regression analysis, in order to ensure that symptoms were new in nature. The outcome of persistence of symptoms at 12 months was defined as having at least one symptom in the past 2 weeks preceding the questionnaire. The outcome of functional impairment was defined as having mild, moderate, or severe functional impairment using the Sheehan Disability Scale . Multivan = 703 (20%) with a positive test result and 2812 (80%) with a negative test result. Of those invited to participate, 1639 persons answered the questionnaire (46.6% response rate), with a mean age of 45.4 years , 60.8% were women, and 29.2% had a positive SARS‐CoV‐2 test, compared to nonresponders, who had a mean age of 47.8 years  (p < 0.001) and of whom 56.2% were women (p < 0.001). Of the 1639 persons answering the questionnaire, 192 reported a positive test between the initial test and the follow‐up and were excluded because of lack of a laboratory‐confirmed test date and result. Ultimately, 1447 persons were included in the analysis  had a laboratory‐confirmed SARS‐CoV‐2 infection and n = 1160 (80.2%) had a negative RT‐PCR test, n = 194 (13.4%) self‐reported hospitalization since their test date, with n = 33 (11.5%) reporting their hospitalization was due to SARS‐CoV‐2 infection. The median time from testing to survey was 367 days  overall, 388 days  for individuals who tested positive, and 364 days  for individuals who tested negative.The mean age of the included participants was 45.2 years SD, 14.5) and 61.2% were women. When considering baseline characteristics (Table 4.5 and 6p < 0.001). Significant differences according to SARS‐CoV‐2 infection were seen in the prevalence of fatigue and several neurologic, psychiatric, upper respiratory, and cardiac symptoms (Table p < 0.001). Table At 12 months, an estimated 33.4% of individuals post SARS‐CoV‐2 infection still had at least one symptom compared to 6.5% in the control group (p < 0.001). In the week preceding the questionnaire, participants reported to have experienced one or more days of reduced productivity in the week preceding the questionnaire in 11.8% of cases compared to 3.9% in the control group (p < 0.001).Functional impairment was more important in individuals who tested positive compared to those tested negative for SARS‐CoV‐2, and so were the numbers of days lost and days with reduced productivity in the week preceding the follow‐up . The association was significant in the subgroups of women, men, individuals younger than 40 years, and those between 40–59 years of age. The association was also significant for individuals without any past medical or psychiatric conditions . The mean weighted mental health score using the SF‐12 questionnaire was 41.8  for individuals post SARS‐CoV‐2 infection compared to 41.0  for the control group (p = 0.041). Additionally, the mean HAD anxiety score was 5.5  for individuals post SARS‐CoV‐2 infection compared to 6.2  for the control group (p = 0.008). The mean HAD depression score was 3.4  for individuals post SARS‐CoV‐2 infection versus 3.7  for the control group (p = 0.189). Further details are presented in Table Self‐rated health was reported as very good to excellent in 47.4% of individuals at 12 months post SARS‐CoV‐2 infection, as good in 38.8% of individuals, and as fair to poor in 13.8% of individuals  and are associated with more functional impairment and loss of productivity when compared to other common upper respiratory infections or pandemic‐related effects in general. Individuals suffering from PASC should be cared for long term, with a multidisciplinary approach addressing the range of their symptoms and any potential impact on their productivity and activities of daily living. These findings also underline the importance of continuing to follow public health recommendations that aim to reduce infection, regardless of age, sex, or underlying conditions in order to avoid persistent symptoms.Limitations in this study include the nature of the follow‐up with self‐reported items as well as the relatively low response rate, which, although similar to other general survey questionnaires , may resTo conclude, SARS‐CoV‐2 infection causes persistent symptoms at 12 months from diagnosis in addition to the COVID‐19 pandemic effects, and more than other potential upper respiratory infections. As the COVID‐19 landscape continues to evolve with new treatment options, vaccination, and new variants, physicians should continue monitoring their patients and encouraging them to avoid infection and re‐infection regardless of age, sex, and underlying conditions in order to reduce the risk of PASC.The authors declare no potential conflict of interest. Furthermore, we confirm that the manuscript has been read and approved by all the named authors, and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that this work was conducted with the ethical approval of the Cantonal Research Ethics Commission of Geneva, Switzerland, and that the approvals are acknowledged in the manuscript.Mayssam Nehme: Conceptualization; formal analysis; investigation; methodology; writing – original draft; writing – review and editing. Olivia Braillard: Conceptualization; data curation; writing – review and editing. François Chappuis: Conceptualization; writing – original draft; writing – review and editing. Laurent Kaiser: Data curation; writing – review and editing. Idris Guessous: Conceptualization; data curation; funding acquisition; methodology; project administration; resources; supervision; writing – original draft; writing – review and editin. All authors of the CoviCare Study team contributed to the study design and reviewing the manuscript.This study is funded by the Leenaards Foundation, the Geneva University Hospitals Private Foundation, and the Private Research Funds of the Division of Primary Care Medicine at the Geneva University Hospitals. The funders of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the manuscript.Supplement 1. Survey instrumentClick here for additional data file.


The Wireless Medium Access Control (WMAC) protocol functions by handling various data frames in order to forward them to neighbor sensor nodes. Under this circumstance, WMAC policies need secure data communication rules and intrusion detection procedures to safeguard the data from attackers. The existing secure Medium Access Control (MAC) policies provide expected and predictable practices against channel attackers. These security policies can be easily breached by any intelligent attacks or malicious actions. The proposed Wireless Interleaved Honeypot-Framing Model (WIHFM) newly implements distributed honeypot-based security mechanisms in each sensor node to act reactively against various attackers. The proposed WIHFM creates an optimal Wireless Sensor Network (WSN) channel model, Wireless Interleaved Honeypot Frames (WIHFs), secure hash-based random frame-interleaving principles, node-centric honeypot engines, and channel-covering techniques. Compared to various existing MAC security policies, the proposed model transforms unpredictable IHFs into legitimate frame sequences against channel attackers. Additionally, introducing WIHFs is a new-fangled approach for distributed WSNs. The successful development of the proposed WIHFM ensures resilient security standards and neighbor-based intrusion alert procedures for protecting MAC frames. Particularly, the proposed wireless honeypot methodology creates a novel idea of using honeypot frame traps against open wireless channel attacks. The development of a novel wireless honeypot traps deals with various challenges such as distributed honeypot management principles , dynamic network backbone management principles (On Demand Acyclic Connectivity model), and distributed attack isolation policies. This effort provides an effective wireless attack-trapping solution in dynamic WSNs. The simulation results show the advantage of the proposed WIHFM over the existing techniques such as Secure Zebra MAC (SZ-MAC), Blockchain-Assisted Secure-Routing Mechanism (BASR), and the Trust-Based Node Evaluation (TBNE) procedure. The experimental section confirms the proposed model attains a 10% to 14% superior performance compared to the existing techniques. Wireless Sensor Networks (WSNs) are diversely used for significant applications such as industrial automation, animal-tracking systems, ocean-monitoring systems, defense support systems, rural surveillance systems , and othThe sensed data are processed and communicated to respective sink nodes through multi-hop-routing procedures. The data on the wireless medium are more vulnerable compared to dedicated wired channels. WSNs need optimal protection via channel security for their application-specific network architectures. As wireless sensor nodes operate under limited internal resources with respect to their memory, battery source, and processor efficiency, the security algorithms must provide efficient and lightweight solutions against unexpected channel attacks. Secure wireless data communication is essential against both active and passive attackers. The intrusions and attacks that infiltrate into data sequences on air create opportunities for data theft, identity theft, packet drops, wormholes, sinkholes, and other functional irregularities in WSNs. Attacks on wireless channels are more frequent than on wired channels. In the absence of steadfast secure servers and protected channels, the wireless sensor nodes meet open threats to live data transmissions. The technical contributions of global research are growing and thereby enabling the construction of flexible network security models for WSNs. Generally, the current wireless security models use cryptography techniques, key distribution models, blockchain