Further univariate analysis with Kaplan–Meier calculations demonstrated that antibiotic administration was strongly linked with both reduced DFS (p = 0.04) at significant statistical levels and OS (p = 0.088) at borderline statistical levels. Antibiotic administration was identified as a significant independent prognostic factor for DFS  and OS  by Cox proportional hazards model analysis. Antibiotics that initiated reduced efficiency of chemotherapy were more noticeable in the HER2-positive subgroup for both DFS  and OS , as well as in the T3-4 subgroup for both DFS  and OS  by stratified analysis. Antibiotic administration might be associated with reduced efficacy of neoadjuvant therapy and poor prognosis in BC patients. As a preliminary study, our research made preparations for further understanding and large-scale analyses of the impact of antibiotics on the efficacy of neoadjuvant therapy.Mounting evidence suggests that microbiota dysbiosis caused by antibiotic administration is a risk factor for cancer, but few research reports focus on the relationships between antibiotics and chemotherapy efficiency. We evaluated the influence of antibiotic administration on neoadjuvant therapy efficacy in patients with breast cancer (BC) in the present study. BC patients were stratified into two groups: antibiotic-treated and control based on antibiotic administration within 30 days after neoadjuvant therapy initiation. Disease-free survival (DFS) and overall survival (OS) were assessed using the Kaplan–Meier method, and the Cox proportional hazards model was used for multivariate analyses. The pathologic complete response rate of the control group was significantly higher than that of the antibiotic-treated group (29.09% vs. 10.20%,  The treatment of breast cancer has entered the era of individualized treatment with increasing systemic treatment, especially neoadjuvant therapy—preoperative systemic therapy for breast cancer without distant metastasis. The application of neoadjuvant therapy reduces the tumor burden and increases the likelihood of breast conservation to improve BC patients' quality of life2. It also enables clinicians to obtain individual drug sensitivity information on BC patients to guide further treatment3. The phenomenon of pathological complete response (pCR) was defined as no residual invasive tumor on pathologic assessment after therapy showing an associated survival benefit4.The latest cancer data in 2020 indicates that breast cancer is the most prevalent type of cancer worldwide. In China, breast cancer is the most common malignant tumor and the fifth leading cause of female cancer mortality5. Antibiotics are effective for treating various infections. Nevertheless, their application cannot be located as precisely as targeted drugs, which in turn leads to intestinal dysbacteriosis. The gut microbiota participates in many aspects of the human physiological process, from producing nutrients and vitamins to fighting pathogens and protecting immune system development and epithelial mucosa homeostasis6. Accumulating evidence suggests that gut microbiota has an impact on the efficacy of anti-tumor therapies—including chemotherapy, immunotherapy, radiotherapy, and surgery—used to treat solid tumors  with numerous mechanisms, including xenometabolism, immune microenvironment, and changed microbial community structure6. Antibiotic-treated mice displayed oxaliplatin (OXA) chemoresistance for colon carcinoma and lymphoma compared with specific pathogen-free (SPF) mice, which suggested that antibiotic exposure was associated with reduced chemotherapy efficacy7. Further research is needed in breast cancer to study the relationship between the effect of neoadjuvant therapy and antibiotics.Antibiotics are required to prevent and treat infectious bacterial diseases, mainly in BC patients with febrile neutropenia (FN), which is a severe adverse effect induced by chemotherapyIn the present study, we aimed to evaluate the influence of antibiotic administration on neoadjuvant therapy efficacy and prognosis in BC patients. We expect that our findings will provide a basis for future therapeutic concepts in BC patients who require antibiotics during neoadjuvant therapy.p = 0.924, Table 8. We defined pCR as no residual invasive tumor in either the tumor bed or lymph node on pathologic assessment after therapy.Patients with BC who received new adjuvant chemotherapy followed by surgery at the Fourth Hospital of Hebei Medical University between January 2013 and September 2015 were enrolled in this retrospective study. All methods were performed in accordance with the relevant guidelines and regulations for human participants and were supervised and approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University (No. 2020KS001). Written informed consent was provided by participants. Neoadjuvant therapy consisted of docetaxel (T), anthracycline (A), cyclophosphamide (C), and Herceptin (H), and the types of neoadjuvant therapy were balanced between the two groups  was the time from operation to the date of disease progression or death. Overall survival (OS) was defined as the time from neoadjuvant therapy initiation to the date of death. At the end of follow-up, 3 (2.5%) patients and 9 (7.5%) patients were lost to follow-up, separately in DFS and OS analysis. We used the Kaplan–Meier method to plot survival curves and the log-rank test to evaluate the prognosis of patients. Univariate and multivariate analyses were done using the Cox proportional hazards model to identify the risk factors for survival. We did statistical analyses by using RStudio, version 1.3.1093  and SPSS software, version 19.0 , and p = 0.987). Dose intensity  and frequency of dose delay  were not significantly different between the two groups , with the proportion of Miller-Payne grades 2 and 3 increasing in the antibiotic-treated group and Miller-Payne grades 4 and 5 increasing in the control group   and a borderline significant difference in OS (p = 0.088) between antibiotic-treated and control BC patients  and OS . Furthermore, HER2 status was also identified as a significant independent prognostic factor for DFS  and OS . These data demonstrated that antibiotic-induced microbiota dysbiosis might modify BC patient outcomes by decreasing chemotherapy treatment efficiency.The potential outcome predictors were analyzed using the Cox proportional hazards model for multivariate analysis. As shown in Tables p = 0.003) and OS , as well as in the T3-4 subgroup for both DFS  and OS  by stratified analysis. The p-values for interaction were less than 0.05 in both the HER2 subgroup and T3-4 subgroup, indicating that antibiotic administration might change the outcome in HER2-positive BC patients and those whose primary tumor was larger than 5 cm.The influence of antibiotic administration on DFS and OS was further investigated within individual subgroups of BC patients by stratified analysis, and the antibiotic-treated group displayed a trend of reduced DFS Fig. A and OS 10. Antibiotics induce dysbacteriosis by changing the intestinal flora environment involved in tumorigenesis, causing inflammation and disorders of the immune system12. The gastrointestinal microbiota could influence cancer immunotherapy on both the prognostic and therapeutic sides13, and the application of antibiotics could reduce the clinical benefit of immune checkpoint inhibitors (ICIs) in renal cell carcinoma (RCC) and non-small-cell lung cancer (NSCLC)14. A previous article also found that antibiotics could affect the efficiency of chemotherapy in esophageal cancer15. Consistent with a previous report, we discovered that antibiotic-induced dysbacteriosis might also modified neoadjuvant therapy efficiency in BC patients.We estimated the effect of antibiotic administration on neoadjuvant therapy and prognosis in BC patients. We found that patients who administrated antibiotics might have a decreased treatment efficiency of neoadjuvant therapy referring to the Miller-Payne system and ORR. It is known that the composition of the intestinal flora of cancer patients is different from that of individuals without cancer. The intestinal flora plays an important role in the human immune system7. Similar results were observed with oxaliplatin (OXA) treatment7. In addition to immune modulations, bacterial translocation can also directly modulate chemotherapy efficacy. In colorectal cell lines (HCT116 and HT29), autophagy of Fusobacterium nucleatum was associated with chemoresistance to 5-fluorouracil (5-FU) and oxaliplatin (OXA)16.Changes in the microbial community structure may affect the efficacy of anti-tumor therapy, including chemotherapy. Intestinal dysbacteriosis reduces regulatory T cells (Tregs) and increases Th1 and Th17 cells to modulate the immune microenvironment of tumors, resulting in cyclophosphamide (CTX) chemoresistance17. Researchers used antibiotic administration or fecal microbiota transplantation from antibiotic-treated donors to change the community structure of gut microbiota to influence treatment efficiency17. Fecal microbiota transplantation provided an opportunity to correct antibiotic-induced chemotherapy failure. Our study was retrospective and lacked information on the intestinal flora of patients at the beginning of neoadjuvant therapy. It is vital to identify the gut microbiota responsible for dysbacteriosis-related treatment inefficiency in BC patients. The discovery of targeted bacteria capable of rescuing antibiotic-associated dysbiosis made it feasible to promote anti-tumor efficacy by modulating microbiome diversity, especially for HER2-positive BC patients.A recent study showed that gut microbiota was directly involved in the immune-mediated trastuzumab anti-tumor efficacy in 24 BC patients and micep = 0.017). However, the pCR rate was not a significant independent prognostic factor for DFS and OS. The pCR rate is an effective indicator of short-term treatment in neoadjuvant therapy4. However, most studies show that the pCR rate is higher in hormone receptor (HR)-negative breast cancer patients after neoadjuvant therapy and improves DFS and OS outcomes compared patients with less than a pCR. However, there was no advantage in overall survival compared with HR-positive patients18. As this study was small and there were 82 (68.33%) HR-positive patients in our study, the pCR rate was not a significant factor for determining DFS or OS.In our study, the pathological complete response (pCR) rate was significantly higher in the control group than in the antibiotic-treated group (29.09% vs. 10.20%, 19. We hope that our research draws clinicians’ attention to optimizing management of cancer patients during chemotherapy to reduce the incidence of chemotherapy-related side effects. As this study was small and retrospective, no conclusion for clinical practice is possible. More trials are necessary to assess the benefits and risks of antibiotics in breast cancer patients receiving neoadjuvant therapy.HER2 status was a significant independent prognostic factor of breast cancer, and we obtained the same conclusion in our study. In the subgroup analysis, the use of antibiotics dramatically decreased the treatment efficiency in HER2-positive and T3-4 subgroups of BC patients. Cancer patients may be treated with antibiotics because of conditions such as chemotherapy-related agranulocytosis, malnutrition, and cachexiaAntibiotic administration might be associated with reduced chemotherapy efficacy and poor prognosis in BC patients, especially in HER2-positive BC patients and patients whose primary tumor was larger than 5 cm. As a preliminary study, our research made preparations for further understanding and large-scale analyses of the impact of antibiotics on the efficacy of neoadjuvant therapy.Supplementary Information.


