Automatic segmentation of infected lesions from computed tomography (CT) of COVID-19 patients is crucial for accurate diagnosis and follow-up assessment. The remaining challenges are the obvious scale difference between different types of COVID-19 lesions and the similarity between the lesions and normal tissues. This work aims to segment lesions of different scales and lesion boundaries correctly by utilizing multiscale and multilevel features. A novel multiscale dilated convolutional network (MSDC-Net) is proposed against the scale difference of lesions and the low contrast between lesions and normal tissues in CT images. In our MSDC-Net, we propose a multiscale feature capture block (MSFCB) to effectively capture multiscale features for better segmentation of lesions at different scales. Furthermore, a multilevel feature aggregate (MLFA) module is proposed to reduce the information loss in the downsampling process. Experiments on the publicly available COVID-19 CT Segmentation dataset demonstrate that the proposed MSDC-Net is superior to other existing methods in segmenting lesion boundaries and large, medium, and small lesions, and achieves the best results in Dice similarity coefficient, sensitivity and mean intersection-over-union (mIoU) scores of 82.4%, 81.1% and 78.2%, respectively. Compared with other methods, the proposed model has an average improvement of 10.6% and 11.8% on Dice and mIoU. Compared with the existing methods, our network achieves more accurate segmentation of lesions at various scales and lesion boundaries, which will facilitate further clinical analysis. In the future, we consider integrating the automatic detection and segmentation of COVID-19, and conduct research on the automatic diagnosis system of COVID-19. According to statistics from the World Health Organization (updated 23 February 2021), there have been 110.7 million global cumulative cases and more than 2.4 million deaths since the start of the pandemic2. Rapid screening of suspected patients plays a crucial role in preventing and controlling this global pandemic3. Reverse transcription-polymerase chain reaction (RT-PCR) is currently considered the gold standard for diagnosing COVID-19. However, with the rapid spread of the virus, RT-PCR testing faces a massive shortage of test kits and high false negative rates4. Computed tomography (CT) imaging can provide quantitative measurement of disease progression and has become an essential supplementary tool for RT-PCR tests to screen suspected patients and diagnose diseases5.In early 2020, coronavirus disease 2019 (COVID-19) broke out and quickly became a global epidemic, causing infections, deaths, and economic losses on a massive scale6. In recent years, deep convolutional neural networks (DCNNs) have become an important tool to assist radiologists in diagnosis8. Khan et al.9 explained that various CNNs have been widely used in medical image processing problems, because CNN has hierarchical feature extraction capabilities for extracting features at different levels, such as higher, mid and low-level features. For example, Chen et al.10 used the U-net++ network to obtain the infected regions and then classified these infected regions. Wang et al.11 used a more complex 3D U-Net++ network to segment the lesion regions and then used a classifier to determine whether each region is COVID-19-alike. Hassantabar et al.12 proposed a deep neural network and a convolutional neural network to diagnose COVID-19 patients, then a segmentation method is designed for the location of COVID-19 infected tissues in lung X-ray images. Ahmadi et al.13 used the Quantum Matched-Filter Technique method to find noise of MRI images and reduce it, then integrated the deep spiking neural network with conditional random field to segment brain tumors from MRI images. Khan et al.14 proposed a CNN-based two-stage method for classification and segmentation of COVID-19 infected areas, in which a CoV-CTNet was proposed to classify COVID-19 samples, and a segmentation model was provided to segment and analyze the infectious regions. Considering that high-quality labeled data and clean labels are usually difficult to acquire, Fan et al.15 proposed a segmentation network that utilizes the attention mechanism to help the network identify infected regions. Wang et al.16 proposed a novel framework introducing a noise-robust Dice loss function to learn from noisy labels to segment the infected regions from CT images. Zheng et al.17 proposed a weakly supervised deep learning framework using 3D CT volumes to detect COVID-19. Ahmadi et al.18 used robust principal component analysis to find brain tumor location and separate them from MRI images, then used the resulting images as ground truth images of convolutional neural network to segment brain tumors. Hussain et al.19 built a large dataset of chest X-ray images of COVID-19 patients, and proposed a CNN-based method to discriminate COVID-19 patients from healthy individuals. Moreover, in the work proposed in20, the authors use machine learning algorithms to evaluate the effect of statins on the severity of COVID-19 based on clinical characteristics, and concluded that decision tree is an effective method for predicting the severity of COVID-19.In practice, segmenting lesions form CT images can provide crucial information for doctors to diagnose and quantify lung diseases. The manual segmentation of infected regions is performed by radiologists based on their experience and suffers from inter- and intra-observer variabilities. Compared with manual segmentation, deep learning methods can automatically learn more distinguishable features from the input image, avoiding human subjectivity and other factors22, which have a great influence on the segmentation accuracy23. These multiscale features will determine the accuracy of pixel classification during the lesion segmentation. In fact, the above methods do not fully consider the multiscale feature information of infected lesions. Moreover, it can also be seen from Fig. Although some methods have been proposed to segment infected lesions from CT images, the difficult problems have not been completely solved. The scale of different infected lesions varies greatly in CT images. As shown in column 4 of Fig. 26, shown in Fig. In this paper, we propose a MSDC-Net for precise segmentation of infected lesions from COVID-19 chest CT. Our motivation stems from the fact that the multiscale features contribute the network to accurately segment objects of different sizes and detailed information in low-level features promotes network to segment object boundaries accurately. Inspired by the excellent performance ofWe propose a multiscale feature capture block (MSFCB) which employs a series of dilated convolutions to capture contextual features at different scales and a MSFF module to fuse the learned features of different convolutional layers in a more effective way.A MLFA module is used to aggregate feature learned in several different blocks. Therefore, a reinforced aggregation of low- and high-level features is used to improve the accuracy of segmentation, especially for lesion boundaries.We propose a novel MSDC-Net for precise segmentation of infected lesions at different scales and lesion boundaries with a combination of MSFCB and MLFA module. Extensive experiments on COVID-19 CT segmentation dataset demonstrate the effectiveness of the proposed MSDC-Net.In this work, we focus on multiscale and multilevel feature learning. To sum up, the contributions of this paper are as threefold:The rest of the paper is organized as follows. The framework of the proposed MSDC-Net and the structure of various modules are presented in section “In this section, we first introduce the dilated convolution in details. We then present the architecture of our MSDC-Net and MSFCB and clarify how to use them to extract multiscale features. Finally, we provide the details of our MSFF module and MLFA module and illustrate the working process.28 that only use regular convolution for extracting features, we introduce a new MSFCB, which uses dilated convolution to gather context information of different scales. Then, we add an MSFF module at the bottleneck of the MSFCB, where the MSFF module introduce parallel inter-linking among dilated convolutions to fuse multiscale features. Finally, to better segment boundaries of lesions, we add a MLFA module at the bottleneck of the encoder–decoder structure to aggregate low- and high-level features though using inter-linking among different MSFCBs.Our proposed MSDC-Net is shown in Fig. r enlarges the kernel size to As shown in Fig. The architecture of our MSDC-Net is shown in Fig. 11 which simply use two 3 × 3 regular convolutions, we use a novel MSFCB to effectively capture multiscale features of lesions, as shown in Fig. In contrast to previous studiesxl be the output of the last layer in the block:H is defined as a batch normalization (BN), followed by a 3 × 3 convolution and a ReLU, and Compared with the existing methods, we add a MSFF module to fuse the output of all the layers in the block