technology, Intrusion Detection Systems (IDS), and channel-aware-data-hiding mechanisms.The dedicated wired network infrastructures use firewalls, secure private network policies, centralized IDS engines, honeypots, and key distribution servers to handle attacks efficiently. Among these security models, honeypots are a collection of decoy network systems positioned among legitimate network resources. The honeypot security mechanism provides a virtual system trap with fake utilities to attract both internal and external attacks. The honeypot environment receives the incoming events and analyzes the features to decide the attacks. Here, the attackers may utilize and harm the fake resources. The results generated from honeypot engines help to enrich the network security policies gradually.Honeypots are classified under various categories as highly interactive models, lowly interactive models, research models, and production models. Unlike wired network security models, wireless networks need more flexibility in their security layer functions. Particularly, a WSN requires completely distributed and lightweight security mechanisms that are controlled at each sensor node. The proposed WIHFM is aimed at developing a distributed honeypot environment in each sensor node to secure wireless mediums. According to the proposed idea, each sensor node holds fake internal resources to trap the attackers. The proposed model has been created for developing lightweight honeypot resources in each node with IHF contents. In this newly developed environment, the attacker trap has been created by a WIHFM that is shared among legitimate data frames on the wireless channel.The wireless MAC 802.11) policies and legitimate data frames are vulnerable to attacks. The combination of node-centric honeypot engines and the IHF traps increases the possibility of attracting various attacks from other sensor nodes. The currently accessible secure data communication models are widely anticipated and estimated by intellectual attackers. Regarding the choice of WSN security solutions, Mezrag et al. [.11 policOn the other hand, this technique was not enabled over random WSNs with deeply analyzed results. Guimaraes et al.  comparedCorrespondingly, this technique targeted the benefit of energy-aware-routing principles. Based on security concerns, Huanan et al.  shared vGenerally, the application of WSNs decides the levels of security features to be configured in the real-time conditions. In this case, Majid et al.  deeply aAt the research stage, security concerns are seriously evaluated, and various solutions are proposed. Salau et al.  describeThe channel protection schemes proposed in this work are equipped with node validity schemes, secure channel profile-sharing principles, signal strength indicators, and cluster management principles. Similarly, a few research methodologies found possible solutions to provide secure data transmission solutions under IoT-based WSNs . Anyhow,Wang et al.  found a In the same manner, Lygerou et al.  proposedOn the basis of motivation, Veluchamy et al.  proposedConsidering the computation load and time complexities, Acosta et al.  developeIn the sequence of security considerations, Pashaei et al.  stated tOn-Demand Acyclic Network Connectivity model (ODAC);Interleaved Honeypot-Framing Traps;Distributed Neighbor-based Honeypot Engines;Secure Hashing with Random Interleaving procedures;Confidential and Authenticated Channel Protection.As discussed in this section, various security models and honeypot frameworks are suggested to improve network security metrics. Furthermore, the honeypot-based security limitations are identified for MAC principles. As many research techniques consider network-level security challenges, the identification of channel security and MAC security provides an important and novel suggestion for innovative honeypot strategies. In this regard, this article finds the crucial limitations of the existing systems such as the lack of novel secure MAC models, distributed honeypot engines, Interleaved Honeypot Frame (IHF) solutions, and open traps in WSN. These are considered major research problems under growing attack styles and novel threats against WSNs. In this case, the newly developed security mechanism should be estimated with complex wireless channel traps. Considering these ideas, the proposed WIHFM is motivated to develop a neighbor-based distributed honeypot environment with IHF traps to attract attackers through the open channel. Mainly, the proposed WIHFM contributes the following security features for WSNs:As per the proposed model and technical contributions, the environment of WSNs is created to optimize the network backbone and dynamic link management principles. As mentioned in the list of major contributions, the proposed model configures an ODAC for managing the associativity rules according to uncertain node links and node abilities with respect to time. According to the positioned ODAC-based network backbone and routing principles, the wireless communication shall be operated feasibly. In the next phase, wireless honeypot frames are modelled with appropriate fake data (trap frames) and internal security features among other legitimate data frames in each sensor node. The ordered inclusion of legitimate data frames and honeypot frames on the wireless channel is designed with the help of honeypot-interleaving procedures (interleaving and de-interleaving Algorithms) and randomly generated trap-slot positions. Similarly, data communication is secured by optimal channel security procedures . Hence, the proposed model creates dynamic and distributed honeypot traps in each node to decoy attackers.According to the contributions listed above, the proposed article has been systematized from Analyzing and understanding the current security solutions against various wireless channel attacks provides necessary future frameworks. In this regard, this article considers significant related contributions of recent research works. Additionally, this section provides the specific limitations of different research works and the need for a newly developed WIHFM. Pietro et al.  deliberaAt the same time, the development of current WSN models and their variances receives notable industrial considerations. Khan et al.  proposedSimilarly, the recent advances and security requirements for WSNs and IoT were discussed by Gupta et al.  and BharHere, the attacks such as replay attacks, nonce attacks, and routing attacks were investigated under WSN conditions. On the other hand, the need for improvised solutions was unexploited in this development area.Boyle et al.  and KarlSingh et al.  proposedElshrkawey et al.  and KumaKhashan et al.  suggesteAlmansoori et al.  developeThis work concluded that the secure SZ-MAC model confirmed optimal elimination of vulnerable entries and maximum network throughput attainment. The technical aspects of the work limit the uncertain channel handling and the development of random attacker traps.Awan et al.  proposedWith the same scope, Meena et al.  introducFrom the literature analysis, the proposed article finds the overall limitations of existing techniques such as the lack of node-centric IDS engines, attacker traps, MAC confidentiality, interleaved honeypot solutions, and dynamic sensor connectivity models. These are considered major research limitations to be developed to secure MAC principles. Based on providing the solutions to the above research expectations, the proposed WIHFM has been designed and developed with novel technical aspects. Channel-aware security principles and medium protection in WSN are creating major impacts in data communication. WSN contains a vast number of sensor nodes and independent links for establishing each channel. Wireless sessions established from each sensor node are naturally vulnerable to suspicious activities and attackers. The wireless medium needs protection in terms of secure MAC policies, secure link establishments, and secure data transmissions.The security needs of the wireless medium can be designed and developed based on various cryptography techniques and Distributed IDS (DIDS) procedures. The secure medium places appropriate counterattacking or protection schemes against unique attack patterns. Mostly, wireless channels establish a secure MAC model using different types of cryptography techniques. The cryptography mechanisms such as public key models, private key models, signature validation models, and other integrity-checking models are employed. The provision of standard cryptography techniques are circumvented based on an attacker’s efficiency for extracting the contents of MAC frames. Here, the deployment of a novel MAC-framing solution yields unanticipated protection against channel attackers ,37.Thus, the proposed model develops randomly generated Interleaved Honeypot Frames (IHFs) for protecting the streams of WSN frames. Based on this idea, the network model, attacker model, suspicious event model, and proposed channel-aware security principles have been designed as follows.Let the model of WSN be assumed to have sensor node quantity denoted as Equation (1) shows the basic channel model of WSN. As shown in Equation (1), from  channel . At the Equation (2) denotes The proposed system creates the On Demand Acyclic Connectivity model (ODAC) for enabling the logical links between active sensor nodes. The proposed ODAC model enables energy-sensitive connection establishment rules and association policies among the sensor nodes in an active channel. According to the model, ty model ,40. AccoIn this case, In the same manner, Equation (5) depicts the relationship between As given in Equation (6), As discussed, Equation (7) measures the impact of