The host–guest interactions of cucurbit[7]uril (CB[7]) as host and amphetamine (AMP), methamphetamine (MET) and their enantiomeric forms (S-form and R-form) as guests were computationally investigated using density functional theory calculations with the recent D4 atomic-charge dependent dispersion corrections. The analysis of energetic, structural and electronic properties with the aid of frontier molecular orbital analysis, charge decomposition analysis (CDA), extended charge decomposition analysis (ECDA) and independent gradient model (IGM) approach allowed to characterize the host–guest interactions in the studied systems. Energetic results indicate the formation of stable non-covalent complexes where R-AMP@CB[7] and S-AMP@CB[7] are more stable thermodynamically than R-MET@CB[7] and S-MET@CB[7] in gas phase while the reverse is true in water solvent. Based on structural analysis, a recognition mechanism is proposed, which suggests that the synergistic effect of van der Waals forces, ion–dipole interactions, intermolecular charge transfer interactions and intermolecular hydrogen bonding is responsible for the stabilization of the complexes. The geometries of the complexes obtained theoretically are in good agreement with the X-ray experimental structures and indicate that the phenyl ring of amphetamine and methamphetamine is deeply buried into the cavity of CB[7] through hydrophobic interactions while the ammonium group remains outside the cavity to establish hydrogen bonds with the portal oxygen atoms of CB[7]. In 1887, the Romanian chemist, Lazăr Edeleanu discovered synthetic psychostimulants which are known as amphetamines ,3. This As reported by the World Health Organization, amphetamine-type stimulants (ATS) belong to a class of drugs that include amphetamine (AMP), methamphetamine (MET) and some stimulants which are structurally related .Methamphetamine is commonly referred to as “meth” or “crystal meth”. It is a very strong stimulant of the central nervous system (psychostimulant) which is derived from phenethylamine and amphetamine group of substances. Similarly to amphetamine, methamphetamine effects result in an increased level of wakefulness, attention and energy; however, taking high doses will expose users to higher levels of euphoria, sexual desire and self-esteem ,11. MethImmunochromatography and enzyme-linked immunosorbent assay (ELISA) are the most-used techniques to identify ATS using oral fluid . ClassicMacrocyclic compounds are important structures and key building blocks in supramolecular chemistry ,17,18. AIn their study about the development of new point-of-use, portable and wireless drug sensor, Jang et al. showed the high sensitivity of the host molecule CB[7] (combined with organic field-effect transistors) toward ATS, thus providing a viable methodology for the fabrication of selective and sensitive drug sensors . The potHerein, we present a theoretical study on the structural, electronic, biosensing and energetic properties of the inclusion of amphetamine and methamphetamine inside the cavity of CB[7]. The present work aims to investigate the stability of the formed complexes and to examine the nature of the intermolecular interactions involved in the complexation process using the new DFT-D4 dispersion correction model. The initial molecular structure of CB[7] was taken from the crystal structure determined by X-ray diffraction methods CCDC 1542694) . Both S 2694 . Bocomplexation represents the complexation energy, Ecomplex, Efree guest and Efree CB[7] are respectively the optimized energies of the complex, the free amphetamine or methamphetamine and the free cucurbit[7]uril.The center of AMP, MET and CB[7] was set as the center of the coordination system, then AMP and MET guests, as well as their enantiomers, were translated along the Z-axis from −10 to +10 Å with 2 Å step as shown in The aqueous phase geometries were used to characterize the nature of the interactions occurring between the guest molecules (AMP and MET) and CB[7] by performing an independent gradient model (IGM) analysis based on Hirshfeld partition of electron density . The nonThe complexation energies calculated in gas phase as a function of Z coordinate are reported in The S-enantiomer complexes (S-AMP@CB[7] and S-MET@CB[7]) are respectively slightly more stable by −9.36 and −1.13 kJ/mol in the gas phase than their corresponding R-enantiomers (R-AMP@CB[7] and R-MET@CB[7]).For higher accuracy, we reoptimized each of the four most stable complexes at BLYP-D4/def2-TZVP-gCP level of theory in gas and aqueous phases. The calculated complexation energies are reported in In gas phase, we note that as the basis set improves from def2-SVP to def2-TZVP, the computed complexation energies of R-AMP@CB[7], S-AMP@CB[7], R-MET@CB[7] and S-MET@CB[7] are −338.85, −349.58, −334.01 and −334.53 kJ/mol, respectively. The improvement of basis set quality does not lead to significant variation of the complexation energies .Complexation at temperature 298K for each complex and compared it with the experimental values. The experimental ΔGComplexation values of AMP@CB[7] and MET@CB[7] are −34.7 and −33.8 kJ/mol, which are very close to the computed values . Therefore, our calculations results are in good agreement with the experimental data.In aqueous phase, a significant decrease in the complexation energy values was noted ; howeverIt is also clear that complexes of R-AMP@CB[7] and S-AMP@CB[7] are more stable than their analogs R-MET@CB[7] and S-MET@CB[7] in gas phase while in water solvent the stability is reversed, and the latter complexes become more stable. The water solvent significantly decreased the complexation energy, most likely due to the large dipole moments of the complexes in water. Indeed, the calculated dipole moments of R-AMP@CB[7], S-AMP@CB[7], R-MET@CB[7] and S-MET@CB[7] in water are 18.03, 14.93, 17.32 and 17.88 D, respectively, and are larger than their corresponding values of 9.23, 8.73, 9.26 and 9.19 D in gas phase.Further, single-point energy calculations were performed on BLYP-D4/def2-TZVP optimized geometries at B3LYP-D4/def2-TZVP level of theory , both fuSol for the following guest exchange reaction in solution (2):Using a simple thermodynamic study, it is possible to determine which guest interacts most strongly with CB[7]. The idea will be based on the computation of standard free energy change ΔΔG*Sol can be determined from its components by introducing a thermodynamic cycle using Equation (3):0g is the change of free energy in the gas-phase reaction; ΔG*sol(AMP@CB[7]), ΔG*sol(MET), ΔG*sol(MET@CB[7]) and ΔG*sol(AMP) are, respectively, the solvation free energies of AMP@CB[7], (MET), MET@CB[7] and (AMP) species in water.As shown in A] emphasizes calculation over the population of the two enantiomers of each guest. Gas phase free energies of all species were estimated at T = 298.15 K and p = 1 atm through the computation of their energy and geometry at BLYP-D4/def2-SVP-gCP level of theory. Gibbs free energies of aqueous solvation were computed for all species using the conductor-like polarizable continuum model (CPCM) using BLYP-D4/def2-TZVP-gCP. sol is 12.7 kJ/mol. The positive value of ΔΔG*sol indicates that the exchange reaction moves in the direction of the formation of MET@CB[7] complex, therefore, CB[7] forms complex with methamphetamine more favorably than with amphetamine. MET and AMP species exhibit several isomers, their Gibbs free energies have been evaluated using a Boltzmann distribution according to the following relation (4):The geometries of fully optimized structures of all inclusion complexes calculated at BLYP-D4-def2-TZVP-gCP level in water solvent  are visuDFT simulations show that AMP and MET are encapsulated inside CB[7]. Indeed, the benzyl moiety of AMP and MET is deeply inserted into CB[7] due to hydrophobic interactions between the CB[7] cavity and the phenyl ring, whereas the ammonium group of AMP and the methylammonium group of MET are found outside the cavity, at the top of CB[7] leaning to the closest carbonyl groups at the portals, in agreement with the experimental structures. The calculated shortest N…O bond lengths for AMP@CB[7] and MET@CB[7] are both equal to 2.85 Å and are close to the experimental bond values of 2.76 and 2.78 Å, respectively, involving effective ion–dipole interactions between CB[7] and the ammonium groups. The characterization of non-covalent interactions (NCI) is of great importance in molecular recognition systems. We have employed NCI analysis to understand the nature of intermolecular interactions occurring in AMP@CB[7] and MET@CB[7] complexes through the isosurface map of the independent gradient model (IGM) approach. IGM Plots for all compounds were generated with an isovalue of 0.005 a.u. and shown in As observed in The real-time application of drug biosensing based on organic field-effect transistors sensors is a promising platform for detecting illegal drugs such as amphetamine and methamphetamine. Jang et al. showed that OFET based sensors functionalized with CB[7] derivatives for the binding of amphetamine and methamphetamine exhibit highly sensitive and selective sensing properties in water, urine and physiological buffers . AnalysiAccording to the energies of HOMO and LUMO levels reported in Frontier molecular orbital (HOMO and LUMO) plots for the studied complexes are shown in The CDA and ECDA analysis methods can provide insight into the charge transfer (donation and back donation) between fragments in non-covalent complexes. The calculated results indicate that the CDA and ECDA analysis of R-AMP@CB[7], S-AMP@CB[7], R-MET@CB[7] and S-AMP@CB[7] follow the same trend as shown in DFT calculations including D4 dispersion correction were carried out to investigate the inclusion process between CB[7] and amphetamine-type stimulant drugs. The conclusions are as followed:According to the computed complexation energies, the inclusion process of amphetamine and methamphetamine drugs in CB[7] is favorable in gas phase and in water solution.In gas phase, R-AMP@CB[7] and S-AMP@CB[7] are thermodynamically more favored than R-MET@CB[7] and S-MET@CB[7], while the latter are more favored than the former in water solvent.The presence of a mixture of R and S enantiomers was evidenced by DFT-D4 calculations, with a small energy difference in favor of the S enantiomers.X-ray data support the theoretically predicted inclusion conformations of amphetamine and methamphetamine in the CB[7] with the phenyl ring moiety deeply inserted inside the CB[7] cavity and the ammonium group outside the cavity.Structural analysis indicated that van der Waals forces, intermolecular hydrogen bonds, charge transfer interactions and ion-dipole interactions between CB[7] and amphetamine and methamphetamine play a major role in the process of complex stabilization. Frontier molecular orbital analysis of all complexes revealed that the LUMO is shifted to lower energy and stabilized upon complexation, the resulting decrease in the HOMO-LUMO gap leads to a variation of the conductivity, making them suitable for biosensing applications. These theoretical results highlight the high affinity of cucurbiturils for cationic guests through cooperative non-covalent interactions and their potential as stabilizing hosts and a good sensing candidates for the detection of drugs in real samples for practical applications.