after the last layer. Let Different from the simple fusion method of existing methods, we propose a more effective fusion method. As shown in Fig. Several works have shown that detailed information in low-level features helps network to segment object boundaries. However, most existing methods usually use all low- and high-level features in the upsampling path. Hence, we aggregate the features of different levels in the downsampling path with a MLFA module, as shown in Fig. As shown in Fig. 29, which contains 100 axial CT images from 40 different COVID-19 patients. The resolution of images is 512 × 512. Segmentation was performed by a radiologist using three labels: dark gray, bright gray, and white. To avoid overfitting, we augment the original dataset using flips and rotations, and the resolution of images is still 512 × 512. We randomly divide the dataset into three sub-sets: training set, test set and validation set, the proportions of which are 50%, 30%, and 20% respectively.In this work, we use the publically available “COVID-19 CT Segmentation dataset”M denotes the number of samples, N denotes the number of pixels, C denotes the total number of categories, and y corresponds to the one-hot encoding of the sample label. pij is calculated by Softmax, which denotes the probability of assigning the label i to the pixel j. The network is trained by using adaptive moment estimation (Adam) to minimize the loss function. Compared with stochastic gradient descent, the Adam optimization algorithm designs independent adaptive learning rates for different parameters by calculating the first-order and second-order moments of the gradient.We adopted the categorical cross entropy loss function:k represents the number of pixel categories, pii represents the number of pixels whose actual category is i and the predicted category is also i, pij represents the number of pixels whose actual category is i but predicted category is j, and pji represents the number of pixels whose actual category is j but predicted category is i. Furthermore, TP, FP, and FN represent true positive, false positive, and false negative predictions, respectively.For a quantitative evaluation, we use the four widely adopted metrics, i.e., the mean intersection-over-union (mIoU), Dice similarity coefficient, sensitivity, and specificity.In this subsection, we conduct several experiments to evaluate the effectiveness of key components of our MSDC-Net, including the dilated convolution, MSFCB, and MLFA module.First, we trained a network containing only regular convolutions as the baseline. Then, we replace the regular convolutions of the last convolution block with dilated convolutions. Quantitative results are reported in Table In addition, the advantage of the dilated convolution is also confirmed by Fig. To further analyze the effectiveness of the dilated convolution, we conduct experiments on how many dilated convolutions can achieve the best segmentation results. In the encoder of our network, there are five blocks, as shown in Fig. When excessively dilated convolutions are used in the encoder, the overlarge receptive field of the shallow convolution will weaken its ability to capture local information. In addition, when the receptive field size of the deep convolution is larger than the size of the input image, the filter will degenerate to a 1 × 1 convolution, leading to a decrease in the network performance. In the following experiments, we will use the network in which dilated convolution is utilized within the last two blocks unless stated otherwise.To explore the contribution of the proposed MSFCB and MLFA module, we train a network that only uses the original two 3 × 3 convolutions and skip connections as the baseline. Then, we sequentially add the MSFCB, MLFA module, and their combination for joint learning. The experimental results are shown in Table 30 achieves a significant increase in Dice and mIoU values but has a lower specificity score than U-Net24 and SegNet28, which indicates that the methods proposed by28 are more conservative. In Table We compare the proposed MSDC-Net with four state-of-the-art networks of semantic segmentation. Quantitative comparison results of these networks are shown in Table Moreover, quantitative performances on multi-class lesion segmentation, including separate ground-glass, consolidation and pleural effusion region, are summarized in Table The segmentation results of our MSDC-Net and other methods, shown in Fig. 27, the encoder–decoder structure of our method could recover the detailed information during the upsampling process, where the mIoU and Dice scores increased from 62.5% and 67.9% to 78.2% and 82.4%, respectively. Although U-Net24 and SegNet28 are also based on encoder–decoder structures, MSFCB helps to extract and analyze lesion features of different scales in CT images. Compared with U-Net and SegNet, the mIoU of the proposed model is increased by 11.1% and 12.6%, respectively. Furthermore, unlike Dilated-1030, we use a combination of regular convolution and dilated convolution to reduce the computational complexity and add two modules to segment the lesions of different scales more accurately. It is worth noting that compared with other networks, the proposed MSDC-Net has much faster inference speed (0.59 times the average inference time) while providing a great performance improvement . For example, the proposed MSDC-Net provides the best achievable Dice score (82.4%) and mIoU (78.2%) while consisting of 0.63 × parameters of Dilated-10. The inference time is reduced by 0.093 s compared with FCN. In the case of obtaining the highest performance, the significant increase in inference speed is mainly achieved by frequently adding cross-layer and cross-block connection operations in the network. Therefore, this scheme has a greater advantage over other networks in terms of inference speed.In summary, several key components are proposed to enable our network to achieve significant improvements in lesions segmentation of different scales and lesion boundaries. Compared with only recognizing COVID-19 in CT images, lesion segmentation can quantify the number of lesions, lesion volume changes, and lesion density changes, allowing radiologists to quickly understand changes in lesions, and greatly improve the efficiency of radiologists in analyzing changes in the patients' condition. Compared with FCNOne of the limitations in this work is that our MSDC-Net focuses on lesion segmentation of COVID-19. Although the accurate segmentation of infected lesions is critical to making treatment decisions, it is often necessary to identify COVID-19 patients before this. Therefore, in the future, we will study a computer-aided diagnosis system that consists of the following three stages: (a) automatic detection of COVID-19 lesions, (b) segmentation of lesions, and (c) quantitative analysis of lesions.This paper proposed a MSDC-Net for precise segmentation of infected lesions from CT images. The goal of this paper is to provide an effective and economical tool for faster infection analysis to greatly reduce the spread and massive death toll of COVID-19 through mass-screening and quickly grasp the changes of lesions by quantifying the number, volume and density of lesions. The significant scale difference between different types of COVID-19 lesions and the similarity between the lesions and normal tissues make it different to accurately segment infected lesions. Therefore, we proposed an MSFCB with a series of dilated convolutions to gathering more multiscale context information and introduced an MLFA module for the effective integration of captured multiscale features. Moreover, a MLFA module is used to aggregate features of different levels, which not only effectively replenish context information loss in the repeated downsampling operations but also substantially reduce the semantic gaps between subsequent encoder–decoder. Extensive experiments have been conducted on COVID-19 CT Segmentation dataset analyze the effectiveness of the proposed key modules. The proposed MSDC-Net with several key modules overcomes the limitations of traditional methods that achieved a significant improvement of performance. The results demonstrate that MSFCB can obtain multiscale features of lesions and improve the segmentation accuracy of lesions at different scales, while MLFA module can reduce the information loss in the downsampling process and provide more spatially detailed information when upsampling. Quantitative comparison results showed that our MSDC-Net achieved the best results in the Dice, sensitivity and mIoU and increased the Dice and mIoU values by 10.6% and 11.8%, respectively, on average when compared with the other methods. Qualitative comparison results showed that our MSDC-Net is superior to most existing methods in the segmentation of lesion at various sizes and lesion boundaries. Moreover, it is found that the proposed network is not only effective in COVID lesion segmentation, but also provides a new method and idea for accurately segmenting objects of different sizes at the same time.