adversarial data extraction functions and frame-corrupting functions The novel wireless honeypot-framing practices generate the chances of attackers accessing the honeypot frame instead of an original data frame. Each MAC frame sequence contains randomly inserted honeypot frames to deceive the attackers so as to make them feel as if they are accessing the original frame contents. The interleaved honeypot frame consists of an internal Message Validity Block (MVB), Honeypot Boot Block (HBC), and Intrusion Analysis and Validation Block (IVB). As shown in As mentioned in The proposed honeypot frames are inserted based on the frame-interleaving algorithm at the sender’s end. A sender node activates a honeypot frame-interleaving algorithm before it transmits the data on a wireless medium. As given in The technical problem and solution to implementing random honeypot slot generation are illustrated in procedure A and procedure B. Procedure A shows random bit string generation using the sender’s internal node attributes, SHA-3 (512 Bit) computations, and random bit elector function. This approach ensures absolute slot randomness in honeypot frame insertions.Algorithm 1. Procedure A.Procedure (A):Output: 1. Get node MAC address (48 bits), 2. Set random seed matrix 3. Computer,                 4. Call the SHA-3 (512 Bits) function and do               5. Start binary bit string computation,        6. Redo for all 7. Construct binary 8. Generate random slot bits, 9. Store 10. Recall the steps from 1 to 10.Input: End of Procedure (A)At the end of Procedure A (shown in Algorithm 1), the sender node produces these random slot identifiers as a sequence of keyed bit strings n in WSN ,46. In tAlgorithm 2. Procedure B.Procedure (B):Output: Interleaved Honeypot Frames On Channel 1. Set2. Get the stream of3. Set Interleaved Honeypot Frame (IHF) queue4. Do for each transmission,                                                                                5. Set Deinterleaver Honeypot Frame (DHF)queue6. Do for each reception,                                                                                7. Do for all active channels,8. Call Procedure (A) for each9. Set the number ofInput:End of Procedure (B)Based on the sequence of contiguous bit strings, the parity bits are computed for managing bit-level similarity at the sender and receiver sides. The node-centric parity-checker function finds odd parity combinations of bit strings to insert the frames.In WSN, there are various open channels available for sensor data transmissions. However, the active channels are being attacked by malicious activities. Here, the proposed system initiates the IHF solutions for all active wireless channels,The ICFs have a major role in acting against malicious attacks and all suspicious injections on the wireless channel. As WSN distributes the data from various sender nodes via multi-path-routing procedures, the need for sender-originated IHFs is generated for the original frame sequence. In this process, each IHF holds a random number of fake data and other lookalike network credentials to thwart the attackers ,48.At the same time, the interleaving and de-interleaving functions are called at the sender point and receiver point, respectively. The data-forwarding nodes and neighbors of sender and receiver are associated with exchanging the data and not extracting the contents. In the same manner, the activation of the node-centric honeypot-based DIDS engine depends on the successful malicious access to random IHF on the channel. The proposed system develops locally reactive honeypot DIDS engines in each sensor node against the attacker’s events. The proposed honeypot DIDS procedures are called due to HBC activation based on attackers’ interruptions of IHF. Procedure C (Algorithm 3) illustrates the secure wireless MAC practices based on IHF and node-centric honeypot DIDS calls.The proposed wireless honeypot frames, Algorithm 3. Procedure C.Procedure (C):Output: Attacker logs and report1. Do for all attacker access over MAC frame sequence,                     Go to step 2.                     Go to step 3.2. Call HBC and IVB functions (Procedure (D))          Create an alert to all neighbours,          Make an entry in the attacker’s record,3. Continue4. Do for all active channels,5. Call procedure (A) and procedure (B) for activating eachInput: Attacker accessEnd of Procedure (C)Procedure C shown in Algorithm 3 shows the active-monitoring steps to track the attacker’s access over wireless MAC frames.Algorithm 4. Procedure D.Procedure (D):Output: Suspicious events detection1. From Procedure C (step 2)2. Set node’s internal intrusion dataset3. Get4. Call                        Call IDS rule engine functions             Start              Validate             Validate             Validate              5. Start rule-based classification function,          6. Set event classifier logs for each node7. Update the logs for each session 8. Redo for all channelsInput:End of Procedure (D)Compared to other existing mechanisms, the proposed wireless honeypot principles ensure real-time data security only on active channels rather than entire channel connections. The reactive security support system provides a novel solution against various attacks with minimal energy consumption. The attack alert system for each channel works based on the attacker database and rule-based intrusion detection policies ,50. ThesThe rule-based attack classifiers analyze the sensor node’s request at the receiver node depending on successful IHF access by any attacker node. Usually, the legitimate sensor nodes are restricted from accessing IHF as they have been known to interleave functions. From the technical discussion of the proposed model, the novel wireless IHF techniques are identified as more reliable security solutions ,52,53. ACipher Frames,Decrypted Frames,Equations (10) and (11) provide the details of channel frame encryption and decryption models support for secure data transmission. The data confidentiality on wireless channels is achieved by blocking specific AES functions. In the same manner, the participant authentications are ensured with the help of ECCDS principles. As sensor nodes are limited to energy resources and other memory constraints, device-specific authentication principles are required. Based on this issue, ECCDS has been loaded inside the sensor node to authenticate insiders and outsiders. Equation (12) provides the technical aspects of ECCDS with message authentication codes.Thus, the proposed security principles and wireless IHF models protect vulnerable WSN channels from attackers. The experimental test has been created based on the technical specifications listed in As the simulation environment has been created for a worst case WSN scenario, in the configured geographical area (1000 m × 1000 m), the deployment of 300 sensor nodes with a random mobility model create appropriate complexity levels in terms of population, channel establishment time, and link management. Generally, the maximum transmission range of a sensor node is configured up to 100 m. In this scenario, the transmission range has been increased up to 150 m to acquire the actual performance of a real-time sensor environment. In this case, a constant position mobility model can also be used for deploying WSNs. However, the mobility model leads to network uncertainties and random options for constructing the links and channels for each iteration in the simulation model. This practice helps to observe the performance of the proposed system under complicated geographical conditions rather than constantly deployed sensor nodes.Here, the proposed experimental setup expects a wormhole attacker, a packet-dropping attacker, an identity theft attacker, a MAC frame-eavesdropping attacker, and a misrouting attacker. Apart from legitimate sensor nodes, attacker nodes are created in order to initiate the attacks on various channels. These attacker functions are called from selected attacker nodes to initiate malicious events randomly, as per the simulation setup. The wormhole nodes  are created around legitimate sensor nodes in the network. The packet-dropping attacker node calls the functions to collect the data from a legitimate node and drop the same data at the receiving point itself. In this simulation, the identity theft attacker is considered to initiate the functions to obtain the neighbors’ identifiers and manipulate these identifiers due to the attacker’s lack of network reputations. The eavesdropping attacks in the sensor networks are configured with the simulated functions for recording the nearest data transmissions of any node. Finally, the misrouting attack calls the simulated functions that change the legitimate routing table entities into fraudulent entities. The legitimate sensor nodes shall expect any attack from other nodes to be intercepted by the wireless channel. The network simulation arrangement has been deployed using Network Simulator (NS-3.35) tool. In this work, we have implemented network setup and data transmission scenarios using the C++ options of NS-3. Similarly, the internal functions of the existing techniques and proposed models were created with the help of python library files. The proposed model has been developed and compared with the notable existing techniques such as SZ-MAC, BASR, and TBNE. The proposed work and the works acquired for the experimental comparison are implemented in the NS-3 tool under the specified network environment  and netwEach session consumes a unique communication duration based on the currently available communicating parties in the network . As giveAs shown in Based on these discussions, the practical abilities of the proposed WIHFM are recognized