In a Wi-Fi indoor positioning system (IPS), the performance of the IPS depends on the channel state information (CSI), which is often limited due to the multipath fading effect, especially in indoor environments involving multiple non-line-of-sight propagation paths. In this paper, we propose a novel IPS utilizing trajectory CSI observed from predetermined trajectories instead of the CSI collected at each stationary location; thus, the proposed method enables all the CSI along each route to be continuously encountered in the observation. Further, by using a generative adversarial network (GAN), which helps enlarge the training dataset, the cost of trajectory CSI collection can be significantly reduced. To fully exploit the trajectory CSI’s spatial and temporal information, the proposed IPS employs a deep learning network of a one-dimensional convolutional neural network–long short-term memory (1DCNN-LSTM). The proposed IPS was hardware-implemented, where digital signal processors and a universal software radio peripheral were used as a modem and radio frequency transceiver, respectively, for both access point and mobile device of Wi-Fi. We verified that the proposed IPS based on the trajectory CSI far outperforms the state-of-the-art IPS based on the CSI collected from stationary locations through extensive experimental tests and computer simulations. Owing to an increase in the number of portable devices, such as mobile phones and tablets, over the past few years ,2, an inAs a state-of-the-art technology, Wi-Fi fingerprinting indoor localization systems (IPS) have been extensively researched for both localization ,7 and acA Wi-Fi fingerprinting IPS can utilize either the received system strength indicator (RSSI) or channel state information (CSI) as fingerprints for each location. For Wi-Fi-based IPSs using RSSI, ref.  demonstrDespite all the merits and preferences in localization applications, the Wi-Fi fingerprinting IPS still requires significant improvements to be practical. To resolve these problems in CSI-based IPS, we consider the following three key aspects in this paper.First, we investigate data collection procedure to provide specific information representing the unique characteristics of each location of interest—the collected data should be stable and trustworthy, such that the system utilizing the data can be sufficiently robust. The conventional method of collecting data for Wi-Fi fingerprinting IPS measures the CSI from arbitrarily stationary locations predetermined in advance ,15,16,17Second, we designed a system that learns the underlying information from the inputs as effectively and efficiently as possible. Many IPS-related papers ,19,20 thThird, we explore methods to efficiently acquire more training data at a reasonable cost. With more training data, a more accurate neural network system that provides better mapping between the input and output without overfitting can be obtained; however, acquiring more data is time-consuming and labor-intensive . Many stIn this paper, we address the three key aspects to optimize the IPS by proposing a novel method of measuring CSI along trajectories instead of collecting the CSI from predetermined stationary locations. Compared to the CSI collected from a stationary location, the continuous CSI collected from a trajectory provides both the CSI from the current location and the CSI from previous locations. To exploit both the spatial and temporal information from the trajectory CSI, we adopted a one-dimensional convolutional neural network–long-short term memory (1DCNN-LSTM) neural architecture to enhance the accuracy of the proposed IPS. We also employed a generative adversarial network (GAN) to resolve the challenges of obtaining more training data. The excellent performance of the proposed IPS is observed through extensive experiments using a testbed implemented with multicore digital signal processors (DSPs) and a graphics processing unit (GPU) for the Wi-Fi emulator and neural network system, respectively.Proposal of adopting continuous CSI captured along each trajectory instead of stationary RPs in a given indoor environment as a novel feature of creating a fingerprinting map for IPS.Hardware implementation of the entire IPS using DSPs as an emulator of Wi-Fi APs and mobile devices (MDs), and a GPU as a trainer for the 1DCNN-LSTM deep learning architecture.Performance analysis of a 1DCNN-LSTM deep learning architecture as a function of the filter size, number of layers, batch size, etc.Application of the GAN to enlarge the dataset such that a large amount of synthetic trajectory CSI can be generated to be added as input data of the 1DCNN-LSTM deep learning architecture without actually collecting the trajectory CSI, which consequently enhances the performance of the proposed IPS with limited dataset size.The main contributions of this paper can be summarized as follows.The remainder of this paper is organized as follows. In CSI contains fine-grained information of the wireless channel, especially for OFDM-based systems, because it can be separately obtained for each subcarrier, where RSSI simply provides coarse-grained information for the entire frequency band . FurtherLet The Only the amplitude of the channel, nsceiver .The IPS implementation procedure can be divided into two phases, offline and online, as shown in During the offline phase, the CSI at each predefined RP was collected and used as training data for the deep learning neural network. Note that, depending on various performance requirements, a deep learning network structure can be customized in many ways . After tDuring the online phase, the CSI at each predefined TP, which is generally different from the RPs, is collected and used as test data to evaluate the performance of the entire IPS designed during the offline phase, as described above.w in measurement n at each RP In this paper, only a single AP with a single Tx antenna was considered, along with several predefined RPs in an area of interest. Each RP The conventional method of collecting channel data is based on the CSI measured from each stationary location ,18,19,20For an extensive comparison, fficient  correspoIn this subsection, we propose a novel paradigm of collecting the channel data, which makes it possible to collect channel data from the trajectory in between each pair of RPs. As the conventional method of collecting the channel data is focused on stationary RPs only, it can only collect channel data from the predetermined location points, which causes the channel data in between RPs to be ignored. Consequently, the proposed technique allows channel data captured continuously between each pair of adjacent RPs, instead of separately collecting the channel data at each RP.The proposed data collection process comprises four steps to capture the channel data along with each trajectory between two RPs  to extract useful spatial features from the trajectory CSI needed to design the target IPS. Unlike a two-dimensional convolutional neural network that processes 2D data such as images, the 1DCNN is used for processing 1D data such as the trajectory CSI.While the 1DCNN is exploited to extract the spatial features of the trajectory CSI, the proposed IPS employs the long and short-term memory (LSTM)  to extraT segments, each of which has a dimension of T is the number of segments that determines the number of 1DCNNs for a given input dataset.Based on the abovementioned characteristics of 1DCNN and LSTM, the 1DCNN-LSTM architecture was adopted in this paper, as shown in After the trajectory CSI in a route from itigated .The proposed neural network should be trained in such a way that the loss function dea from  to trainT segments, are used to train the LSTM. The loss function for the second phase is still the MSE for the T segments of the MIMO-LSTM model [The outputs of the 1DCNNs, {TM model  as:(6)LlAlthough it is desirable to collect as much trajectory CSI as possible for an accurate training of the target neural network, data preparation requires considerable time and effort. To increase the number of training samples without increasing the number of actual observations, we adopted the GAN to efficiently enlarge the training dataset so that the number of observation samples can be significantly increased even with a limited amount of trajectory CSI.GANs were originally developed  to learnM is the number of time steps per trajectory and K is the number of data measurements per trajectory, with L is the dimension of the latent space.To generate the synthetic trajectory CSI based on the GAN, we consider the dataset of trajectory CSI obtained from actual observations as follows:z being p(x) and p(z), respectively. The loss function comprises two terms, including the generator and discriminator function denoted by The Adam optimizer  was usedThe proposed deep learning models of 1DCNN-LSTM and GAN were implemented with TensorFlow 2.0 on an NVidia RTX 2080Ti GPU using the Ubuntu 20.04 operating system. The dataset for training and testing the neural networks was obtained from the proposed IPS—the hardware implementation is detailed in As mentioned in Among the 100 measurements for each trajectory, one out of every five trajectory CSI measurements were selected to build the test dataset, whereas the remaining measurements were the training dataset. Consequently, our dataset comprises 80 measurements of the trajectory CSI for the training dataset and 20 for the test dataset. The 100 measurements for each trajectory must be shuffled well enough such that the training data can be allocated as uniformly as possible over the entire observation period. Consequently, the total number of samples in the training data set is 8,960,000 per trajectory (=80 measurements of 2000 samples for each of 56 subcarriers) while that of the test dataset is 2,240,000 per trajectory (=20 measurements of 2000 samples for each of 56 subcarriers). By using the abovementioned training and test datasets, the proposed deep learning networks are trained and tested to construct the optimal IPS.To achieve the best-optimized model for the 1DCNN, we conducted experiments to evaluate the impact of the convolutional filter dimension on the 1DCNN performance based on the number of convolutional layers and filters.We also observed the performance of the IPS as a function of the number of convolutional layers with various numbers of convolutional filters employed at each layer. Through extensive experiments, we found that the 256-128-64-32 configuration provides the lowest mean distance error, i.e., 1.34 m, among all the observed configurations. However, the training time for this configuration, i.e., 268 s, was much longer than that for most other configurations because of its four-layered structure with numerous convolutional filters. In the case of both the mean distance error and training time, we found that the configuration of 64-32 provides reasonably good performance with a mean distance error of 1.35 m, which is only 0.75% higher than that of the configuration of 256-128-64-32, and the training time is 134 s, which is approximately 50% faster than that of the configuration of 256-128-64-32.From the above experiments, we observed that a double-layered model with 64 filters in the first layer and 32 filters in the second layer provided the best performance in terms of both the mean distance error and training time in the indoor experimental environment described in T as described in T increases, more 1DCNNs are utilized to extract features from the corresponding segment. To find the optimal T for the 1DCNN-LSTM architecture, we conducted extensive experimental tests on different values of T. The detailed parameters for each LSTM of the proposed IPS are listed in The number of segments T is summarized in The performance of the IPS based on For each LSTM, the number of units, which is related to the capacity of the LSTM for learning the input data, must