Cytomegalovirus (CMV) infection and graft versus host disease (GVHD) both contribute to increased morbidity and mortality following allogeneic hematopoietic cell transplantation . Since the development of GVHD can increase a patient’s risk of developing CMV infection post-allo-HCT, the aim of our retrospective study was to assess the effectiveness of letermovir prophylaxis in preventing CMV infection in these patients at high risk for CMV reactivation. Letermovir is an antiviral approved for the prevention of CMV infection. This study demonstrated that patients with GVHD had significantly fewer CMV infections when they received letermovir prophylaxis compared to patients who did not receive letermovir.n = 119) and who developed aGVHD compared to a control group (n = 143) who did not receive letermovir. Among aGVHD patients, letermovir prophylaxis decreased CS-CMVi in patients with aGVHD , reduced non-relapsed mortality (p = 0.04) and improved overall survival (p = 0.04). This data suggests that letermovir prophylaxis improves outcomes by preventing CS-CMVi in patients with aGVHD.Cytomegalovirus (CMV) is the most clinically significant infection after allogeneic hematopoietic-cell transplantation  and is associated with increased mortality. The risk for CMV reactivation increases with graft versus host disease (GVHD). GVHD contributes to significant morbidity and mortality and is treated with immunosuppressive therapies that can further increase CMV infection risk. Prophylaxis with letermovir, an oral antiviral approved to prevent CMV, has been shown to decrease the incidence of CMV infection post-allo-HCT in patients at high risk of CMV reactivation, but there is a lack of data confirming this benefit in patients with GVHD. In this single-center, retrospective study, we assessed the incidence of clinically significant CMV infection (CS-CMVi) in allo-HCT patients who received letermovir prophylaxis ( Primary infection or reactivation of CMV occurs with highest incidence in CMV-seropositive patients undergoing allogeneic hematopoietic cell transplantation , especially if they received stem cells from CMV-seronegative donors . PatientLetermovir is an oral antiviral agent that inhibits the CMV-terminase complex to prevent CMV replication. Letermovir’s novel mechanism has no cross-resistance with other antiviral agents and has a favorable side effect profile, which makes it ideal for use in the prophylactic setting ,14. In cGraft versus host disease is a serious but common complication associated with allo-HCT as a result of donor T-cell mediated stem cells that attack immunocompromised host tissues. Treatment of acute GVHD involves high dose corticosteroids, which suppress the immune system and put patients at even greater risk of infectious complications . There iThis was a single-center retrospective cohort study of allo-HCT patients comparing the use of letermovir prophylaxis to a historical control group. Patients were included in this study if they were at least 18 years of age or older, CMV seropositive, and received an allo-HCT at The Ohio State University Comprehensive Cancer Center–James Cancer Hospital between 1 June 2016, through 30 June 2020. Letermovir use was incorporated into institutional infection prophylaxis guidelines at The Ohio State University for allo-HCT patients in July 2018. Therefore, patients who received allo-HCT from July 2018 through June 2020 comprised the letermovir group, while patients who received allo-HCT from June 2016 to July 2018 were included in the control group. This time frame encompasses a significant number of patients who received letermovir as standard of care and a comparable number in the control group who received an allo-HCT before letermovir was approved and widely used. Patients were followed through day +200 post-transplant.The primary objective of this study was to determine the incidence of CS-CMVi within the first 200 days post-allo-HCT in patients who developed aGVHD grade ≥ 2. CS-CMVi was defined as CMV disease or CMV viremia leading to preemptive treatment with ganciclovir, valganciclovir, or foscarnet, as described previously . SecondaPatient demographic and disease characteristics were summarized using descriptive statistics and compared using Wilcoxon rank sum test for continuous data and Pearson’s chi-squared test or Fisher’s exact test for categorical data, respectively. Time to CS-CMVi was calculated from the date of transplant to the initiation of preemptive treatment, and time to CMV viremia was calculated from the date of transplant to the first date of detectible CMV DNA. The cumulative incidence of CMV infection was estimated and compared using Gray’s test accounting for the competing risk of early death without CMV. The proportional sub-distribution hazards models were used to evaluate the associations between the use of letermovir and the risk of CMV adjusting for potential confounding factors. Non-relapse mortality (NRM) was defined as the time from the date of transplant to the date of death due to reasons other than relapse, treating relapse as a competing risk. NRM was analyzed similarly as described above for CMV outcome. Overall survival (OS) was calculated from the date of transplant to the date of death, censoring those alive at the date of last contact. The OS rate was estimated using the Kaplan–Meier method and Cox proportional hazard models were used to evaluate the associations between the use of letermovir and the risk of death. Letermovir was treated as a time-dependent covariate in all the models to account for letermovir’s impact on the risk of CMV only during the time period where patients were actually taking the drug. Stata 14 was used for the analyses and all the tests were two-sided with significance level at 0.05.p = 0.01), had more males (p = 0.048), had more ATG use or T-cell depleted grafts (p = 0.004), and less post-transplant cyclophosphamide (p = 0.003).Between 1 June 2016 and 30 June 2020, a total of 262 allo-HCT events were assessed with 119 events including patients who received letermovir prophylaxis and 143 events as part of the control group. Four of the included patients received two allo-HCTs within the study time frame, and in these patients, each transplant event was assessed separately. During the first allo-HCT for these four patients, letermovir was used in one allo-HCT event, and during the second allo-HCT, letermovir was used in three allo-HCTs. The two groups were similar with regard to diagnosis, donor class, stem cell source, donor positive CMV serostatus, conditioning regimen, and development of aGVHD grade ≥ 2 . There wp < 0.001) within 200 days post-allo-HCT, adjusting for age, gender, GVHD prophylaxis and use of ATG or T-cell depleted graft (p < 0.001)  in the letermovir group and 78 patients (54.5%) in the control group. Of the aGVHD patients, 26 (37.7%) in the letermovir group developed CS-CMVi compared to 71 patients (91%) in the control group. Among all transplant events, treatment for CS-CMVi was initiated in 110 allo-HCTs, which included 81 of 143 (56.6%) who did not receive letermovir compared to 29 of 119 (24.4%) who received letermovir . The inced graft a. A simi< 0.001) c. No oth< 0.001) b,d. InciIn a multivariable analysis among all patients, letermovir reduced the risk of developing CS-CMVi, while the development of aGVHD and use of post-transplant cyclophosphamide for GVHD prophylaxis increased the risk of CS-CMVi d.p < 0.001 and HR 2.05 [95% CI 1.35–3.13], p = 0.001, respectively) and increased risk of NRM .Non-relapsed mortality occurred in 8.4% of patients in the letermovir group compared to 9.7% of patients who did not receive letermovir a. OveralThe median duration of letermovir therapy was 95 days in all patients who received letermovir and 94 days in patients with acute GVHD grade ≥ 2. Nine patients were continued on letermovir indefinitely for recurrent CMV viremia. The most common reason for discontinuation of letermovir was due to completion of therapy at day +100 post-transplant.This single-center retrospective study showed that letermovir prophylaxis significantly reduced CS-CMVi in allo-HCT patients with aGVHD. Among patients with aGVHD, letermovir prophylaxis was the only variable that reduced CS-CMVi in a competing risk model. There was also significantly less CS-CMVi and CMV viremia in all patients who received letermovir prophylaxis. In addition, letermovir prophylaxis was also associated with significantly less risk of NRM as well as improved OS.Current published literature has shown similar benefits with the use of letermovir for prevention of CS-CMVi, but our study adds additional analysis of patients who developed aGVHD post-transplant. The phase 3 trial of letermovir prophylaxis by Marty and colleagues included 373 patients who received letermovir and 192 patients who received placebo and found that 37.5% of patients who received letermovir developed CS-CMVi compared to 60.6% of patients who did not receive letermovir . AndersoMost of the previously published studies did not report the outcomes of GVHD patients, and some only included a small number of these patients ,11,14. ALetermovir is approved for use up to day +100. Patients in our study were assessed over an extended time frame through day +200 post-transplant. This was intentional to capture the period of time after letermovir is typically discontinued in order to examine if the duration of letermovir was affected by CMV infection and/or the need for GVHD treatment. This study did not find any difference in the letermovir duration between patients who developed GVHD and those who did not. There were patients who received extended durations of letermovir with a range of up to 531 days of therapy. These extended durations occurred most commonly due to recurrent CMV viremia. The most appropriate duration of letermovir for patients who develop aGVHD is not well defined. It is possible that there would be benefit in continuing letermovir or considering reinitiation of letermovir while treating aGVHD. Further prospective trials are warranted to determine the extent of benefit of letermovir for CMV prophylaxis in patients who develop GVHD. We also showed that patients who received letermovir had improved OS and NRM. The improvement in OS is likely driven by the improvement in NRM. This is to be expected given that letermovir is an intervention that decreases infectious mortality.The rates of aGVHD grade ≥ 2 reported in this study in both the letermovir group and control group  are relatively high compared to averages previously published in the literature. The majority of our patient population (78%) received matched unrelated, mismatched unrelated, or haploidentical transplants, which are known to have a higher risk of GVHD compared to a matched related donor transplant ,21. PeriThere are several limitations worth considering in this study. This was a retrospective chart review conducted at a single site, which inherently reduces generalizability and can be influenced by confounding factors. However, with minimal exclusion criteria, we were able to include a large sample size of patients and achieved results similar to multicenter trials, which suggests greater generalizability. There was an imbalance in gender between the two groups, which is unlikely to have impacted our results as there is no data to suggest a difference in outcomes, development of GVHD, or incidence of CS-CMVi based on gender. The median age of the control group was slightly older than the letermovir group , which could have contributed to the lower mortality seen in the letermovir group as older patients have been shown to have a higher risk of mortality post-transplant. However, published literature has reported improved mortality rates with the use of letermovir regardless of age, which is consistent with our data and suggests that the age imbalance in our two groups is not clinically meaningful .With inclusion of a historical control group, this group could have been influenced by changes in clinical practice over time. Changes in clinical practice during this study period included the decreased use of ATG and T-cell depleted graft and increased use of post-transplant cyclophosphamide for GVHD prophylaxis. Post-transplant cyclophosphamide has been shown to increase the incidence of CMV infection in CMV-seropositive recipients and was more commonly used in our letermovir group ,24,25. ALetermovir prophylaxis was effective at reducing the incidence of CS-CMVi among patients with aGVHD who are at an increased risk for CMV reactivation. Similar to other studies, letermovir prophylaxis also significantly reduced the incidence of CS-CMVi in all patients, the incidence of CMV viremia, and improved non-relapsed mortality and overall survival. This data suggests that letermovir prophylaxis should be considered in patients with aGVHD for the prevention of CMV infection.