during different iterations. The functional aspects of the proposed WIHFM are compared with SZ-MAC, BASR, and TBNE. Each existing technique uses unique channel security principles such as secure MAC protocol, blockchain commutations, and key-based trust evaluation models, respectively. In this regard, Compared to other channel security models, the proposed WIHFM creates complex random honeypot trap possibilities uniquely . ConsequIn this routing strategy, the AODV optimizes the reactive route updates and OLSR optimizes overall network updates latencies. Based on distributed WIHFM functions, the need for optimal routing fusion confirms a minimal routing delay compared to other existing techniques. In this scope, a secure and trusted routing protocol is another choice for notable research works ,65.In this experiment, the proposed WIHFM and BASR produce almost bordering routing delay (msec). However, the proposed WIHFM has a smaller routing delay than BASR between 102 msec and 127 msec. Other techniques attain a maximum routing delay between 153 msec and 160 msec. This result shows the optimal handling of data routing through multiple channels. The proposed functional internals of each security technique consume significant computation overhead from 12.6% to 16.8%. In these cases, the proposed model achieves a slightly improved overhead compared to other techniques between 12.6% and 14.9%. Similarly, BASR attains its maximum overhead of 15.6%. Compared to other security techniques, the proposed model has lightweight IHF procedures, distributed honeypot management principles, and attack detection rules in each node. Hence, the proposed WIHFM manages the finest computation overhead from among the other systems, as the velocity of the sensor node varies from 10 m/s to 50 m/s. In the same manner, the important feature of the proposed security framework is examined using a secure throughout probability rate as given in Complexity evaluation experiments and encryption quality analysis tasks are essential for optimizing the performance of tiny wireless sensor nodes. As the proposed method uses AES and ECCDS for establishing channel confidentiality and neighbor authentication, respectively, Likewise, In this evaluation, the proposed WIHFM-ODAC system achieves an optimal channel recreation time (msec) during attacker interruptions  and otheIn this manner, the resiliency of ODAC has been justified. Thus, the proposed WIHFM ensures secure wireless communication through significantly developed IHF functions . In addiThe security principles and cryptography mechanisms used for protecting data communications are widely practiced among various types of wireless networks. However, the identification of a novel MAC security management system supports resilient WSN protection against various attackers. In this article, the proposed system was implemented with distributed WIHFM and ODAC virtual link management features. As per the newly developed model, the IHF features were identified as novel distributed solutions implemented in each sensor node of the WSN. Similarly, the WIHFM enabled IHF computations and distributed wireless honeypot engines to create random attacker traps to coax the attackers into forged honeypot resources of the sensor node. Based on the proposed model, the attackers and the malicious activities were effectively trapped in IHFs. Consequently, the trapped accesses were recognized as either intrusions or attacks. Finally, the proposed model was compared with the existing frameworks such as BASR, SZ-MAC, and TBNE, as shown in the experimental section. Moreover, the proposed WIHFM outclassed other techniques, as discussed through various performance metrics. However, the proposed WIHFM has limitations regarding its uncertain IHF latency and honeypot response time as the network dynamics change continuously. Future technical aspects, such as the intelligent network dynamic analysis model, dynamic programming model, and uncertainty computation model, are expected to compensate the limitations of WIHFM.As the current network MAC models are not optimally enriched with low-powered dynamic programming solutions against channel uncertainties, the need to improve the static limitations of WIHFM is required in the future. In addition, the next-generation honeypot solutions in the wireless environment are expected to be improved with these distributed trap management policies for application-specific sensor platforms. Notably, the next-generation WSN models are widely sensitive to the energy utilization rate with respect to their application platform. Considering the internals of notable WSNs’ applications, the proposed novel WIHFM can be implemented for military sensor networks, healthcare sensor networks, and other deployment strategies exclusively in the future.


Lung cancer is one of the most lethal cancers worldwide. Cisplatin, a widely used anti‐lung cancer drug, has been limited in clinical application due to its drug resistance. Medicines targeting mitochondrial electron transport chain (ETC) complexes may be effective candidates for cisplatin‐based chemotherapy.In this study, the small molecule drug library from Food and Drug Administration FDA was used to screen for medicines targeting ETC. MTT and colony formation assays were used to investigate cell proliferation. Flow cytometry was employed to analyze cell cycle, apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential. Wound scratch and transwell assays were used to detect migration and invasion abilities. The activities of the ETC complex were tested using kits. Western blot analysis was used to investigate the expressions of related proteins. A mouse xenograft model was constructed to verify the antitumor effect in vivo.The results showed that mubritinib can reduce the activation of the PI3K/mTOR signal pathway, disrupt mitochondrial function, significantly increase ROS levels and induce oxidative stress, and ultimately exert its antitumor effect against non‐small cell lung cancer (NSCLC) both in vivo and in vitro. In addition, the combination of cisplatin and mubritinib can improve the tumor‐suppressive effect of cisplatin.Mubritinib can upregulate intracellular ROS concentration and cell apoptosis, inhibit the PI3K signaling pathway and interfere with the function of mitochondria, thus reducing cell proliferation and increasing ROS induced apoptosis by reducing the activation of Nrf2 by PI3K. In addition to directly causing DNA damage, cisplatin can also lead to DNA damage by inducing ROS production, thus leading to apoptosis. However, ROS can activate Nrf2, which in turn leads to the increase of HO‐1, GPX4 and other antioxidants, which feedback the inhibitory effect of ROS. In addition, the highly expressed PI3K/Akt/mTOR signaling pathway in tumor cells promotes energy generation and cell proliferation by promoting mitochondrial function. PI3K also inhibited Nrf2 activation. This might be one of the important mechanisms of endogenous drug resistance of cisplatin. Mubritinib can upregulate intracellular ROS concentration and cell apoptosis on the one hand, while on the other hand, it can inhibit the PI3K signaling pathway and interfere with the function of mitochondria, thus reducing the cell proliferation and increasing ROS induced apoptosis by reducing the activation of Nrf2 by PI3K. Lung cancer (LC) is the second most common cancer and one of the most lethal cancers worldwide.Cisplatin, a first‐line medicine for the treatment of NSCLC, is thought to block cell cycle progression and induce cell death by binding to nuclear DNA and inducing cisplatin‐DNA cross‐linking.A recent study found that mubritinib, a Food and Drug Administration (FDA) approved drug initially used as a human epidermal growth factor receptor 2 (HER2) inhibitor, could significantly reduce the development of leukemia through inducing mitochondrial respiration‐associated ROS overproduction and cell apoptosis instead of targeting HER2 protein.2 incubator at 37°C.Human lung adenocarcinoma cell lines NCI‐H1975, A549, HCC‐827, and NCI‐H2170 were purchased from ATCC and cultured in a Dulbecco's Modified Eagle's Medium (HyClone) containing 10% fetal bovine serum and 1‰ tertiary antibody in a COThe small‐molecule drug library from FDA was used to investigate the sensitivity of NCI‐H1975 using the MTT assay. The antitumor effects of mubritinib on NCI‐H1975, A549, HCC‐827, and NCI‐H2170 cells were observed using MTT assay after different doses of mubritinib treatment for 24, 48, and 72 h. NCI‐H1975 cells seeded in 6‐well plates were treated with mubritinib and/or cisplatin for 15 days and stained with crystal violet solution.After 48 h of treatment with mubritinib and/or cisplatin, NCI‐H1975 cells were harvested and washed with PBS. Some cells were stained with Annexin V‐FITC and PI for 15 min in the dark at room temperature (RT) and then analyzed for apoptosis using FACScan (Becton Dickinson). Some cells were fixed with 75% cold ethanol at −20°C for 1 h, stained with 200 μl PI (50 μg/ml) for 30 min at RT in the dark, and the cell cycles were analyzed using FACScan. Some cells were stained with 500 μl JC‐1 (20 μg/ml) for 20 min at 37°C and analyzed for the mitochondrial membrane potential using FACScan. Other cells were stained with 500 μl of DCHF‐DA working solution for 30 min at 37°C and analyzed for ROS using FACScan.NCI‐H1975 cells seeded in 12‐well plates were scratched using a sterile 0.4–0.5 mm wide pipette tip. After 48 h treatment with mubritinib and/or cisplatin, the wounds were observed at 0 and 48 h after scratching.For NCI‐H1975 cells, the serum‐free cell suspension was added to the upper chamber and the serum‐containing medium was added to the lower chamber. After being treated with mubritinib