be set up with an appropriate value in such a way that it causes neither over-fitting with an excessively large value nor under-fitting with an excessively small value. Thus, the tuning of this hyperparameter is generally determined empirically through experiments.The number of samples in the training dataset used to estimate the gradient of the loss before the model weights are updated is denoted as the batch size . It is aThe objective of this subsection is to verify that the route prediction accuracy is improved as we increase the amount of input data of the proposed neural network by concatenating several consecutive trajectory CSI of a given route. With the five routes predetermined, as shown in The objective of this subsection is to present the performance of the proposed IPS, which employs the synthetic data provided by the GAN model discussed in To observe how the synthetic data provided by the GAN model enhance the performance of the proposed IPS, we performed two experiments as follows. In the first experiment, 20%  of the entire training data was randomly selected and presented to the GAN model to generate a set of synthetic data, which was added to the dataset of the 16 real measurements to increase the total quantity of the training dataset. Subsequently, the performance of the proposed IPS was analyzed as a function of the number of synthetic data added to the 16 real measurements. In the second experiment, we performed the same experiments as the first, except that all the training data  were used along with the synthetic data as the training dataset. State-of-the-art neural networks, i.e., ConFi , DeepFi The objective of this subsection is to present the performance of the proposed IPS with a few different selections for the spacing between RPs in two different signal environments. The two different indoor signal environments are (1) narrow laboratory with various furniture, and (2) relatively wide corridor, of which the photos are shown in In this paper, we proposed a novel IPS using the trajectory CSI continuously collected along each trajectory. Compared to the traditional method of collecting CSI measurements at predetermined fixed locations, our proposed trajectory CSI collection method can collect continuous CSI existing in the trajectories, and utilize it to achieve high accuracy and reliability in localization. The proposed IPS was implemented using multicore DSP and USRP as a modem and RF transceiver, respectively, to emulate Wi-Fi APs and MDs supporting the IEEE 802.11ac protocol, and using a GPU as a training device for the proposed system. For the deep learning solution, we adopted a 1DCNN-LSTM deep learning architecture, which can extract both spatial and temporal information from the given trajectory CSI to predict the MD’s current location with the help of the information provided from previous data. Additionally, to tackle the challenge of obtaining sufficient trajectory CSI, the GAN was employed in this paper to generate synthetic data with only a small amount of real trajectory CSI measurements. Through extensive onsite experiments in an indoor environment, the results consistently demonstrate that our 1DCNN-LSTM deep learning structure trained with the training dataset comprising both real and synthetic data generated by the GAN achieves an average localization error of 0.74 m, which outperforms the state-of-the-art algorithms ConFi by 46.0%, DeepFi by 47.9%, and Horus by 61.9%.


Trypanosoma brucei rhodesiense and T. b. gambiense. Animal trypanosomoses have major effects on livestock production and the economy in developing countries, with disease management depending mainly on chemotherapy. Moreover, only few drugs are available and these have adverse effects on patients, are costly, show poor accessibility, and parasites develop drug resistance to them. Therefore, novel trypanocidal drugs are urgently needed. Here, the effects of synthesized nitrofurantoin analogs were evaluated against six species/strains of animal and human trypanosomes, and the treatment efficacy of the selected compounds was assessed in vivo. Analogs 11 and 12, containing 11- and 12-carbon aliphatic chains, respectively, showed the highest trypanocidal activity (IC50 < 0.34 µM) and the lowest cytotoxicity (IC50 > 246.02 µM) in vitro. Structure-activity relationship analysis suggested that the trypanocidal activity and cytotoxicity were related to the number of carbons in the aliphatic chain and electronegativity. In vivo experiments, involving oral treatment with nitrofurantoin, showed partial efficacy, whereas the selected analogs showed no treatment efficacy. These results indicate that nitrofurantoin analogs with high hydrophilicity are required for in vivo assessment to determine if they are promising leads for developing trypanocidal drugs.African trypanosomes cause diseases in humans and livestock. Human African trypanosomiasis is caused by  Trypanosoma brucei gambiense and T. b. rhodesiense. The disease has devastating socio-economic impacts, such as reduced income generation, negative effects on the education of children, increased healthcare costs, and long-term health consequences [T. congolense, T. vivax, and T. brucei brucei, represents a major health concern in animal development, and its deleterious impacts can be manifested in terms of reduced livestock productivity, costly disease control, and trade implications, as well as negative impacts on agriculture and food security [T. evansi infection. It is mechanically transmitted by hematophagous biting flies such as Tabanus spp., Stomoxys spp., and species of tsetse flies [African trypanosomes, transmitted by tsetse fly, cause several diseases in both humans and livestock on the Sub-Saharan African continent. Human African trypanosomiasis (HAT) is caused by equences ,2. In cosecurity ,4. Surrase flies . Animal se flies ,5.T. b. gambiense [T. b. rhodesiense and AT. Therefore, the screening and development of safe and effective compounds for treating HAT/AT are urgently required.HAT/AT control requires an integrated approach involving the use of trypanocidal drugs ,6, reducambiense . Particuambiense . In contNitrofurantoin is a hypoxic agent with activity against a myriad of anaerobic pathogens. This nitrofuran compound contains a Schiff base derived from 5-nitrofuraldehyde, which is known to be effective against a wide spectrum of gram-positive and gram-negative bacteria and various pathogens, including trypanosomes ,11. NitrT. cruzi. More recently, the combination of nifurtimox and eflornithine was evaluated for the treatment of late-stage T. b. gambiense sleeping sickness [The clinical drug furazolidone belongs to the group of nitrofuran antibiotics and has been widely used as an antibacterial and antiprotozoal feed additive for poultry, cattle, and farmed fish ,14. Nifusickness ,16,17. Fsickness . OverallA summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in T. congolense IL3000 and T. b. rhodesiense Chirundu were more sensitive to these nitrofurantoin analogs than the other trypanosome strains/species.Trypanocidal activities differed among the trypanosome species. However, 50 < 0.031 µM) against T. congolense IL3000 and T. b. rhodesiense Chirundu was observed with the long chain analogs 9–12, bearing side chains of 9–11 carbons, respectively (n = 9–12), in sub-series 1. These analogs showed relatively moderate (0.16–0.65 µM) activity against T. evansi Tansui, T. b. brucei GUTat3.1, T. b. gambiense IL1922, and T. b. rhodesiense IL1501.The strongest trypanocidal activity  was stronger than that of analog 14, featuring a neutral group and those of analogs 15 and 18 possessing electron-donating groups. In contrast, electronegativity had no effect on cytotoxicity within sub-series 2.Furthermore, variation in the trypanocidal activity for analogs in sub-series 2 was found based on the electronegativity of 9, 11, and 12) showed strong trypanocidal activity and were used to confirm the results of in vitro assay and nitrofurantoin. These three analogs were evaluated. All mice treated with nitrofurantoin and its analogs by intraperitoneal injection at 0.1 mg/kg, died within 9 dpi, because of high parasitemia  in sub-series 1 decreased with increasing chain lengths up to four carbons  (5–12) increased with increasing chain lengths up to 12 carbons  (9–11) in sub-series 1 may be related to their higher lipophilicity, which increased the cLogP value. This feature is typically associated with better permeation of a molecule through biological tissues/membranes, as well as interactions with transporter proteins and enzymes [T. cruzi parasite [In the present study, the trypanocidal activity of 19 nitrofurantoin analogs was evaluated against six human and animal trypanosomes in vitro. Investigation of the structure–activity relationship within the series revealed that the trypanocidal activity of short alkyl analogs  a. The tr 12, 12) a. The cy 12, 12) a. The st enzymes . Similarparasite . Variousparasite ,22.16, 17, and 19) was stronger than that of the compounds containing a neutral group (14) and those bearing electron-donating groups (15 and 18) in sub-series 2  is an antibiotic that is orally used to treat bladder infections. Nitrofurantoin shows extremely low lipophilicity (clogP −0.22), which may explain its poor trypanocidal activity, as it cannot efficiently permeate parasitic biomembranes. In contrast, analogs 1 and 2 have low cLogP values of −0.04 and 0.36, respectively [T. b. rhodesiense Chirundu and T. congolense IL3000 strains. HAT and AT are endemic diseases, mainly occurring in rural areas of developing countries, and persist because of the lack of healthcare and veterinary medicine infrastructure. Therefore, oral drugs are highly convenient for treating these diseases. Although they showed strong trypanocidal activities in vitro, no treatment efficacy was observed with nitrofurantoin analogs 9, 11, and 12 against T. congolense infection in vivo, after both oral and intraperitoneal administration. Oral treatment with nitrofurantoin, however, demonstrated partial efficacy. nitrofurantoin possessed weak trypanocidal activity in vitro, but dissolved in water with a lower clogP than that of the tested analogs, which were more lipophilic and thus had higher clogP values. This result supports that high-hydrosolubility nitrofurantoin analogs should be developed and evaluated for their in vivo trypanocidal activity, in further studies. Analogs 1 and 2 showing moderate in vitro trypanocidal activity and higher solubility (lower cLogP) than the current selectees may be viable starting points for developing oral treatments for HAT and AT in endemic areas [Nitrofurantoin  [1–12, whereas sub-series 2 comprised benzyl-substituted analogs 13–19. The latter sub-series featured analogs 15 and 18, which bore the electron-donating groups CH3 and OCH3, respectively, whereas analogs 16, 17, and 19 possessed the electron-withdrawing groups CF3, NO2, and Br, respectively. Analog 14 bore a neutral group H, and analog 13 possessed an ethyl bridge between the nitrofurantoin scaffold and the benzyl group.The nitrofurantoin analogs  were synon (SN2) . The ser8, which was 30-fold more potent than nitrofurantoin [The analogs possessed lipophilic properties, with lipophilicity coefficients (clogP) in the targeted range of 1–5 ,32. Theyurantoin .These analogs were dissolved at 10 mg/mL in dimethyl sulfoxide and stored at −30 °C until use.T. b. brucei GUTat3.1, T. b. rhodesiense IL1501, T. b. rhodesiense Chirundu, T. b. gambiense IL1922, and T. evansi Tansui strains were propagated in culture at 37 °C, whereas the T. congolense IL3000 strain was propagated at 33 °C in Hirumi’s modified Iscove’s medium-9, supplemented with 20% heat-inactivated fetal bovine serum [The bloodstream forms of 3 cells/mL , 1 × 104 cells/mL (T. evansi Tansui and T. b. rhodesiense Chirundu), and 1 × 105 cells/mL (T. congolense IL3000), were cultivated in Nunc® MicroWell 96-well