Caregivers experience social, physical and psychological burdens in caring for people with dementia. A study was conducted to assess the efficacy of a multimodal comprehensive care methodology training programme for the family caregivers of people with dementia.This research was an intervention trial with a quasi-experimental design. A total of 148 family caregivers of people with dementia participated in a multimodal comprehensive care methodology training programme for 6 hours (three times for 2 hours) in 3 months, which was followed by weekly delivery of information via postcard. The care burden of the caregivers was evaluated by the Japanese short version of the Zarit Burden Interview (J-ZBI) before the training, 1 month post-training and 3 months post-training (primary outcome). Each caregiver assessed the symptoms of the people with dementia for whom they provided care with the Behavioral Pathology in Alzheimer’s Disease (Behave-AD) (secondary outcome).P < 0.001). The mean care burden scores before, 1 month after, and 3 months after the intervention were 13.3, 10.9 and 10.6, respectively. The mean Behave-AD score of 101 people with dementia (68%) 3 months post-training was lower than that at pre-training, but the difference was not statistically significant .A total of 117 family caregivers (79%) were assessed 3 months after training. Over the course of the programme, the care burden significantly decreased from pre-training to 3 months post-training , Registered 4 February 2021 – Retrospectively registered Dementia is a major health problem that causes physical and mental burdens on patients and caregivers. Informal caregivers, primarily spouses and children, play a central role in the care and health preservation of people with dementia who live at home. Although cognitive deficits are the clinical indication of dementia, behavioural and psychological symptoms of dementia (BPSDs) are almost ubiquitous and can dominate disease presentation .Managing BPSDs is one of the most challenging aspects of care, causing caregiver burden and upset . CaregivA previous systematic review by Feast et al.  revealedPre-preliminaries), 2) Preparation (Preliminaries), 3) Integration of communication (Sensory circle), 4) Emotional consolidation , and 5) Next appointment (Appointment). The goal of Notification (Stage 1) is to announce the presence of the caregiver, avoid surprise approaches and respect the patient’s privacy and autonomy. Preparation (Stage 2) represents the initial establishment of a relationship through the relationship pillars , and it allows the caregiver to obtain consent for the relationship from the person receiving the care. Integration of communication (Stage 3) includes the provision of care with a consistent positive emotional environment between the caregiver and the patient. At the end of the care, Emotional consolidation (Stage 4) is a stage of cognitive and mental stimulation that leaves a positive impression of the relationship and care in the emotional memory of the person receiving it, which facilitates consent to the relationship and the acceptance of future care. Next appointment (Stage 5) is the final moment of the relationship, in which commitment to future care is affirmed because the emotional memory is functioning even if patient has advanced dementia. Goodbyes are said during this stage, and a new meeting is scheduled, which prevents a feeling of abandonment [The French care methodology of Gineste-Marescotti, called Humanitude™, was performed extensively in several settings, including nursing homes and hospitals, in the last 40 years , 15. Ginndonment .The multimodal comprehensive care methodology training programme includes skills that may be used at home. The programme includes lectures, demonstrations and role-playing workshops to teach participants to use this methodology for the care of people with dementia at home. After each training session, participants received weekly postcards with tips of daily care based on the methodology for 3 months. The 12 postcards included tips with illustrations that consisted of 2 for sequence of care, 6 for general communication skills and 4 for communication skills depending on pathophysiology of dementia.The present study determined whether the multimodal comprehensive care methodology training programme would reduce family caregiver burden in caring for people with dementia. We also investigated BPSDs in people with dementia.An intervention trial with a quasi-experimental design was performed. This study was designed as one of the geriatric friendly city projects in Fukuoka city. Participants were consecutively recruited via a public relations magazine of Fukuoka city, which is the 5th largest city of Japan, in November 2016. Participants were non-randomly sampled. Inclusion criteria included: 1) primary family caregiver for a patient who was clinically diagnosed with dementia; 2) residing with the homebound person with dementia; 3) caring for a person with dementia who was over 65 years of age; and 4) having no experience with multimodal comprehensive care methodology training prior to this research. The family caregivers and people with dementia were mailed an information leaflet and informed consent, which they were asked to fill out and return. If people with dementia were unable to provide informed consent, proxy consent from the family caregiver was obtained. A total of 148 participants were recruited. A monthly 2-h training programme was held three times in a conference room of ACROS Fukuoka, which is a public complex in Fukuoka city, from December 2016 to Feb 2017.Participants who consented to the study were sent pre-training survey forms as the baseline assessment. Participants completed the Japanese short version of the Zarit Burden Interview (J-ZBI) to assess caregiver burden from a few weeks before the training to the first day of the training , 17. TheA monthly 2-h multimodal comprehensive care methodology training programme was provided 3 times. The training programme was delivered to approximately 20 family caregivers at a time. Three certified instructors, i.e., two nurses and a physiotherapist, who had 10 weeks of training on teaching Humanitude™, performed the training to participants.One month and 3 months after the training, post-training surveys were performed to obtain the post-intervention J-ZBI scores of the participants and the post-intervention Behave-AD scores of the people with dementia Fig. . The samParticipants provided pre-training data directly to researchers on the first day of training, including the pre-intervention J-ZBI scores of the participants, the pre-intervention Behave-AD of the person with dementia and the characteristics data of family caregivers and the people with dementia. One month and 3 months after the training, participants sent post-training data to researchers, including the post-intervention J-ZBI scores and post-intervention Behave-AD scores via mail.The ZBI is one of the most widely used measures of caregiver burden and assesses the impact of caregiving on caregivers, including physical, mental, social and economic aspects . In thisThe Behave-AD is the most widely used instrument for the evaluation of dementia-related behavioural changes based on informant interviews . The queThe primary outcome was caregiver burden as measured using the J-ZBI scores from before the training (pre-training) to 3 months later (post-training). The secondary outcome was behavioural changes in people with dementia as measured using the Behave-AD scores between pre-training and post-training.P < 0.05.These analyses were performed using R statistical software (version 3.5.3). The characteristics data of family caregivers and people with dementia were analysed using descriptive statistics. Analytical statistics were used to address the primary and secondary outcomes. The normality of all data was verified by the Shapiro-Wilk test. A Wilcoxon signed rank test was used to test for significant differences between the pre-training and post-training J-ZBI scores and Behave-AD scores. The paired-samples t-test was used to test for significant differences in the categories of the J-ZBI scores. The baseline characteristics of the people with dementia were compared using the Fisher’s exact test for categorical variables and the Mann-Whitney U test for continuous variables. Statistical significance was defined when A total of 148 family caregivers were enrolled in the study to complete the multimodal comprehensive care methodology training programme. The family caregivers were an average age of 59 years (SD = 11.7). Most family caregivers were female (81%). Seventy-five caregivers (64%) were children of the people with dementia. Twenty-three caregivers (20%) were a spouse of the person with dementia.Among the 148 participants, the post-training response rate was 131 (89%). In the analysis of the J-ZBI scores, 14 participants were excluded due to missing data. A total of 117 (79%) patients were assessed after these adjustments.p < 0.05) from pre-training to post-training. Decreases in scores from pre- to post-training were observed among the participants regardless of age, gender, and spouse-child relationship. In particular, there were significant reductions in the care burden among participants younger than 65 years, participants older than 65 years, female participants, and children of people with dementia.The distribution of the participants and the results of the pre-training and the post-training J-ZBI scores are shown in Table n = 42, 41.6%). The people’s support and care need levels were collected. The public long-term care insurance system in Japan classifies frail older adults according to seven levels  using a nationally standardized and validated algorithm. The level is determined according to older adults’ physical and mental care needs [Among the 148 people with dementia, the post-training response rate was 82% (122 patients). The characteristics of the 148 patients are described in Table re needs . In Japare needs . Forty-sp < 0.05) from pre-training to post-training. There were significant improvements in dementia-related behavioural changes among people who needed high levels of care and people who had taken sleep medications or antipsychotic medications.The results of the pre-training and post-training Behave-AD scores are shown in Table The findings of this study show that multimodal comprehensive care communication training for family caregivers decreased the care burden of caring for people with dementia. There were a greater number of communication training studies that examined care burden, psychological distress and challenging behaviour in samples of family caregivers. However, few RCT studies demonstrated a reduction in family caregiver burden , 23. PreOur findings are similar to two previous communication training interventions that were evaluated using RCTs that were particularly effective in reducing caregiver burden , 23. TheOur study revealed that there was a reduction in the care burden among family caregivers regardless of age, gender, and spouse-child relationship. A majority of studies on the gender of caregivers have reported a higher burden among females . There iThis study documented post-training diminution of behavioural and psychological symptoms of people with dementia as well as a reduction of the family caregiver burden. However, to the best of our knowledge, no communication training studies resulted in statistically significant changes in the behavioural and psychological symptoms of people with dementia and the family care burden. Our study showed that the post-training Behave-AD scores of people with dementia who needed high levels of care were lower than their pre-training scores. Considering that behavioural problems and psychological symptoms were found to be the primary factor associated with caregiver burden for caregivers of people with dementia , the preSeveral limitations should be discussed. First, this study was an intervention trial with a quasi-experimental design, not a randomized controlled trial. Therefore, it is possible that confounding factors influenced the association between the training and the J-ZBI and Behave-AD scores. Further randomized controlled studies with larger sample sizes are required. Second, the sample size of this study was small. Third, the outcome assessment time frame in this study might be a weakness with respect to the assessment of care burden. Our study showed that the Behave-AD scores 3 months after training were slightly higher than those 1 month after training. While family caregiver burden may have been significantly reduced immediately after training, the study provides limited insights about the long-term efficacy of the training.This study was conducted to assess the effectiveness of a multimodal comprehensive care methodology training to reduce the care burden of family caregivers. The multimodal comprehensive care methodology training was associated with a reduction of the care burden of family caregivers. Because this was a single-arm, pre-post study, the findings suggest that randomized controlled trials with larger sample sizes are needed.