and/or cisplatin for 48 h, the lower surface was fixed with 70% methanol solution for 30 min, stained with crystal violet, and examined under the microscope.NCI‐H1975 cells treated with 80 nM mubritinib for 24 h were harvested and subjected to RNA sequencing (RNA‐seq) performed by Huada Gene Corporation.After being treated with mubritinib and/or cisplatin for 48 h, NCI‐H1975 cells were harvested and resuspended with mitochondrial isolation reagent. Cellular mitochondria were extracted and the activities of mitochondrial respiratory chain complex I‐V were detected according to the manufacturer's instruction.After being treated with mubritinib and/or cisplatin for 48 h, NCI‐H1975 cells were collected and lysed in RIPA lysis buffer containing phosphatase and protease inhibitors. After BCA quantification, proteins were separated by 12% SDS‐PAGE and transferred to methanol‐activated PVDF membranes. Membranes were blocked with 5% skimmed milk for 1 h, followed by incubation with primary antibodies  (Affinity company) overnight at 4°C, with HRP‐conjugated secondary antibodies for 1 h at RT, visualized using ECL, and quantified with Image J Software.6 were subcutaneously injected into the right flank of each mouse, and the growth of the transplanted tumor was continuously monitored. When the tumor volume reached 100–150 mm3, the mice were divided into four groups with five mice in each group: (1) control (Ctr): intraperitoneal injection of 100 μl of PBS once a day; (2) cisplatin (Pt): intraperitoneal injection of 100 μl of cisplatin (5 mg/kg), once a week; (3) mubritinib (Mu): intraperitoneal injection of 100 μl of mubritinib (10 mg/kg), once a day; and (4) combination (Com): combined injection of cisplatin and mubritinib as the monotherapy group.6‐week‐old male BALB/c‐Nu mice were purchased from Beijing Unilever Laboratory Animal Co., Ltd., and raised under specific nonpathogenic conditions. The feeding and operation of experimental animals followed regulations on the management of experimental animals of Jilin University, and the experimental protocol was approved by the Experimental Animal Ethics Committee of Jilin University. Animal care was carried out following the recommendations in the ARRIVE guidelines.NCI‐H1975 cells 2 × 102 × long diameter/2. At the end of the treatment, mice were sacrificed by inhalation of an overdose of ether. The tumor, heart, liver, spleen, lung, and kidney were dissected and fixed with formalin.During treatment, the long and short diameters of the transplanted tumor were measured, and the volumes were calculated: volume = short diameterFixed tissues were dehydrated and embedded. The resulting paraffin blocks were prepared and sliced into 4 μm slices. After being dewaxed and hydrated, these slices were stained with hematoxylin and eosin. Later, the slices were destained in ethanol and viewed under a microscope.For immunohistochemical staining, hydrated slices were heated in citrate antigen, incubated with drops of peroxidase blocker, sealed with goat serum, and incubated in primary antibody overnight and biotin‐labeled secondary antibody for 20 min. After incubation in antibiotic peroxidase solution and DAB solution for the color development, nuclei were stained with hematoxylin.For TUNEL staining, hydrated slices were incubated with DNase‐free protease K and TUNEL solution, and visualized under the immunofluorescence microscope. Five fields were randomly selected and the number of cells was counted. Apoptosis rate = the number of apoptotic cells/the total number of tumor cells ×100%.p < 0.05 was considered statistically significant.All data are expressed as mean ± standard deviation (mean ± SEM). One‐way ANOVA in SPSS software was used for comparison between multiple groups, and The FDA drug library search results showed that 42 out of 2992 small molecules had significant inhibitory effects on NCI‐H1975 lung neoplasms cells Figure . MubritiNext, the inhibitory effects of mubritinib on NCIH1975, A549, HCC‐827, and NCI‐H2170 were detected by MTT. The results showed that the antitumor effects of mubritinib on NCI‐H1975 and A549 were better than that of HCC‐827 and NCI‐H2170 Figure . In addip < 0.01)   Figure .p < 0.01)   Figure  as shownq vaule ≤0.001) in NCI‐H1975 cells treated with mubritinib, respectively , suggesting that mubritinib can cause mitochondrial dysfunction and lead to abnormal energy metabolism , indicating that mubritinib increased the production of ROS, which in turn promoted the reduction of mitochondrial membrane potential.The abnormal activity of mitochondrial complex enzymes affects electron transport in the respiratory chain and induces the production of ROS. As shown in Figure p < 0.05)  , but there was no significant difference between the combination group and the single‐dose administration group (p > 0.05) , and was further reduced in the combined group (p < 0.01)  Figure . In addi) Figure .p < 0.01), and further increased in the combined group (p < 0.05)  and further decreased in the combined group (p < 0.01)  Figure .In addition, the activity of complexes I, III, IV, and V was decreased in the mubritinib group, the activity of complexes I and III was decreased, and complex V was increased in the cisplatin group. The activity of complex I, III, IV, and V was reduced in the combined group compared to the cisplatin group Figure .p < 0.05), and was further downregulated in the combined group compared with the cisplatin group (p < 0.05)  Figure .p < 0.05)  Figure .HE staining showed tumor tissue damage, cell reduction, intracellular nuclear pyknosis and nucleolysis, and homogeneous red‐stained necrotic areas were observed in the treatment group with the most obvious damage in the combined group Figure . In addiTUNEL fluorescence staining showed that compared with the control group, the apoptosis rate of cells increased in the treatment groups and further increased in the combined group Figure . ImmunohLung cancer (LC) is one of the most common malignant tumors with an increasing number of cases and deaths worldwide.In this study, 42 drugs with inhibitory effects on NCI‐H1975 lung neoplasms cells were screened from the FDA drug library. Mubritinib, which has similar tumor suppressor activity to docetaxel, was selected for the following experiments. Grygielewicz et al.To further verify the antitumor effects of mubritinib on LC cells, their apoptosis, proliferation, invasion, and migration abilities were examined. The results showed that mubritinib can promote apoptosis, inhibit proliferation by blocking the cell cycle in the G2 phase, and inhibit the invasion and migration of NCI‐H1975 cells, suggesting that mubritinib may be a candidate drug for lung neoplasms treatment. However, the potential mechanism of action of mubritinib is still unknown. Similarly, Stephenson et al.To further explore the mechanism of action of mubritinib on LC cells, RNA‐seq, also known as transcriptome sequencing technology, was used to reflect the types and amounts of mRNA, small RNA, and noncoding RNA in NCI‐H1975 cells at the transcription level. The analysis showed that the most obvious genetic alteration was thioredoxin interacting protein (TXNIP), which is closely related to the production of cellular ROS and oxidative stress, and further affects the process of apoptosis.Next, a series of experiments were performed to verify the possible mechanisms mentioned by RNA‐seq. Results indicated that the level of PI3K, p‐Akt, and mTOR in NCI‐H1975 cells were significantly reduced by mubritinib, indicating that mubritinib may inhibit cell proliferation by blocking the PI3K‐Akt–mTOR signaling pathway. The expression of p‐ERK further confirmed this speculation.Considering that mTOR is a key factor affecting cellular energy metabolism and synthesis,Akt is a serine/threonine kinase that binds the mammalian target of rapamycin (mTOR) complex 2.In addition, abnormal electron conduction in mitochondrial respiratory chain complexes can increase ROS levels. The detection of intracellular ROS showed that the level of ROS in the mubritinib group was significantly increased, and the expression of Nrf‐2 was upregulated, indicating that NCI‐H1975 cells were subjected to a higher degree of oxidative stress, which in turn activated the antioxidant pathway. The decrease of HO‐1 and GPX‐4 may be due to the increase of ROS and lead to their depletion. Similar to this study, it has been reported that mubritinib can inhibit acute myeloid leukemia by disrupting mitochondrial respiration and inducing oxidative stress and apoptosis.Cisplatin, as a key drug in the treatment of LC, delays the survival and life quality of LC patients. However, several disadvantages of cisplatin, including drug resistance, dose‐dependent toxicity, and poor specificity, seriously affect the prognosis of patients.ROS are byproducts of oxygen consumption and cellular metabolism, formed by partial reduction of molecular oxygen.Mitochondrial dysfunction can lead to the release of apoptotic factors such as endonuclease G (Endo‐G) and cytochrome C, and therefore activate mitochondria‐mediated apoptosis.During the experiment, no significant difference in bodyweight and organ morphology was observed among all groups, indicating that the combination of mubritinib and cisplatin was well tolerated.In conclusion, mubritinib has strong antitumor effects in vivo and in vitro. A better inhibitory effect was observed when it was used in combination with cisplatin by inhibiting the PI3K/Akt/mTOR signaling pathway, increasing the level of cellular ROS, destroying mitochondrial function, and causing cell energy metabolism disorders in NSCLC.The authors declare that there are no conflicts of interest.Appendix S1 Supporting InformationClick here for additional data file.