optical bottom plates  and exposed to various analogs at seven different concentrations for 3 days, in an incubator at 37 °C (33 °C for T. congolense IL3000). After 3 days of incubation, the condition of the bloodstream forms was observed with phase-contrast microscopy. Twenty-five microliters of the Cell-TiterGlo®  reagent was added into each well, and bioluminescence was measured using a GloMax®-Multi+Detection System plate reader (Promega).The trypanocidal activity and cytotoxicity of nitrofurantoin analogs were evaluated according to previous reports ,35, with4 cells/mL) in a 96-well cell culture plate (Thermo Fisher Scientific) with the analogs, at seven different concentrations for 3 days at 37 °C. Next, 10 µl of the Cell Counting Kit-8 solution  was added into each well before and after 4 h of incubation. Absorbance was measured at 450 nm using a GloMax®-Multi+Detection System plate reader.Additionally, Madin–Darby bovine kidney cells were cultivated  of each analog in trypanosomes and the Madin–Darby bovine kidney cells was calculated using the GraphPad Prism version 8 software .The half maximum inhibitory concentration  were used in this study. All animals had ad libitum access to normal chow and water.T. congolense IL3000 strain was propagated in a mouse and used for infection. The parasites were passaged once in a mouse before the experiment. The experimental mice were intraperitoneally infected with 100 μL of T. congolense in phosphate-buffered saline, containing 10% glucose at 1 × 103 cells/mouse. First, the mice were randomly divided into six groups of three mice, each as follows. Group I (no treatment), mice were not infected and were only treated with solvent . Group II received 3.5 mg/kg of diminazene aceturate treatment. Groups III (9), IV (11), V (12), and VI were treated with nitrofurantoin and the mice were infected and intraperitoneally treated with 0.1 mg/kg of the compound.The virulent 9), IV (11), V (12), and VI received nitrofurantoin—the mice were infected and orally treated with 10 mg/kg of the compound.In the second experiment, the mice were randomly divided into six groups of three mice, each as follows—group I received no treatment; group II received 3.5 mg/kg of diminazene aceturate treatment; and groups III (Treatment was initiated at 2 days post-infection (dpi) and continued for seven consecutive days. The treatments were freshly prepared each day. Parasitemia was observed using a cell counting chamber until 14 dpi.T. congolense and some confined endemic acute HAT infections caused by the T. b. rhodesiense Chirundu strain. Our preliminary in vivo experiments also showed that NF and its analogs with high water solubility should be evaluated to determine their treatment efficacy in mouse models, in further studies.In conclusion, our results indicate that nitrofurantoin may be suitable as a lead compound for developing novel trypanocidal drugs, provided that compounds with good oral bioavailability and in vivo activity can be designed, particularly against animal African trypanosomosis caused by


Val158Met; rs4680) of the catechol-o-methyltransferase gene (COMT), influence working memory performance.Despite the widespread use of oral contraceptives (OCs), and the well-documented influence of estrogens, notably 17β-estradiol (E2), on cognition, research relating OCs to working memory is limited and mixed. Two factors may contribute to these mixed findings: 1) pharmacokinetics of oral contraceptives, which drive fluctuations in synthetic hormone levels; and 2) genetic polymorphisms related to dopamine degradation and working memory, which interact with E2. This research investigated whether the pharmacokinetics of oral contraceptives, in concert with the single nucleotide polymorphism (COMT. If they were not taking OCs (n = 62), sessions occurred in the early follicular (low E2) and late follicular (high E2) phase. If they were taking OCs (n = 52), sessions occurred 1–2 hours after  and ~24 hours after (low EE) pill ingestion. Working memory was tested using the N-back, AX-CPT, Digit Span, and Digit Ordering Tasks. Data were analyzed using multilevel models with estrogen condition, COMT, and group as predictors, controlling for mood and practice effects.University-age women taking and not taking OCs were tested for working memory and genotyped for COMT val/val (low dopamine) were less accurate on 2-back lure trials than those with COMT met/met (high dopamine). For women not taking OCs, cycle phase moderated COMT’s influence on lure accuracy. When compared, women taking OCs had higher AX-CPT proactive control indices than those not taking OCs.For women taking OCs, time of pill ingestion did not influence performance. However, the subgroup with COMT in women taking OCs suggests that, in women taking OCs, suppressed endogenous E2–not fluctuating EE levels–may be more relevant for working memory. Future studies are needed to differentiate effects of endogenous versus synthetic estrogens on working memory.These findings suggest that oral contraceptives are not detrimental for young women’s working memory and that they may increase proactive control. The more pronounced effects of  Oral contraceptives (OCs), a widely used form of hormonal birth control, are also often prescribed for treating endometriosis and polycystic ovarian syndrome, and managing symptoms related to the menstrual cycle ,2. From Indeed, there are many reasons to expect OCs to influence women’s cognition, as OCs contain synthetic estrogen, and ample evidence suggests estrogens influence the brain and behaviour. For example, 17β-estradiol (E2) binds to and activates estrogen receptors throughout the brain, influencing neurotransmission, gene transcription and neurogenesis . AccordiWhile research comparing women taking OCs (OC) to those naturally cycling and not taking OCs (NOC) is sparse, what exists in younger women shows a correlation of taking OCs with higher prefrontal cortical grey matter volume , and redOne factor that may contribute to these inconsistent findings are the pharmacokinetics of OCs which have not been considered in experimental design. Endogenous hormone levels in OCs are continually suppressed and are comparable to or lower than the levels in early follicular phase of NOC, the menstrual cycle phase associated with lowest circulating sex hormone levels  of the gene coding for catechol-o-methyltransferase (COMT), the protein that degrades dopamine at synapses in the prefrontal cortex  and a negative one when dopamine levels are high (COMT met/met). Thus, OC studies grouping women with different COMT genotypes and hence, dopamine levels, might mask high EE’s (or low E2’s) relationship to working memory.Also overlooked when considering OCs effect on cognition is their possible interaction with dopamine—a neurotransmitter that strongly modulates working memory. The relationship between dopamine and memory is complicated, with some research suggesting an inverted-U dopamine response curve, in which working memory performance is highest when dopamine levels are neither very high nor very low ; Fig 1)..Fig 1). ex , interaex  would influence cognitive performance. We also hypothesized that, if OCs have the same effect on working memory as endogenous E2, higher EE 1–2 hours after pill ingestion would improve working memory in low dopamine conditions  and reduce it in high dopamine conditions (COMT met/met). On the other hand, since endogenous E2 remains stably low in women taking OCs [COMT met/met) to outperform OC with low dopamine , independent of EE levels.Therefore, we asked the following questions: 1) do the pharmacokinetics of EE–as measured by pill ingestion time–influence OC’s working memory?; and 2) does EE interact with dopamine–as measured by king OCs ,23–25, aThis study was approved by the University of Toronto Social Sciences, Humanities and Education Research Ethics Board (Research Information System Protocol Number: 35242) and was in accordance with the declaration of Helsinki. Women ages 18–30 were recruited through the University of Toronto’s Introductory Psychology course and community advertisements. Potential participants were phone screened prior to study enrolment and provided written informed consent prior to testing.Exclusion criteria were: not fluent in English, currently pregnant or pregnant within the past 6 months, history of neurological or psychiatric disorders including concussion with loss of consciousness, regular cigarette smoking, currently taking psychoactive medications or exogenous hormones (other than OCs), and a diagnosis of an endocrine disorder. Inclusion criteria were: for NOC, not using hormonal contraception, and an average menstrual cycle length of 21–35 days, with regular menstrual cycles for the past 6 months, and for OC, use of a combined pill for at least 3 consecutive months prior to participation. Type of synthetic hormones in the OCs was neither an exclusion nor an inclusion criterion. Initially, 167 participants (95 NOC and 72 OC) were recruited for our study. After exclusions, the final cohort had 119 participants: 62 NOC and 57 OC . Since tWe used a within-subjects design. Each person participated in two testing sessions, timed according to their menstrual cycle phase (NOC) or pill ingestion (OC). For NOC, one session occurred during the early follicular phase (cycle day 1–5) and another during the late follicular phase (cycle day 9–14), determined by forward counting from day of menstruation, and when possible, referencing period tracking applications. OC were scheduled 1–2 hours after pill ingestion and again, roughly 2 weeks later approximately 24 hours after pill ingestion. The order of sessions was counterbalanced for both groups.COMT Val158Met SNP.Participants were tested from 10:30am to 6:00pm, an optimal time of day for younger adults to undergo cognitive assessment . ParticiCOMT Val158Met SNP) and E2 as measured in blood [This task was chosen because it tests verbal working memory . It was in blood . Additioin blood , and reqParticipants completed a computerized 1-back and 2-back task with letter stimuli using Inquisit 5 software . Prior to data collection, participants completed a practice 1-back and 2-back block. Each letter was presented for 1000 ms followed by a 1000 ms fixation cross. Participants were instructed to make a button press for target trials when the current letter was the same as ‘N’-positions back in the sequence . A different button response was required for non-target trials. Consistent with others, the 2-back condition included hard-to-reject lure trials that were either a 1-back or 3-back match to the current stimulus . In the This task was selected because it also tests verbal working memory. In the Forward DS, participants were instructed to repeat back sequences of digits read aloud by the experimenter; in the Backward DS, participants were asked to repeat the sequence in reverse order. There were 2 trials for each sequence length, and the maximum sequence length was 8 digits on the Forward task and 7 digits on the Backward task . The ForThis task was selected because it requires working memory and engages the dorsolateral PFC ; it requThis task was selected because it requires the maintenance of goal-relevant information and inhibition of prepotent responses, important components of working memory , and becParticipants completed 2 blocks, each consisting of 150 trials, of which 70% were AX, 10% were AY, 10% were BX, and 10% were BY. A high proportion of AX trials was used to create a prepotent response tendency to AX. The reaction time and accuracy of responses were recorded.COMT genotype and E2 levels [COMT, group (NOC or OC), estrogen condition, and their interactions. “Estrogen condition” referred to cycle phase  in NOC and to pill ingestion time  in OC. Random slopes were modelled for estrogen condition, grouped by participant, and for each participant a random intercept was modelled. Session and estrogen