In March 2020, England went into its first lockdown in response to the COVID-19 pandemic. Restrictions eased temporarily, followed by second and third waves in October 2020 and January 2021. Recent data showed that the COVID-19 pandemic resulted in reduced transmission of some invasive diseases. We assess the impact of the COVID-19 pandemic on pertussis incidence and on the immunisation programme in England.We assessed trends in pertussis cases from 2012 to 2020 by age group and month. Incidence from the time that England eased its initial lockdown measures in July 2020 through to summer 2021 was calculated and the incidence rate ratios of pertussis cases from five years prior to the pandemic (July 2014 – June 2019) compared to the same time period during the pandemic (July 2020 – June 2021). Vaccine coverage estimates for pertussis containing vaccines were reviewed for the maternal and childhood programmes.A substantial decline in pertussis cases was observed from April 2020 onwards, marking the lowest number of cases in the last decade. Pertussis incidence dropped in all age groups, particularly among infants less than one year old . The incidence rate ratio was 0.02 (95% CI 0.01 to 0.02) for July 2014 to June 2019 (pre-pandemic) compared to the pandemic period of July 2020 to June 2021. None of the cases had a co-infection with SARS-CoV-2. Vaccine coverage for infants born between January to March 2020 with three doses of pertussis vaccine by 12 months of age decreased by 1.1% points compared to infants born between January to March 2019 . Prenatal pertussis coverage for the 2020 to 2021 financial year was 2.7% points lower than the year prior to the pandemic .Lockdown measures due to the COVID-19 pandemic have had a significant impact on pertussis transmission. With the easing of restrictions it is important to continue monitoring pertussis cases in England alongside coverage of the maternal and childhood immunisation programmes. Bordetella pertussis (pertussis) is a vaccine-preventable respiratory infection caused by the bacterium Bordetella pertussis. The World Health Organization (WHO) reported over 151,000 cases of pertussis in 2018, with the highest burden of disease among infants [In October 2004, a less reactogenic acellular pertussis vaccine was introduced in the United Kingdom (UK) routine infant schedule, replacing the whole cell pertussis vaccine offered at 8, 12 and 16 weeks , 4. An aIn England, a cyclical pattern of pertussis cases is observed every 3–4 years with an annual peak occurring during the months of July to September. A cyclical peak in pertussis cases was expected in the 2020 calendar year after increasing case numbers were observed during the later months of 2019. However, in March 2020, the UK went into its first COVID-19 lock-down where physical distancing measures were implemented which included school closures, the prohibition of gatherings and non-essential use public transportation and advise to work from home [Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis globally and for influenza in England [Recent data showed that after COVID-19 pandemic control measures were introduced there was reduced transmission of invasive disease due to  England –13.0) ranges from 2 to 2.5 [0 estimates based on a next generation matrix of 5.5 across five countries including England and Wales [Like pertussis, SARS-CoV-2 (the virus causing COVID-19 disease) is transmitted via respiratory droplets. SARS-CoV-2 spreads from an infected individual during either the symptomatic or pre-symptomatic phase of infection and has an incubation period of 2 to 14 days . SARS-Cond Wales  .By 30 June 2021, there were 4,853,425 people in the UK who had tested positive for SARS-CoV-2 either by laboratory-confirmed PCR test in the UK and/ or by a rapid lateral flow test, in England only .The aim of our study was to summarise and assess the impact of measures introduced to control COVID-19 on pertussis incidence and vaccine coverage in England.The UK Health Security Agency (UKHSA) is responsible for the national surveillance of vaccine-preventable infections with detailed individual follow-up of all laboratory-confirmed pertussis cases in England. Laboratory confirmation occurs at the local hospital microbiology laboratory (culture); through the UKHSA Specialist Microbiology Service laboratory network ; or at the national reference laboratory . Both serology and oral fluid testing are based on demonstrating significant anti-PT immunoglobulin G titres above a predefined threshold considered indicative of recent infection (with the caveat that results may be confounded by pertussis vaccination within the previous year). All laboratory-confirmed cases are followed up with the patients’ general practitioner (GP) to collect clinical and epidemiological data including vaccination history and, for infants born after 1 October 2012, maternal vaccination status . We asseFinally, to assess whether the number of pertussis cases was impacted by testing, the percent positivity of serology, PCR and oral fluid tests was calculated from January 2014 to June 2021 by dividing the total number of positive cases by the total number of oral fluid, PCR and serological pertussis tests reported to UKHSA each month. Culture negative samples were not included in the percent positivity analysis, but overall culture samples accounted for a small proportion of positive tests 573/26,808 (2.1%).UKHSA is responsible for monitoring and evaluating routine immunisation programmes, including vaccine coverage for all pertussis containing vaccines . We summPrenatal pertussis vaccine coverage among women who have delivered and were vaccinated prior to delivering. Data were collected from GP records from ImmForm .Completed course of DTaP/IPV/Hib/HepB  vaccine evaluated at 12 months from the COVER collection, which evaluates coverage from the Child Health Information Systems (CHIS) across England , 23.Dose 3 coverage of Hexavalent vaccine evaluated at 6 months from ImmForm , which eAll pertussis cases from July 2020 to June 2021, were linked to all SARS-CoV-2 positive test results from Pillar 1 and 2  from the official line list on 10 August 2021. Cases were linked using National Health Service (NHS) number, name of the individual and date of birth. Individuals who tested positive for SARS-CoV-2 and had onset of confirmed pertussis within a 28-day period were considered to have a co-infection. When the pertussis symptom onset date was not available, date of sample was used.The overall number of laboratory-confirmed cases of pertussis cases drastically dropped after the outbreak in 2012/2013, with an expected cyclical increase in 2016/2017 and the beginning of a peak observed again in 2019/20. Cases in 2019 remained consistently higher from March to December, than during the same months in 2018. The number of cases remained high with 311, 290 and 205 cases in January, February and March 2020, respectively, compared to 209, 158 and 237 cases in January to March 2019, consistent with the possibility of 2020 becoming a peak year ) reduction in the overall rate of pertussis cases from July 2020 to June 2021 compared to the same annual periods from 2014 to 2019  and their percent positivity dropped among all age groups from July-August 2020 onwards to the lowest positivity rates observed since before 2014. However, an increasing number of tests was observed, particularly among those under 5 and in those aged 15 years and above, from September 2020 onwards, although the percent positivity remained very low Fig. .The prenatal pertussis vaccination programme introduced initially as an emergency programme as a result of the large outbreak in 2012 has remained consistently around 68 to 71% since 2016/17 when the denominator or eligible women was amended  showed the first upward trend in coverage in the five years  subsequently had SARS-CoV-2. No individuals had both infections within 28 days, therefore there were no SARS-CoV-2 – pertussis co-infections. Furthermore, there were no individuals with pertussis succeeding a SARS-CoV-2 infection.Based on epidemiological observations of pertussis trends in England, an anticipated peak year was expected in 2020. However, with the first national COVID-19 lockdown implemented in England from 23 to 2020, the number of pertussis cases in all age groups declined sharply, from April onwards.When evaluating the incidence and incidence rate ratios from June 2020 to July 2021 compared to the same time periods in the previous five years, there was a significant drop temporally associated with the COVID-19 pandemic. Our findings concur with observations of pertussis cases in France where researchers observed an overall decrease in adjusted incidence rate ratios of 0.102 (95% CI 0.040–0.256) in outpatient laboratories and 0.216 (95% CI 0.071–0.656) in 41 paediatric hospitals from 2013 to 2020 across France due to the COVID-19 pandemic .S. pneumoniae cases in 26 countries and territories [The sustained low numbers of pertussis cases are likely to be a result of measures to prevent the spread of COVID-19. The highest rates of pertussis are typically seen in infants and children. It is possible that school and nursery closures, social distancing and mask-wearing policies caused the large reduction in cases among children and infants and subsequent infection in