Hispanic/Latina women are less likely to be diagnosed with local stage breast cancer than White women. Additionally, foreign‐born women have lower mammography rates than US‐born women. We evaluated the combined effect of birthplace and race/ethnicity on screening habits of women at higher‐than‐average risk of breast cancer.recently (≤2 years ago), formerly (>2 years ago), and never screened. Adjustments included sociodemographic, socioeconomic, and health variables.Multinomial logistic regression was used to evaluate breast cancer screening in 44,524 women in the Sister Study cohort. Screening methods ascertained at enrollment (2003–2009) included mammography, ultrasound, and magnetic resonance imaging. Timing of screening was assessed as Most women in the sample were US‐born non‐Hispanic/Latina White (92%), were ≥50 years old (73%), had one first‐degree female relative with breast cancer (73%), and were screened in the past two years (97%). US‐born Hispanic/Latina women had higher odds  than US‐born non‐Hispanic/Latina White women of not having received a breast cancer screening in the past 2 years, relative to a recent screening. Similarly, foreign‐born Hispanic/Latina women had higher odds  than US‐born non‐Hispanic/Latina White women of never having received a breast cancer screening.We observed that Hispanic/Latina women have higher odds of never and dated breast cancer screenings compared to US‐born White women. Birthplace and race/ethnicity each contribute to disparities in who receives preventative health care in the United States. It is critical to include birthplace when evaluating health behaviors in minority groups. Multinomial logistic regression was conducted on 44,524 Hispanic and non‐Hispanic White women from the Sister Study cohort to evaluate the associations between combined birthplace and race/ethnicity and timing of most recent breast cancer screening, while controlling for sociodemographic, socioeconomic, and health factors. Foreign‐born Hispanic women were found to have higher odds of never receiving a breast cancer screening compared to US‐born non‐Hispanic White women. Similarly, US‐born Hispanic women were found to have higher odds of having an outdated breast screening compared to US‐born non‐Hispanic White women. Relative to women with no family history, breast cancer risk is 1.8 times higher in women with one first‐degree relative and 2.93 times higher in women with two first‐degree relatives with the disease.Breast cancer screening is an important preventive health measure used to detect breast cancer in an early, easy‐to‐treat stage. Early detection is associated with more successful treatment and higher rates of survival in women.et al.et al.The United States is currently home to around 44.8 million immigrants, 44.3% of whom are Hispanic/Latino and over half of whom are female.In the United States, Hispanic/Latino individuals experience lower rates of many cancers than non‐Hispanic/Latino White individuals, including female breast cancer.Given the rapidly growing US Hispanic/Latino population, the known health risks associated with acculturation, and the benefits of adherence to screening guidelines, it is essential to understand the disparities in breast cancer screening utilization observed in the United States. This need is even more pronounced among women with family history of breast cancer due to their heightened risk. Efforts to increase screening and reduce stage‐of‐diagnosis disparities among Hispanic/Latina women in the United States may be more effective if we better understand the composite effects of birthplace and race/ethnicity on breast cancer screening. To address this need, we analyzed the Sister Study cohort baseline data to investigate the associations between combined birthplace and race/ethnicity and timing of most recent screening, while accounting for important risk factors such as family history and behavioral exposures. Our study contributes to the breast cancer disparity literature as one of the few to use national cohort data to assess screening use by birthplace/race/ethnicity among a higher‐than‐average‐risk population.22.1https://sisterstudy.niehs.nih.gov and in past publication.We used data collected by the Sister Study, a longitudinal prospective cohort study of 50,884 women residing in the United States, including Puerto Rico. Women were eligible for enrollment if they were between the ages of 35 and 74 and had a full‐ or half‐sister who had been diagnosed with breast cancer but had never been diagnosed with breast cancer themselves. Participants provided written informed consent at the time of enrollment (2003–2009). A detailed description of the Sister Study design and methods can be found at n = 2 women due to study withdrawal, we restricted our analysis to 44,815 women who identified as non‐Hispanic/Latina White or Hispanic/Latina. We further excluded women due to incomplete breast cancer screening information (n = 8), prophylactic mastectomy (n = 228), and previous diagnosis of invasive breast cancer or ductal carcinoma in situ or otherwise ambiguous diagnosis information (n = 55). Therefore, information from 44,524 women contributed to this analysis.Due to small numbers, women who identified as foreign‐born non‐Hispanic/Latina and either Black, American Indian/Alaskan Native, Asian, and Native Hawaiian or other Pacific Islander were excluded. Therefore, after omitting 2.2recently screened, which consisted of women who had received a mammogram in the past two years and/or received an MRI or ultrasound;  US‐born non‐Hispanic/Latina White; (2) foreign‐born non‐Hispanic/Latina White; (3) US‐born Hispanic/Latina; and (4) foreign‐born Hispanic/Latina. Age was divided into three categories: (1) under 40; (2) 40–49; and (3) 50 and older. Finally, marital status was considered a binary variable with 2.3.2The socioeconomic factors included in our analysis were annual household income ((1) under $20,000; (2) $20,000 to $99,999; and (3) $100,000 and above) and highest educational attainment ((1) high school/GED or less; (2) some college or associate degree; (3) bachelor's degree; and (4) graduate degree).2.3.32); (2) overweight (25–29.9 kg/m2); and (3) obese (30 kg/m2 and above). Lastly, we used three levels for number of first‐degree female relatives with breast cancer: (1) one; (2) two; and (3) three or more. We considered first‐degree female relatives to be full‐ or half‐sisters, mothers, and daughters.The health factors included in our analysis were smoking status, alcohol consumption, BMI, and number of first‐degree female relatives with breast cancer. Smoking status response categories were (1) never; (2) past; and (3) current smoker. Alcohol consumption was characterized by (1) current drinker or (2) not‐current drinker. The BMI categories were (1) underweight/normal weight (under 25 kg/m2.4n = 1764). Analyses were conducted using StataSE 16. All analytical files are available by reasonable request to the Sister Study.We conducted multinomial logistic regression using the Sister Study cohort baseline data to examine the associations between sociodemographic, socioeconomic, and health characteristics and timing of most recent breast cancer screening in non‐Hispanic/Latina White and Hispanic/Latina women with a family history of the disease. Our multinomial logistic regression models had timing of most recent breast cancer screening as the dependent variable, with recently screened as the reference group. The following independent variables were included: sociodemographic indicators (Model 1); socioeconomic indicators (Model 2); and health indicators (Model 3). All variables were retained in subsequent models. Sensitivity analyses were conducted in which women under 40 years of age were removed from the sample , married or living as married (77%), had an annual household income between $20,000 and $99,999 61%), and had attained a bachelor's degree or higher (51%). Additionally, most women reported drinking alcohol (82%), never smoking (55%), and having one first‐degree