condition were partially correlated within participant, so session was only included as a covariate to control for practice effects and not a random effect. CES-D score was also a covariate in order to control for mood, as premenstrual dysphoric disorder, although only in a minority of women, was not an exclusionary criterion, and use of OCs has been associated with first diagnosis of depression and increased use of antidepressants [All analyses were conducted in R 3.6.1 . For the2 levels . Trial-bressants . The modressants .Trial-by-trial reaction time on correct trials was modeled in the same way using the lmer function from the lme4 package . HoweverIf the full models for trial-by-trial performance yielded significant effects, reduced models were run separating NOC from OC to understand the source of the effect. Additional exploratory analyses were run examining trial-by-trial accuracy and reaction time on target and non-target trials in the 1-back and 2-back tasks. The False Discovery Rate (FDR) correction for multiple comparisons was applied to exploratory analyses using the p.adjust function from the stats package.For the AX-CPT, a proactive behavioural index (PBI) was calculated to quantify the extent of proactive control (planning to respond after seeing an A) vs. reactive control (considering responding after seeing an X) used by participants . The comAX-CPT PBIs, number of errors on the Digit Ordering Task, and maximum number of digits on the DS Task were also analyzed using multilevel models. COMT, group, estrogen condition with their interactions were included as predictors, and CES-D and session as co-variates. A random intercept was modelled for each participant. Random slopes were not included because these models only contained one summary observation per estrogen condition per participant. Prior to calculating PBI reaction time, reaction times were trimmed using the same criteria as the N-back task and log transformed. The models were estimated with an unstructured covariance matrix using the lmer function from the lme4 package .d = 0.46 , estrogen condition differences as small as d = 0.27 (0.26), genetic differences as small as d = 0.71 (0.25), and interactions as small as d = 1.17 (0.26).Six of the 119 eligible participants completed some, but not all of the tasks, and were therefore excluded from analysis of those tasks . SensitSE, range = 18–28), and the average CES-D score was 13.23 ± 0.77 . The COMT distribution of the sample was in Hardy Weinberg equilibrium , with 20 met/met, 57 met/val, and 42 val/val. There was no significant difference in age , CES-D score , or COMT distribution  between NOC and OC . NOC had an average menstrual cycle length of 28.51 ± 0.31 days .The majority of OCs being taken consisted of a monophasic pill formulation containing 20 ug of EE and 100 ug of levonorgestrel. EE was the synthetic estrogen in all OCs, and doses ranged from 20 to 35 μg. The progestin component was variable in type and dose . Averagemet/met women were significantly more accurate than val/val women on lure trials . Additionally, it showed significant 3-way interactions between COMT, estrogen condition, and group . Therefore, reduced models separating NOC from OC were run to understand gene-estrogen relations within each group.The full multilevel model for 2-back lure accuracy revealed met/met and val/val , or met/met and met/val  on 2-back lure trial accuracy (COMT on 2-back lure accuracy (met/met were significantly more accurate in the high estrogen condition (late follicular) than in the low estrogen condition (early follicular) phase ; however, estrogen condition had less of an effect on met/val  and val/val .The reduced model with NOC revealed no significant differences between accuracy . On the accuracy , such thval/val OC were significantly less accurate than met/met OC on 2-back lures .The multilevel models for 2-back lure reaction times revealed no significant effects of or interactions between None of the 1-back or 2-back task exploratory analyses met the alpha threshold following the liberal FDR correction. Results for the exploratory analyses are reported in the supporting information.COMT, estrogen condition, and group . Therefore, reduced models separating NOC from OC were run.The multilevel model for the Forward DS revealed a trending 3-way interaction between COMT on number of digits remembered  than the low estrogen condition (early follicular) phase, but met/val  and val/val  were comparatively less influenced by estrogen condition. On the other hand, the reduced model with OC revealed no significant effects or interactions between COMT and estrogen condition  or total working memory errors on the Digit Ordering Task .There were no significant effects or interactions between SE = .001, t = 3.33, p = .001; COMT or estrogen condition on PBI reaction time. The multilevel model for PBI accuracy showed no significant effects of or interactions between COMT, group, and estrogen condition  than OC  in low dopamine conditions  and reduce it in high dopamine conditions (COMT met/met). However, we observed no difference in performance between these two proxies for EE levels. Since there was no fluctuation in working memory performance depending on the time of pill ingestion (on its own or in conjunction with COMT genotype), our results suggest that there are no EE effects on working memory.We hypothesized that pill ingestion time would have an effect on working memory on its own, and that it would also modulate the influence of COMT such that met/met had significantly higher accuracy on the 2-back lures than val/val. This finding, compounded with the fact that pill ingestion time did not affect performance, suggests that endogenous estrogens, not exogenous EE, likely mediates working memory performance in OC. OCs suppress ovarian hormone production, such that their levels are comparable to or fall below the range of E2 levels in the early follicular phase of NOC  on 2-back lure accuracy. Further, the one study to date that examined menstrual cycle and COMT-working memory relationships [met/met and val/val polymorphism were amplified in women who were in their low E2 phase. Relatedly, the COMT Val158Met polymorphism affects 2-back performance, with met/met making more correct responses than val/val in postmenopausal women and middle-aged men–both whom have low E2 levels–but has no effect in middle-aged premenopausal women (not stratified by cycle phase) who have higher E2 levels [COMT.Consistent with this possibility, in OC, we found that 2-back lure accuracy only showed a significant relationship with ionships , showed 2 levels . TogetheCOMT. We found that met/met NOC performed significantly better on 2-back lures in the high E2, late follicular phase, while val/val and met/val performance was unaffected by cycle phase. These results are contrary to previous research suggesting that met/met NOC have improved working memory in the low E2 early follicular phase [val/val and 8 met/met [val/val and 7 met/met. It may also be due to ethnic differences of our cohort. Only half of our NOC participants self-identified as Caucasian. In a mixed sex cohort that controlled for sex there was evidence of a working memory advantage to carrying the val allele in a healthy Chinese population [met/met NOC in our study who are not Caucasian may be further from this optimal point and thus, benefit from higher E2. It is also possible that imperfect counterbalancing of estrogen condition could have contributed to these differences; participants were genotyped post-study completion, resulting in a higher proportion of met/met who completed their second session in the late follicular phase . In thel cortex . This subecause they were taking OCs, a proactive measure against pregnancy. Thus, OC may have already been a group with higher proactive control in daily life decisions. Additionally, it is possible that OC may have had higher socio-economic status than NOC. Affordability is one of the main factors that affects a woman’s contraception use [More proactive control could also be explained by a pre-existing group difference. The OC group was recruited tion use . Higher tion use . It woulCOMT, EE condition, or their interactions. However, in NOC, met/met showed better performance than met/val in the high E2 late follicular phase on the Forward DS. A mixed-sex study using the Stroop task found that the met allele was beneficial for cognitive stability of information [In OC, the short term memory test, Forward DS, performance was not directly affected by ormation . Howeverormation . As withCOMT, estrogen condition, and group on the Backward DS or Digit Ordering task, two other tasks that require working memory. Like other young healthy participants, our participants likely devised grouping strategies to improve performance [We did not find any effects or interactions between, formance , ultimatThis study considered endocrine and genetic status as mediators of the effects of OCs on working memory. While these have been underconsidered in memory studies, the effects of OCs in particular, have been understudied given that they are widely used over long terms.COMT Val158Met polymorphism plays a role in the working memory of OC, interacts with E2 in working memory and affects short term memory of NOC. These results highlight that genetic polymorphisms may interact with estrogens and should be considered when studying working and short term memory.This study is the first to consider the effect of the pharmacokinetic properties of OCs and genetics on working memory in women taking OCs. By studying the combination of these different factors on working memory, it demonstrates that the met/met polymorphism, reducing the power of our methods. However, the numbers in this study are within the range of others correlating neuropsychological measures with genetic polymorphisms [As with many genetic polymorphism studies, ours has the common limitation of a low number of participants carrying each polymorphism. Few women had the orphisms ,40,49,50Another weakness is that we did not measure hormones directly. Our method of cycle phase determination was based on forward counting from menstruation onset, which is less reliable. However, NOC in our study were asked to reference period tracking applications or logs when booking sessions and completing questionnaires. This likely increased cycle phase determination beyond forward counting alone. Another weakness of our study, shared by most other studies of OC and cognition, is the variety of OCs formulations used. OCs can contain progestins of varying androgenicity; those with higher androgenicity have been correlated with better visuospatial ability . Future COMT Val158Met genetic polymorphism affected working memory comparing women taking OCs with those not taking them. Surprisingly, we found that the pharmacokinetics of EE did not affect either working or short-term memory. We did find, based on pill ingestion time, that it is likely suppressed endogenous E2–not EE levels–that affect working memory, including interacting with COMT. We also found that in our cohort, OCs contributed to proactive control, which may also be the result of lowered endogenous E2 in women taking OCs. These findings suggest that an important area for further investigation is the interaction between EE and endogenous estrogens in women who take OCs. As well, we hope this project inspires other researchers to investigate other aspects of cognition that may be influenced by the use of OCs.Despite the widespread use of OCs, our knowledge about their impact on the brain and behaviour is limited. There has been no previous study of either the pharmacokinetics of EE on cognition, or its interactions with neurotransmitter systems, two important variables that could affect cognition. We investigated how the pharmacokinetics of EE in concert with the S1 File(DOCX)Click here for additional data file.