adults caring for their children. A similar reduction has been observed throughout the pandemic for invasive ritories . Howeverritories . It is oWe found a concurrent drop in pertussis-containing vaccination rates during the COVID-19 pandemic. A drop in prenatal pertussis vaccination among pregnant women will result in an increased number of infants susceptible to pertussis in the first 8 weeks of life, prior to being eligible for their first dose of hexavalent vaccine . CoveragIn England, pertussis incidence has dramatically declined following the introduction of measures to control the COVID-19 pandemic. These results concur with findings for other diseases in England and globally, even for diseases where viral infections are known to increase susceptibility to bacterial infections such as meningococcal disease and pneumonia , 13.England removed social distancing measures on 19 July 2021 for the first time since late March 2020 with individuals now allowed to visit in large groups indoors and no longer required to wear a mask, though people are encouraged to continue wearing them in enclosed and busy spaces. Subsequent to the COVID-19 lockdowns, an increase in pertussis cases from summer 2021 has yet to be observed, though could occur. COVID-19 has placed a noteworthy impact on the transmission of other infectious diseases. For example, a shift in the seasonal pattern of paediatric respiratory syncytial virus (RSV) has been observed in both the northern and southern hemisphere thus making it difficult to forecast disease patterns and when to expect high disease burden . RSV/perThere have been neither SARS-CoV-2/ pertussis co-infections nor cases of individuals with a SARS-CoV-2 infection followed by pertussis observed in England to date. Whilst there may be less testing for pertussis following a positive SARS-CoV-2 test result, these findings suggest that SARS-CoV-2 infection does not increase susceptibility to pertussis and vice versa. Testing for both pertussis and SARS-CoV-2 should be conducted where symptoms may be suggestive of either disease.Sustained high levels of vaccine coverage are known to reduce pertussis incidence , 34. WitThe COVID-19 pandemic has had a marked impact on pertussis transmission and led to a decline in vaccine coverage for pertussis containing vaccines. As mixing patterns increase, pertussis is likely to re-emerge; however the overlap of cough symptoms in both COVID and pertussis risks pertussis cases being undiagnosed. This highlights the importance of continued vigilance. The continuation of microbiological surveillance and monitoring vaccine coverage in the upcoming months will be critical in assessing the extent of spread of pertussis disease and detecting any new emerging trends in these challenging times.


Studies using either continuous or intermittent access cocaine self-administration procedures showed that cocaine seeking increases during abstinence (incubation of cocaine craving), and that this effect is higher after intermittent cocaine access. Other studies showed that cocaine abstinence is characterized by the emergence of stress- and anxiety-related states which were hypothesized to increase relapse vulnerability. We examined whether incubation of cocaine craving and anxiety-related behaviors are correlated and whether intermittent cocaine self-administration would potentiate these behaviors during abstinence. Male rats self-administered cocaine either continuously (6 h/day) or intermittently  for 14 days, followed by relapse tests after 1 or 21 abstinence days. A group of rats that self-administered saline served as a control. Anxiety-related behaviors were measured on the same abstinence days, using the novelty induced-hypophagia test. Finally, motivation for cocaine was measured using a progressive ratio reinforcement schedule. Lever-presses after 21 abstinence days were higher than after 1 day and this incubation effect was higher in the intermittent access group. Progressive ratio responding was also higher after intermittent cocaine access. Intermittent and continuous cocaine access did not induce anxiety-like responses in the novelty-induced hypophagia test after 1 or 21 abstinence days. Independent of the access condition, incubation of cocaine seeking was not correlated with the novelty-induced hypophagia measures. Results suggest that cocaine-induced anxiety-related states during protracted abstinence do not contribute to incubation of cocaine craving. However, this conclusion is tentative because we used a single anxiety-related measure and did not test female rats. Persistent high rate of relapse is a main characteristic of drug addiction and one of the major obstacles in drug addiction treatment . In humaIn humans, abstinence from chronic cocaine use is characterized by the emergence of negative emotional states such as anxiety, stress, agitation, and depression . These aTogether, these clinical and preclinical studies suggest that exposure to cocaine-associated cues and anxiety-related states during abstinence contribute to relapse vulnerability. However, the link between cocaine craving and anxiety-related behaviors during abstinence is unknown. Therefore, in the present study, we examined whether incubation of cocaine craving and anxiety-related states are correlated and whether intermittent cocaine self-administration would potentiate these behaviors during abstinence. In the study described below, we trained rats to self-administer cocaine for 6-h per day under either intermittent or continuous access and tested them repeatedly on abstinence days 1 and 21 for cocaine seeking and anxiety-like responses, using the novelty-induced hypophagia test . We alsoThe subjects were 29 male Sprague-Dawley rats (Charles River) weighing 300–400 g at the time of surgery. Rats were housed 2/cage for 1–3 weeks before surgery, and individually after surgery. They were maintained on a reverse 12-h light–dark cycle (lights off at 8:00 A.M.). All rats had free access to chow and water in their home cage and free access to water during the self-administration sessions. Procedures were approved by the ACUC of NIDA-IRP and were in accordance with the guide for the care and use of laboratory animals .The rats were anesthetized with isoflurane gas  and a Silastic catheter was inserted into the jugular vein. The distal end of the tubing was passed subcutaneously to the mid-scapular region and the proximal end was fixed to a modified 22-gauge cannula encased in dental cement anchored to a polypropylene mesh , 2017. Tn = 11) received saline infusions according to a yoked procedure. Control rats received a saline infusion each time the paired cocaine rat self-administered a cocaine infusion. Active and inactive lever presses by control yoked rats were recorded but had no programmed consequence. Paired cocaine rats were counterbalanced between rats self-administered cocaine continuously (n = 6) or intermittently (n = 5).Eighteen rats were trained to self-administer cocaine  in opera(-)-Cocaine-HCl (cocaine) was obtained from the NIDA Drug Supply Program via the NIDA-IRP pharmacy and dissolved in sterile saline.During this phase, the rats were housed in the animal facility and were handled two times per week.n = 29) were tested for anxiety-related behaviors on abstinence days 1 and 21 using the novelty-induced hypophagia and defensive burying tests. In a counterbalanced order, one anxiety test was performed before and the other after the relapse test. In the section “Results,” we will only present the results from the novelty-induced hypophagia test for the reason described below. Specifically, there were no significant differences between the continuous access and the saline groups on abstinence days 1 and 21 for either burying time or immobility time in the defensive burying test. For the intermittent access group, immobility time (but not burying time) was significantly higher on abstinence day 21 but not day 1 (data not shown). However, in a subsequent validation experiment  by examining the latency to initiate the intake of a familiar palatable food in a novel (anxiogenic) environment . Three dThis test evaluates response to a threatening or noxious object  by examining two behavioral outcomes, the burying behavior and the time of immobility . The defOn test days, the rats were placed in the same chambers where they previously self-administered cocaine. The relapse test was performed approximately 2 h after the first anxiety test (∼10.30 am). The relapse tests on abstinence days 1 and 21 were conducted under extinction conditions in the presence of the drug-associated cues during a single 30-min session. The session began with the illumination of the houselight and the extension of the active and inactive levers. Active lever presses resulted in contingent presentations of the tone/light cue for 3.5-s, but not cocaine; inactive lever presses had no programmed consequence. The number of non-reinforced active lever presses is the operational measure of drug seeking in incubation studies .On abstinence day 22, the rats were tested under a progressive ratio reinforcement schedule, a measure of the reinforcing effects of drug and non-drug reinforcers . During n = 8) were placed in a chamber equipped with a grid floor through which electric shock was delivered and were exposed to 10 min of footshock stress . Control rats (n = 8) were placed in the same chamber but were not exposed to footshock. Two hours after the footshock procedure, the rats underwent the defensive burying and the novelty-induced hypophagia tests in a counterbalanced order. The tests were separated by 3 h. All the sessions were videotaped, and behavioral responses were measured by an experimenter blind to the experimental conditions. During the novelty-induced hypophagia test, four rats from the intermittent footshock group did not eat the food. For the data analysis of these rats, we assigned the latency to eat value of 15 min (the maximum test duration).To validate that the defensive burying and novelty-induced hypophagia tests induce anxiety-related behaviors under our experimental conditions, we performed a control experiment in which the rats were exposed to aversive uncontrollable and unpredictable footshock before being tested in the two tests. The rats (p < 0.05) with Bonferroni post hoc test. Detailed statistical analyses are described in the section “Results.” Our multifactorial ANOVAs yielded multiple main and interaction effects. Thus, we only report significant effects that are critical for data interpretation . We followed up on significant main and interaction effects  and session  but no significant interaction (p > 0.05). The analysis of daily infusion rate (number of infusions per min) showed significant effects of group  and session  but no significant interaction (p > 0.05).During the acquisition phase in sessions 1–7 (2-h/day continuous access), there were no differences in the number of infusions and the frequency of infusions (infusions/min) between the rats that were assigned to continuous and intermittent cocaine self-administration . The staF1,16 = 16.39, p = 0.0009), abstinence day , lever , and abstinence day × lever interaction . Post hoc analyses of number of active lever presses showed significant differences between abstinence day 1 and day 21 for both groups  and significant difference between the intermittent vs. continuous access groups on both abstinence day 1 and day 21 (p < 0.05), but no significant differences between active and inactive lever presses on abstinence day 1 for both groups and no difference between abstinence day 1 and day 21 for inactive lever presses for both groups .On abstinence days 1 and 21, we measured non-reinforced lever presses (cocaine seeking) in the presence of cues and context associated with cocaine self-administration during the training phase. Independent of the abstinence day, cocaine seeking was higher after intermittent access than after continuous access cocaine self-administration . In addit15 = 2.5, p = 0.03; On abstinence day 22, we tested the rats for their motivation to self-administer cocaine using a progressive ratio reinforcement schedule. The number of infusions earned were higher after intermittent access than after continuous access  but no significant effects of group or interactions group and abstinence day or test order .We assessed anxiety-related behavior in the novelty-induced hypophagia test using two established measures: amount of food consumed and latency to initiate feeding . Intermir values and their p values in r values and their p values in We further examined the relationship between anxiety-related behaviors and incubation of cocaine craving. First, we correlated the food consumed or the latency to start eating with cocaine seeking at day 1 or day 21 of abstinence. The results of this analysis for the intermittent and continuous access group are presented in In summary, we found that continuous and intermittent cocaine self-administration did not induce anxiety-related states in the novelty-induced hypophagia test during either early or late abstinence. Additionally, no correlations were observed between the anxiety-related measures and cocaine seeking or incubation of cocaine craving. These results suggest that incubation of cocaine craving is independent of the emergence of anxiety-related states during abstinence.t14 = 3.3, p = 0.005) and significantly increased latency to initiate food intake . In contrast, unexpectedly, in the defensive burying test  and had no significant effect on immobility time (p > 0.05). This result contrasts with general notion that burying behavior reflects anxiety-related states . Therefore, to verify that under experimental conditions, the two tests can detect anxiety-like states in rats, we expose the rats to intermittent footshock using parameters that induce physiological stress responses  and reinstatement of drug seeking . As expeing test , intermid states  that shoWe examined whether incubation of cocaine craving and anxiety-related behaviors are correlated and whether intermittent cocaine self-administration would potentiate these behaviors during forced abstinence. As in previous studies, we found reliable incubation of cocaine craving after both continuous and intermittent cocaine self-administration and that this incubation was higher after intermittent drug access. In addition, and in agreement with previous reports, progressive ratio responding was higher after intermittent cocaine self-administration. In contrast, we found no evidence that intermittent or continuous cocaine self-administration induced anxiety-related behaviors in the novelty-induced hypophagia test during either early or late abstinence. Finally, independent of the cocaine access condition, incubation of cocaine craving was not correlated with the novelty-induced hypophagia measures. These data should be interpreted with caution because they rely on a single anxiety-related test. However, a conclusion from our study is that the emergence of anxiety- and stress-like states during protracted abstinence does not contribute to the emergence of incubation of cocaine seeking.Both continuous and intermittent cocaine self-administration induced incubation of cocaine craving after forced abstinence. These results confirm those from many previous studies using continuous access cocaine self-administration . Our resHowever, our present and previous results contrast with those from a recent study showing similar incubation of cocaine craving after continuous vs. intermittent cocaine self-administration . The reaFinally, we found that intermittent cocaine access increased inactive lever presses during the relapse tests, the reasons for this effect, which we also observed in our previous study , are unkWe measured anxiety-related behaviors during abstinence after continuous or intermittent cocaine self-administration using the defensive burying and novelty-induced hypophagia tests. In the defensive burying test, intermittent but not continuous self-administration increased immobility time on abstinence day 21 but not day 1 without affecting burying behavior. However, these results are impossible to interpret because, unexpectedly, under our experimental conditions, intermittent footshock decreased probe burying time. Decreased burying time is the classical response to anxiolytic drugs of different drug classes in this test . These pIn the novelty-induced hypophagia test, we found no significant differences between the continuous or intermittent access groups vs. the saline control group for both the food consumed and the latency to start eating on either abstinence day 1 or 21. Our results contrast with those from previous studies reporting increased anxiety-related behaviors in the defensive burying, light-dark, elevated plus-maze, acoustic startle response, and ultrasonic vocalizations tests after non-contingent exposure to cocaine or cocaine self-administration . What maBeyond the use of different anxiety-related tests, methodological differences may account for the discrepant results between our study and previous cocaine self-administration studies. As in our study, Finally, a limitation of our study is the exclusive use of male rats. In this regard, both incubation of cocaine craving and anxiety-related behaviors were shown to be higher in female rats . TherefoWe studied the relationship between the emergence of anxiety-like states and incubation of cocaine craving after intermittent or continuous access cocaine self-administration. Our study provides no evidence that anxiety-like states contribute to incubation of cocaine craving. However, the interpretation of our negative data and their translational implications should be made with caution because we used a single anxiety-related test in male rats.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Animal Care and Use Program (ACUC) of NIDA-IRP.CN and TR carried out the experiments and performed the data analysis. CN, YS, and SI designed the study and wrote the manuscript. All authors critically reviewed the content and approved the final version before submission.