female relative with breast cancer (73%). Most women in the sample (97%) received breast cancer screening in the past two years. See Table %, and ha3.2N = 44,521) included sociodemographic characteristics. This model found that compared to US‐born non‐Hispanic/Latina White women, foreign‐born Hispanic/Latina women had higher odds  of having never been screened for breast cancer than of having been recently screened. Further, the odds of having been formerly screened for US‐born Hispanic/Latina women and for foreign‐born Hispanic/Latina women were 1.62 (95% CI = 1.20–2.19) and 1.66 (95% CI = 1.27–2.17), respectively, compared to US‐born White women.Model 1  retained sociodemographic variables and added income and education. After controlling for socioeconomic indicators, the odds of having never been screened for breast cancer for foreign‐born Hispanic/Latina women was 1.71 (95% CI = 1.16–2.52) compared to US‐born non‐Hispanic/Latina White women. Additionally, compared to recent screening, the odds of former screening in US‐born Hispanic/Latina women were 50% (95% CI = 1.10–2.03) higher than US‐born non‐Hispanic/Latina women. These findings indicate substantial confounding by socioeconomic indicators on the relationship between birthplace/race/ethnicity and breast cancer screening history. See Table Model 2  incorporated health characteristics—smoking status, alcohol consumption, BMI, and number of first‐degree female relatives with breast cancer—and retained sociodemographic and socioeconomic controls from the previous models. Model 3, the most comprehensive model, was selected for interpretation.Model 3 . Similarly, US‐born Hispanic/Latina women were more likely than US‐born non‐Hispanic/Latina White women to receive former screening, relative to recent screening . The differences in odds ratios for combined birthplace and race/ethnicity between models 2 and 3 suggest limited confounding from the health indicators. See Table Removing women under 40 from the sample population did not change our findings. Results from the sensitivity analyses are available online as supplemental information.4Our analysis found distinct birthplace and racial/ethnic differences in screening backgrounds among the women in our study, while controlling for sociodemographic, socioeconomic, and health factors. US‐born Hispanic/Latina women were more likely than US‐born non‐Hispanic/Latina White women to have had their most recent breast cancer screening over 2 years ago. Furthermore, foreign‐born Hispanic/Latina women were more likely to have never received a breast cancer screening than US‐born non‐Hispanic/Latina White women. Despite the relatively low risk of breast cancer among Hispanic/Latina women in the United States, these findings underscore the vulnerability of immigrants and ethnic minorities to late‐stage detection of malignancies.et al.et al.While socioeconomic factors appeared to confound the relationship between birthplace/race/ethnicity and screening history, birthplace and race/ethnicity were significantly associated with breast cancer screening utilization even after controlling for income and education. Compared to US‐born non‐Hispanic/Latina White women, US‐born Hispanic/Latina women were less likely to have received their most recent screening in the past 2 years and foreign‐born Hispanic/Latina women were less likely to have ever received breast cancer screening in their lifetime. These findings are consistent with documented disparities in access to screening among minority ethnic groups and foreign‐born women.et al.Our analysis did not indicate significant confounding from our health indicators—smoking status, alcohol consumption, BMI, and breast cancer family history—on the association between birthplace/race/ethnicity and screening history. However, past research has revealed potential “spillovers”Self‐reported family history of cancer is used by healthcare providers to make screening recommendations. As such, persons who may not have knowledge of, who do not report, or who are not asked about family breast cancer history are vulnerable to underutilization of preventive and screening services that could otherwise reduce mortality and poor health outcomes. Racial and ethnic minorities, including Hispanics/Latinos, as well as immigrants, are less likely than non‐Hispanic/Latino White individuals and the US‐born to report family history of cancer.Immigrant and ethnic minority populations are at higher risk than US‐born non‐Hispanic/Latino White individuals of deteriorating health due to factors associated with migration, acculturation, and structural inequities. Our findings show that Hispanic/Latina women, particularly those in a higher‐than‐average‐risk population, would benefit from tailored public health efforts to improve utilization of breast cancer screening services. To address the underutilization of breast cancer screening services among Hispanic/Latina women, interventions must bear in mind the unique barriers to screening for the foreign‐ and US‐born. Messaging campaigns and interventions aimed at increasing awareness of publicly available screening services should be culturally and linguistically appropriate and should promote intrafamilial conversations about family health history. Breast cancer family history contributes to personal risk and recommendations for preventative services made by physicians. Educational programs for health care providers should include training on the specific risk factors faced by minorities and immigrants.4.1Our study is not without limitations. We conducted a cross‐sectional analysis which does not capture the temporal nature of breast cancer screening history. As we did not have data on the timing of the most recent MRI or ultrasound received by participants, we were limited by an incomplete picture of their screening backgrounds. Additionally, breast cancer screening history was self‐reported which could lead to overreporting of recent screening. The Sister Study cohort only includes women with a sister who has been diagnosed with breast cancer. Therefore, our findings are not generalizable to all US women. Lastly, by not differentiating between Hispanic/Latina subpopulations, our results are generalizations about a highly heterogeneous population. However, ours is the first study to investigate place of birth and racial/ethnic screening disparities in the Sister Study cohort and is an important contribution to the literature on breast cancer screening disparities.4.2Hispanic/Latina women in the United States carry an unequal burden of late‐stage breast cancer diagnosis. Our findings shed new light on the impact of ethnicity and birthplace on screening among a higher‐than‐average‐risk population. Specifically, these results highlight the importance of including birthplace when conducting analyses on minority groups. The observed disparities in our analysis further verify the vulnerability of Hispanic/Latina women to falling through the cracks of our preventive healthcare system. As such, it is of the upmost importance to eliminate disparities in who receives preventive healthcare services in the United States. Additionally, healthcare providers should be aware of potential underreporting of family history among minority and foreign‐born patients so as not to perpetuate inequities.The authors declare no potential conflicts of interest.CJT conducted data analysis, interpreted results, and drafted the manuscript. FW conceptualized and designed the analysis, acquired the data, and supervised manuscript preparation. FAMI helped conceptualized the analysis and prepare and interpret data. KMO validated the data analysis and edited the manuscript. CJT, KMO, and DSP substantially modified the submitted version of the manuscript. All authors read and approved the final version of the manuscript.The Sister Study was approved by the Institutional Review Board (IRB) of the National Institute of Environmental Health Sciences and the Copernicus Group IRB.Table S1Click here for additional data file.