This is also observed in other predominantly IgG4 autoantibody-mediated neurological disorders, such as chronic inflammatory demyelinating polyneuropathy and autoimmune encephalitis with autoantibodies against the Ranvier paranode and juxtaparanode, respectively, and extends beyond neurological autoimmunity as well. Although IgG1 autoantibody-mediated neurological disorders can also respond well to rituximab induction therapy in combination with an autoantibody titer drop, remission tends to be less long-lasting and cases where titers are refractory tend to occur more often than in IgG4 autoimmunity. Moreover, presence of GC-like structures in the thymus of myasthenic patients with predominantly IgG1 autoantibodies against the acetylcholine receptor and in ovarian teratomas of autoimmune encephalitis patients with predominantly IgG1 autoantibodies against the N‐methyl‐d‐aspartate receptor (NMDAR) confers increased the ability to generate LLPCs. Here, we review available information on the short-and long-lived nature of ASCs in IgG1 and IgG4 autoantibody-mediated neurological disorders and highlight common mechanisms as well as differences, all of which can inform therapeutic strategies and personalized medical approaches.As B cells differentiate into antibody-secreting cells (ASCs), short-lived plasmablasts (SLPBs) are produced by a primary extrafollicular response, followed by the generation of memory B cells and long-lived plasma cells (LLPCs) in germinal centers (GCs). Generation of IgG4 antibodies is T helper type 2 (Th2) and IL-4, -13, and -10-driven and can occur parallel to IgE, in response to chronic stimulation by allergens and helminths. Although IgG4 antibodies are non-crosslinking and have limited ability to mobilize complement and cellular cytotoxicity, when self-tolerance is lost, they can disrupt ligand-receptor binding and cause a wide range of autoimmune disorders including neurological autoimmunity. In myasthenia gravis with predominantly IgG4 autoantibodies against muscle-specific kinase (MuSK), it has been observed that one-time CD20 B cells are the major components of the humoral adaptive immune system. Prior to antigenic stimulation, B cells develop in the bone marrow, where V (variable), D (diversity), and J (joining) gene recombination occurs, leading to the formation of the immunoglobulin antigen binding domains and the naïve B cell receptor repertoire. During this process of development and diversity generation, autoreactive clones are physiologically cleared by mechanisms imposed by two tolerance checkpoints: one central and one peripheral . Upon an+ B cell depletion with rituximab , titer decline post rituximab shows that B cell depletion varies from minimal  associated with predominantly IgG4 autoantibodies against muscle-specific kinase (MuSK), where autoreactive ASCs appear to be short-lived . This shituximab –9. As mo minimal  to less  minimal –13. Henc minimal . Of note minimal , 16 but  minimal –24.In this review, we aim to examine whether the MG paradigm extends to other autoimmune neurologic disorders with pathogenic autoantibodies of the IgG1 and IgG4 subclass. Further, we discuss how antigen-experienced B cells differentiate into predominantly IgG1- or IgG4- secreting ASCs and how IgG1 and IgG4 B cell responses generate short- and long-lived autoantibody-producing cells differently. While focusing on human data, we give an overview of how the different subclasses and ASCs contribute to different autoimmune neurological diseases, and in parallel, highlight advances in B cell biology that relate to the development of pathogenic autoantibodies.With the exception of IgM autoantibodies against myelin-associated glycoprotein (MAG) , all patObservations of follicular and extrafollicular responses rely mainly on rodent models. Shortly after exposure to a T-cell-dependent antigen, responding B cells and T cells appear at the B cell follicle/T cell zone border of the lymph node . The inivia Tfh and FDCs in order to select higher-affinity B cell clones while the dark zone facilitates B cell proliferation and somatic hypermutation in order to generate candidates for clonal selection in the light zone  of the lymph nodes or the spleen, which harbor structures known as GCs. After the initial B cell response, the initiation of GCs is orchestrated by various immune cells, including B cells, helper T cells (such as Tfh cells driven by bcl-6), follicular dendritic cells (FDCs), and macrophages, as well as cytokines such as IL-6 –46. Sincght zone  , while LLPCs are described as CD20- CD19- CD38hi CD138+  and IgG4 by far the rarest  –61, IgG4Both IgG4 and IgE class-switch and production are induced by IL-4 and IL-13 –76, howeex vivo via IL-10 and TGF-β  involvement may be different in IgG4 and IgG1 responses. Apart from extrafollicular Tregs that could control GC initiation, follicular regulatory T cells (Tfr) may balance Tfh cells and participate in determining the fate of B cells. Tfr cells may either directly suppress GC B cells through CTLA-4-mediated inhibition of CD80/CD86 co-stimulatory signaling or indirectly do so through IL-10 secretion acting on Tfh cells . Importand TGF-β , 89. Durnd TGF-β , while ind TGF-β . Similarnd TGF-β , 93 and nd TGF-β . Conversnd TGF-β , and furmmune MG . In pempmmune MG  and havemmune MG  are repoClass-switching recombination (CSR) is a fundamental change connected to the maturation of B cells as they evolve towards antibody secretion in response to antigens. CSR is facilitated by AID and materialized by the excision of DNA fragments and the subsequent joining of previously distant regions. As a consequence, CSR can only happen in the 5’-3’ direction of chromosomal topology of the different (corresponding to different classes and subclasses) constant region fragments Figure 2CSR and origins of IgE and IgG4 have been extensively exanimated in allergy and autoimmunity. In allergy, whole-repertoire NGS data from healthy and allergic individuals indicate that indirect switching from IgG1 is the primary source of IgE, while indirect switching from IgG4 is also significant  , 106. InIgG1 and IgG4 autoantibodies bear different mechanisms of pathogenicity because of their molecular characteristics  , 79. ProIn many neurologic diseases, some of them newly defined, pathology is mediated by autoantibodies against surface/extracellular protein antigens. Among those are MG, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), neuromyelitis optica and neuromyelitis optica spectrum disorders (NMOSD), acute disseminated encephalomyelitis (ADEM), relapsing optic neuritis (ON), pediatric acquired demyelinating syndromes, and autoimmune encephalitis (AE) –116. Sucvia MHC II to T cells  B cell depletion after treatment ends  . Circulaent ends –259. Morent ends . Finallyent ends .Evidence from several case series suggests that rituximab is clinically effective in the majority of MG patients; especially in patients with MuSK MG the improvement is more pronounced compared to AChR MG , 16–24. - CD14- CD19med/+ IgD- CD27hi CD38hi SLPBs from the peripheral blood of MuSK MG patients produced MuSK autoantibodies  perivascular B cells to a varying degree in addition to T cells  were CD19+ CD138+ SLPBs, the rest were CD138+.CD19- LLPCs. Production of monoclonal antibodies from these mostly IgG1 (and rarely IgG2) CD138+ SLPBs or LLPCs demonstrated AQP4 specificity and somatic mutations  patients exhibit high titers of autoantibodies with pathogenic properties, while other patients—along with healthy and disease controls—exhibit lower titers , 276. Ofpatients . Moreovepatients . Taken t+ cells in perivascular regions and interstitial spaces that could provide a local source of antibody production, as well as the presence of T cells and the absence of complement deposits and neuronal loss  are predominately of the IgG1 subclass . Patientnal loss , 241. Moma cells , 285. Im- CD14- CD16- CD20+ IgD- CD27+ memory B cells, IgG1 and IgG2 CD3- CD14- CD16- CD27+ CD38+ ASCs (could be both SLPBs or LLPCs)  antibody binding to IgLON5 results in protein internalization and an overall decrease of neuron surface IgLON5 levels. This was not reversed when IgLON5 antibodies were removed, thereby suggesting permanent destruction of the protein’s biological function  antibodies are mainly of IgG4 subclass . A studyposition , 294. Th pathway –299, or  pathway .