Immune checkpoint inhibitors (ICIs) are effective in some cancer patients; however, they may show no efficacy in others. Predictive biomarkers are crucial for appropriately selecting the patients who receive ICI therapy. This study aimed to clarify the predictors of disease progression in urothelial carcinoma (UC) patients treated with an ICI, pembrolizumab.CDKN2A/p16 homozygous deletion.We analyzed the response patterns of 50 UC patients who were treated with pembrolizumab, as well as the association between survival and clinicopathological factors. Clinical factors included age, sex, body mass index, clinical courses, laboratory data, metastases, and adverse events. Pathological factors included special variant, squamous differentiation, programmed cell death ligand‐1 (PD‐L1) expression, CD8‐positive lymphocytes density, and During pembrolizumab treatment, four (8%), 11 (22%), and eight (16%) patients achieved the best‐case scenarios of complete response, partial response, and stable disease, respectively. Twenty‐seven patients (54%) showed progressive disease. In this study, younger age, lower preoperative neutrophil‐to‐lymphocyte ratio (NLR), and positive PD‐L1 expression were significantly correlated with longer progression‐free survival and overall survival. Moreover, lower NLR and positive PD‐L1 expression were independently associated with longer OS in multivariate analysis.Based on our observations, lower NLR and positive PD‐L1 expression may be independent favorable prognostic markers in UC patients treated with pembrolizumab. These results suggest that both host and tumor status can reflect the effectiveness of pembrolizumab among patients with UC. Tumor status of positive PD‐L1 expression, and host immune status of low neutrophil‐to‐lymphocyte ratio, are both prognostic markers for urothelial carcinoma patients treated with pembrolizumab. However, only 20% of UC patients respond to ICIs.Several ICIs recently demonstrated efficacy and survival benefit, and they are widely used in clinical practice. Among them, pembrolizumab clearly revealed overall survival benefits over chemotherapy in the phase III study with a large sample size,Recently, a number of second and later line studies on therapy reported that response rates for anti‐programmed cell death‐1 (PD‐1)/PD ligand‐1 (PD‐L1) antibodies were higher in patients with PD‐L1–high UC than in those with PD‐L1–low UC.CDKN2A/p16 had tended to have no clinical benefit for ICIs.Genetic alterations frequently detected in UC could also be a prognostic marker for ICIs. Nassar et al. reported clinical and seven genomic factors correlated with clinical outcomes in univariable analysis in the ICI cohort, such as neutrophil‐to‐lymphocyte ratio (NLR), visceral metastasis, and single‐nucleotide variant (SNV) count. Especially, in genetic alterations, homologous deletion of Some studies have further suggested associations between special variants and responses to ICIs.CDKN2A/p16 homozygous deletion. In addition, accumulating evidence suggests that the prognostic value of tumor biomarkers may vary according to the patient's characteristics through host–tumor interactions. For example, the association of tumor PD‐L1 expression with outcomes varied among patients with low or high preoperative serum platelet counts in upper tract UC (UTUC).This study explored predictive markers based on clinicopathological data analysis of patients with platinum‐resistant locally advanced or metastatic UC who received pembrolizumab and PD‐1 antibody therapy, pembrolizumab. First, we performed a clinical and histological review. Previously, we reported that squamous differentiation was correlated with tumor progression in patients administered with pembrolizumab for UC.22.1From February 27, 2018, to March 16, 2021, we analyzed 50  of 65 patients with platinum‐resistant locally advanced or metastatic UC who received pembrolizumab in our institution based on availability. All samples of these 50 patients enrolled in this study were acquired via surgical resection or biopsy before the patients received pembrolizumab. When a single case had multiple samples, we chose one sample, the most recent and also a surgical sample rather than a biopsy sample. We extracted the clinical data analyzed in this study from the patients' medical records. Complete blood count was assessed within 7 days of pembrolizumab treatment.This study was approved by the Institutional Review Board of the Saitama Medical University International Medical Center , and informed consent was acquired from all the patients.2.2We analyzed the prognostic factors of progression‐free survival (PFS) and overall survival (OS) using the Cox‐proportional hazards model. PFS and OS were analyzed using the Kaplan–Meier method and a log‐rank test. These analyses were performed using Python software . The interaction was assessed using the least‐squares method for the cross‐product of age, NLR, PD‐L1 expression, and squamous differentiation in PFS or OS. The interaction was analyzed using JMP Pro 16.1 software .2.3Histological review using hematoxylin and eosin‐stained slides was performed by a single genitourinary pathologist (YM) who was blinded to the patients' outcomes. The morphological characteristics analyzed in this study were histological grade and subtype of UC, that is, divergent differentiation shown by squamous differentiation, or types of variant morphologies , according to the 2016 World Health Organization classification system.2.4Formalin‐fixed paraffin‐embedded tumor samples were sliced into 4 μm sections for the immunohistochemical analyses. The immunohistochemistry of each marker was performed using the antibodies: MAC387 , PD‐L1 , and CD8 . Antigen retrieval was performed using Cell Conditioning Solution (CC1‐buffer) (Roche Diagnostics). Visualization was performed using the OptiView DAB Universal Kit for PD‐L1 (Roche Diagnostics) and the iView DAB Universal Kit for the other antibodies (Roche Diagnostics). Hematoxylin was used for counterstaining. Immunoreactivity was assessed by the same pathologist (YM) who performed the histological review.2.5We used the SP263 assay for PD‐L1 expression, which shows PD‐L1 staining that is highly concordant with the 22C3 assay.2.6Image analysis of CD8‐positive cells was performed using Aperio ImageScope software , as previously described.2.7CDKN2A/p16). When ≥15 cells showed zero CDKN2A/p16 signals, it was regarded as homozygous deletion.CDKN2A/p16 signals of tumor cells in FISH.To assess CDKN2A/p16 homozygous deletion in FISH, formalin‐fixed paraffin‐embedded tumor samples were used. Paraffin sections (3 μm) were baked for an hour, placed in xylene for 5 min, and dehydrated in 100% ethanol. The slides were treated with heat (95°C) for an hour with a pretreatment reagent (VP2000) before hybridization. After cooling, deproteinization was performed with 0.8% pepsin lysis buffer and 2X saline sodium citrate (SSC) buffer. Following this, dehydration was performed in 70%, 85%, and 100% alcohol solutions for 5 min each, followed by drying. Next, the ZytoDot 2 SEPC CDKN2A/CEN9 probe  was used. DNA denaturation was performed at 80°C for 5 min and placed overnight in a hybridization chamber at 37°C. The post‐hybridization washes were performed with 2X SSC buffer/0.3% NP‐40. The slides were counterstained with DAPI stain. A minimum of 25 cells were analyzed for copy number changes in chromosome 9p21  and 329 days (10.97 months), respectively. Four (8%), 11 (22%), and eight (16%) patients achieved the best‐case scenarios of complete response (CR), partial response (PR), and stable disease (SD), respectively, according to the patients' computed tomography (CT) findings (37 cases were assessed) and the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST).3.2p = 0.0047 and 0.0042, respectively; Figure p = 0.0056 and 0.0054, respectively, Figure Subsequently, we assessed the relationship between survival and clinicopathological data Table . The medp = 0.021 and 0.0013, respectively, Figure p16 homologous deletion in FISH, were not associated with prognosis.In terms of pathological data, PD‐L1 expression was observed in 13 out of 50 cases (26%) and was associated with better PFS and OS .4In this study, younger age, lower NLR, and positive PD‐L1 expression could be favorable prognostic markers in terms of both PFS and OS. In addition, lower NLR and positive PD‐L1 expression were independently associated with longer OS in the Cox‐proportional hazards model analysis.Several studies have reported that higher NLR is associated with poorer prognosis in patients who received pembrolizumab for UC;We assessed PD‐L1 expression of tumor and immune cells using the SP263 assay scoring algorithm. Positive PD‐L1 expression was associated with better OS and PFS. Consistent with our results, PD‐L1 expression appears to be prognostic in the context of second‐line therapy.Besides NLR and PD‐L1, younger age was also associated with longer PFS and OS. However, in the KEYNOTE‐045 study, Bellmunt et al. reported that the advantage of pembrolizumab in advanced UC seemed to be consistent in spite of age.Pinteraction = 0.025). Among patients with negative PD‐L1 expression, squamous differentiation was not associated with PFS . In contrast, among patients with positive PD‐L1 expression, squamous differentiation tended to have longer PFS . In this context, the combination of histological and immunological assessment is important and further study is needed to confirm the association with squamous differentiation and the effectiveness of pembrolizumab.In this study, squamous differentiation was not associated with prognosis. However, in our previous study, squamous differentiation was associated with shorter OS and PFS in UC patients treated with pembrolizumab.There are some limitations to this study. First, we set the unique cutoff values of age and NLR to maximize statistical power. In NLR, previous studies set higher cutoff values (3.35 and 4),In conclusion, younger age, lower NLR, and positive PD‐L1 expression were associated with better OS and PFS in UC patients treated with pembrolizumab. Moreover, lower NLR and positive PD‐L1 expression were independently associated with longer OS in multivariate analysis. These results suggest that both host and tumor status can reflect the effectiveness of pembrolizumab among patients with UC.This study was approved by the Institutional Review Board of the Saitama Medical University International Medical Center , and informed consent was acquired from all the patients.None to declare.Yu Miyama designed this study, analyzed the data, made the draft, and revised the manuscript. Go Kaneko designed this study, acquired clinical information, made the draft, and revised the manuscript. Koshiro Nishimoto designed this study, acquired clinical information, analyzed statistical data, and interpreted the data. Masanori Yasuda provided suggestions and revised the manuscript. All authors read and approved the final version of the manuscript.Figure S1Click here for additional data file.Table S1Click here for additional data file.Data S1Click here for additional data file.