- CD14- CD16- CD20+ CD27+ memory B cells and CD3- CD14- CD16- CD138+ ASCs (could be both SLPBs or LLPCs); V gene sequences of the LGI1-specific B cells were mutated , contactin-1, or contactin-associated protein 1 (Caspr1), rituximab has been applied to corticosteroid and IVIg-refractory cases . Albeit + CD138+ SLPBs were seen in the CSF, along with CD138+ cells (that could be SLPBs or LLPCs) perivascularly in the brain parenchyma , where IgG1 autoantibodies against the glycine receptor have been found, rituximab has been associated with some improvement and lack of relapse , 302. Inrenchyma .In dipeptidyl-peptidase-like protein-6 (DPPX) encephalitis, autoantibodies are of both the IgG1 and IgG4 subclass, but pathogenicity might be linked to IgG1 and its cross-linking functions , 196. In+ B cell depletion therapy has been applied in a plethora of non-neurological diseases associated with IgG4 autoantibodies such as pemphigus vulgaris (PV), membranous nephropathy (MN), and thrombotic thrombocytopenic purpura (TTP). In all these diseases, B cell depletion has resulted in marked clinical improvement and rapid decrease in autoantibody titers, similar to IgG4 autoantibody-mediated neurological disorders , one can conclude that in disorders where IgG4 autoantibodies are prevalent, there is a marked decrease post-rituximab administration. This is the case in MuSK MG, in CIDP with antibodies against NF155 or contactin1, and in autoimmune encephalitis with LGI1 or Caspr2 antibodies, and also extends beyond the nervous system to disorders such as PV, TTP and MN. This is a clear indication that in these diseases, autoantigen-specific ASCs are short-lived. Further, decreases in post-rituximab titers are seen in disorders where IgG1 and IgG4 autoantibodies coexist, such as DPPX and mGluR5 encephalitis. In disorders where IgG1 autoantibodies are prevalent, rituximab treatment affects a variable titer response, meaning that in some patients, titers are refractory, in some patients, titers mildly decline, and in other patients the reduction of titer or MFI is more pronounced. This variable response is noted in both AChR MG and AQP4 NMO and NMOSD, but in MOGAD MFI decrease seems to be consistent. These results point to the presence of antigen-specific LLPCs in a significant number of patients harboring IgG1 autoantibodies, but also the presence of SLPBs. It should be noted that these studies are complicated by the fact that the disorders in question are rare and therefore the N is low. Moreover, titers are not systematically recorded pre- and post-rituximab. As titers offer valuable information about treatment responses, every effort should be made to record titers more frequently.low antigen-specific cells all point to the existence and ability to generate autoantigen-specific LLPCs. In NMDAR encephalitis, the presence of GC-like structures and CD20- CD138+ ASCs in ovarian teratomas also point to the existence of and ability to produce LLPCs. Moreover, CD138+ ASCs have been observed in the CNS and the CSF of AQP4 NMOSD, NMDAR and GABA-B encephalitis patients, and in the case of AQP4 and NMDAR, the antigen specificity of the CSF ASCs was demonstrated. Unfortunately, and similarly to the case of the IgG4 LGI1 encephalitis, a clear absence of CD19/CD20 staining and negativity prevents the definitive characterization of these cells as LLPCs. In such instances, use of an additional flow cytometric marker in combination with index sorting and the use of an additional immunohistochemical stain would permit capture of this mechanistically valuable piece of information.When collectively interpreting data from experiments that more directly examine autoantigen-specific ASCs, one can conclude that in disorders with IgG4 autoantibodies, the presence of LLPCs is not definitively shown. In MuSK MG, peripheral blood antigen-specific ASCs have been shown to have a SLPB phenotype, whereas in LGI1 encephalitis, CSF antigen-specific ASCs can express CD138. However, it was not specified whether these cells retain CD19 expression. Therefore, these cells could be either SLPBs or LLPCs, which means that the presence of some LLPCs in IgG4-mediated disorders cannot be excluded. In contrast, in diseases with IgG1 autoantibodies there is more definitive evidence for the presence of LLPCs. In AChR MG, production of autoantibodies from bone marrow cells and the presence of thymic GCs and HLA-DRIn conclusion, it seems that the paradigm of the predominantly IgG4 MuSK and predominantly IgG1 AChR MG can be extrapolated to other autoimmune neurological  disorders. Moreover, in disorders with IgG1 autoantibodies, the generation of antigen-specific LLPCs seems to occur to a greater extent as compared to IgG4 disorders. It should be noted, however, that a significant degree of variability exists and that both antigen-specific LLPCs can be generated in some patients with IgG4 autoantibody-mediated disorders, and SLPBs—perhaps more frequently—can be significant producers of autoantibodies in some patients with IgG1 disorders. Variability in relation to the nature of autoantibody-producing cells could also occur at different times in the same patient. This variability underscores the need for personalized medical approaches. Exceptions aside, a generally reduced ability to establish LLPCs in IgG4 responses is strongly supported by immunological observations on the longevity of ACSs from the field of allergy  and IgG4-related disease. It could be the case that IgG4 autoantibody-mediated autoimmunity constitutes a mainly extrafollicular response, but follicular hyperplasia (in the absence of pathogenic antigen-specificity) has been observed in IgG4-RD . The ten2 once a week for 4 to 6 weeks can result in a long-lasting favorable response and is highly recommended. The same induction can be applied in IgG1 disorders. It is important to obtain a pre-rituximab baseline and a post-B cell depletion autoantibody titer in all patients at regular intervals. In the case of a persistence of high titers of either IgG1 or, less frequently, IgG4 autoantibodies, which indicates the presence of LLPCs, repeated rituximab (or other CD20-depleting drug) dosing can be applied to enforce a deeper depletion of lymph node B cells and prevent the formation of new LLPCs, while existing ones slowly wane. An alternative strategy in such refractory cases is the administration of inebilizumab (or other CD19 depleting drug), which would neutralize CD19+ CD20- ASCs that lie more towards the LLPC end of the ASC spectrum, or the application of daratumumab, an anti-CD38 agent more broadly targeting LLPCs. In the case that follicular or extrafollicular reactions within the CNS are suspected , it would be reasonable to apply an agent that, in contrast to monoclonal antibodies, can penetrate the blood-brain barrier and target B cells, such as a Bruton tyrosine kinase inhibitor. Finally, the application of new therapeutic avenues such as blockade of B-T cell interaction (CD40L) or IL-4 in polyrefractory cases warrants investigation. Ultimately and ideally, all these novel approaches should be tested in clinical trials prior to routine application.Differences between IgG4 and IgG1 autoimmune responses are not clinically trivial and can inform therapeutic decisions, especially since IgG4 autoantibody pathology responds impressively well to rituximab induction. More specifically, in IgG4 autoantibody-mediated disorders, prompt induction with rituximab 375 mg/mOur review is not without limitations. First, many of the studies we reference were biased by the use of other immunosuppressants in addition to rituximab and did not have appropriate controls groups, since it is extremely hard to perform randomized controlled trials for rare disorders. Moreover, many of the diseases are aggressive and life-threatening and justify use of more than one immunosuppressant. Second, many of the studies presented and discussed relied on peripheral blood samples, which are easily accessible but not always representative of immunopathological procedures within secondary lymphoid organs or potential tertiary lymphoid structures within the CNS and PNS.Overall, the differences between IgG1 and IgG4 autoimmune responses lead to many interesting new questions that could be explored in future investigations. Is the IgG4 autoimmune response a purely extrafollicular one? Can secondary or tertiary lymphoid structures be located in IgG4 autoantibody-mediated disorders? What are the different features of IgG1 and IgG4 response in the human lymph node? Does the memory cell compartment differ in IgG1 and IgG4 disorders? Is chronic antigenic stimulation necessary for emergence of autoimmunity of the IgG4 type and if yes, is it amenable to tolerization strategies? In IgG1 autoimmune responses, can LLPCs survive in the brain as they do in the bone marrow, and if yes, how can one target all LLPC niches therapeutically? Answering these questions would involve a detailed investigation of IgG1 and IgG4 differential maturation pathways in either extrafollicular spaces or in GC and would improve our understanding of disease mechanisms as well as facilitate development of new therapeutic avenues. New concepts could involve drugs that target crucial cellular interactions that are perceived to be responsible for B cell differentiation and maturation—in particular the T cell and B cell interaction, always keeping in mind that it is the aberrant and not the physiological response that needs to be stopped.CZ: drafting and editing. AV: drafting and editing. PS: concept, design, drafting, and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.