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Head-to-tail cyclized bacteriocins are ribosomally synthesized antimicrobial peptides that are defined by peptide backbone cyclization involving the N- and C- terminal amino acids. Their cyclic nature and overall three-dimensional fold confer superior stability against extreme pH and temperature conditions, and protease degradation. Most of the characterized head-to-tail cyclized bacteriocins were discovered through a traditional approach that involved the screening of bacterial isolates for antimicrobial activity and subsequent isolation and characterization of the active molecule. In this study, we performed genome mining using transporter protein sequences associated with experimentally validated head-to-tail cyclized bacteriocins as driver sequences to search for novel bacteriocins. Biosynthetic gene cluster analysis was then performed to select the high probability functional gene clusters. A total of 387 producer strains that encode putative head-to-tail cyclized bacteriocins were identified. Sequence and phylogenetic analyses revealed that this class of bacteriocins is more diverse than previously thought. Furthermore, our genome mining strategy captured hits that were not identified in precursor-based bioprospecting, showcasing the utility of this approach to expanding the repertoire of head-to-tail cyclized bacteriocins. This work sets the stage for future isolation of novel head-to-tail cyclized bacteriocins to serve as possible alternatives to traditional antibiotics and potentially help address the increasing threat posed by resistant pathogens. Bacteriocins refer to a highly diverse family of bacteria-derived ribosomally synthesized antimicrobial peptides . They arA class of bacteriocins referred to as head-to-tail cyclized bacteriocins are characterized by a cyclic peptide backbone wherein an amide bond is formed between the N- to C-terminal amino acids ,7,8. MorSeveral head-to-tail cyclized bacteriocins are active against a wide range of Gram-positive bacteria while activity against Gram-negative strains was observed when the bacterial membrane integrity was initially compromised with ethylenediaminetetraacetic acid (EDTA) or with the use of higher bacteriocin concentrations ,12,13,14Genes encoding proteins that are involved in the biosynthesis of head-to-tail cyclized bacteriocins are clustered together  either in the chromosomal DNA or plasmid DNA of the producer organism . A typicBacillus pumilus B4107 was discovered through genome mining with the use of the bioinformatics tool BAGEL3 wherein the authors filtered their candidate biosynthetic gene clusters based on the presence of genes encoding a precursor peptide and a SpoIIM protein [Bacillus cereus DDD103 was identified through BLAST analysis using the precursor peptide sequences of known head-to-tail cyclized bacteriocins as search queries [With the rapid advances in DNA sequencing technologies and the development of bioinformatic tools, a plethora of genomic data can now be mined for biosynthetic gene clusters that potentially encode novel natural products, both ribosomal and non-ribosomal in origin ,25,26,27 protein ,29. More queries .To further expand the repertoire of head-to-tail cyclized bacteriocins, we adopted a new genome mining strategy to search for novel head-to-tail cyclized bacteriocins. In particular, transporter protein sequences associated with 19 reported head-to-tail cyclized bacteriocins were used as query sequences in mining for putative head-to-tail cyclized bacteriocins. The Rapid ORF Description and Evaluation Online Tool (RODEO) was then employed to investigate neighboring genes and identify high probability functional biosynthetic gene clusters . A totalThe transporter protein sequences associated with characterized head-to-tail cyclized bacteriocins were used as driver sequences to search the NCBI genome database of non-redundant protein sequences (accessed 30 June 2020) for novel head-to-tail cyclized bacteriocins. The genomic sequences of the protein hits were then analyzed to determine which transporter proteins were clustered with genes encoding a bacteriocin precursor peptide and a SpoIIM protein, since previous studies have shown that biosynthetic gene clusters of experimentally characterized head-to-tail cyclized bacteriocins contain at least these three genes  ,30,32. ICarnobacterium maltaromaticum UAL307, Staphylococcus aureus 4185, and Lactococcus garvieae DCC43, respectively  and 20 putative members. The sequences within this group are produced by 114 different strains, signifying that several members of this group are present in multiple organisms, including the characterized bacteriocin, pumilarin, which is specifically found in 43 different strains.The sequence similarity network analysis resulted in twelve groups and twelve singletons . The larStreptococcus pneumoniae that differ at either the strain or sub strain level. S. pneumoniae strains are reported to produce a major family of bacteriocins known as pneumocins, including a potential circular bacteriocin provisionally named pneumocyclicin, which was found to be present in 34% of pneumococcal genomes [S. pneumoniae are opportunistic pathogens of global renown, understanding how their bacteriocin production confers a competitive growth advantage in the nasopharyngeal environment could be greatly beneficial to medicine.Interestingly, all the previously classified as subgroup ii head-to-tail cyclized bacteriocins  are found in a single group (group 4). This suggests that head-to-tail cyclized bacteriocins are more diverse than previously thought in terms of sequence homology and properties, and may likely not be restricted to the earlier proposed two subgroups. Subgroup i head-to-tail cyclized bacteriocins are defined by their cationic character and relatively high isoelectric point values; while subgroup ii bacteriocins are distinguished by their hydrophobicity, neutral property, and lower isoelectric points ,8. This  genomes . Interes genomes . BecauseStaphylococcus and varies by species and strain, although the majority are from Staphylococcus aureus . Understanding bacteriocin synthesis may be helpful for controlling these populations in pathogenic contexts.Group 5 includes the characterized bacteriocins, thermocin 485 and circularin A, which are identical peptides. Group 5 also has 13 putative bacteriocin members. Groups 6 to 11 do not have any characterized members, and thus, further investigation of these groups via isolation and characterization is desired. Group 7 consists of 4 unique peptide sequences that are found in a total of 43 strains. Each of these peptides come from the genus s aureus . It is aGroup 12 is comprised of two unique peptide sequences, one of which is the well-characterized bacteriocin, carnocyclin A. Lastly, among the 12 singletons (which share the lowest similarity to the rest of the identified peptides) are the experimentally validated peptides, aureocyclicin 4185, leucocyclicin Q, and garvicin ML.Each group in the sequence similarity network  was analThe sequence logos for groups 1 to 8  show thaLactobacillus nodensis DSM 19682 . The sequence logos show that groups 4 and 8 lack positively charged amino acids  and may be regarded as subgroup ii bacteriocins. In addition, members of group 11 and a singleton corresponding to a bacteriocin produced by SM 19682  also disBased on the experimentally validated head-to-tail cyclized bacteriocins, the lengths of the leader peptides can range from 2 to 48 amino acid residues . The varRepresentative biosynthetic gene clusters of the putative bacteriocins are presented in Genes encoding transposases that are associated with horizontal gene transfer can be found across the different groups. Another common occurrence is the presence of a Yip1 protein, which is a membrane protein that may be involved in transport. The presence of Yip1 protein is prevalent especially in group 1. Groups 2, 3, 5, and 11 also have members with Yip1 protein, particularly the gene clusters consisting of a set of three genes that are putative accessory ABC transporter components.One of the longstanding questions about the biosynthesis of head-to-tail cyclized bacteriocins is how the leader peptide is cleaved and the core peptide is cyclized. To date, no enzyme has been identified to facilitate this transformation. It was postulated that genes encoding a peptidase/protease that catalyzes this reaction should be located elsewhere outside the gene cluster . IntriguThe evolutionary relationship of the head-to-tail cyclized bacteriocins was inferred through phylogenetic analysis. The five biggest groups identified in the sequence similarity network analysis  are showIn the phylogenetic tree, members of groups 1 to 5 are shown in blue, orange, violet, teal, and brown, respectively. The results revealed that group 1, which is the biggest group, evolved mainly from the earliest ancestors of head-to-tail cyclized bacteriocins. This group appears to be highly heterogeneous as the members are distributed into different subclades and exhibit varying degrees of hydrophobicity based on the grand average of hydropathicity index (GRAVY) data. Most of the other groups eventually evolved from a common ancestor that diverged from group 1 early in the evolution. As shown in the phylogenetic tree, the next three biggest groups belonging to subgroup i head-to-tail cyclized bacteriocins; namely groups 2, 3, and 5 form more tightly related subclades, and the hydrophobic character is more conserved among members of each group based on the GRAVY analysis. Subgroup ii head-to-tail cyclized bacteriocins, on the other hand, appear to diverge later from subgroup i members. Subgroup ii mainly include groups 4 and 8 members, which are both found to be more closely related to the singleton, leucocyclicin Q, and group 6 members. GRAVY analysis indicates that subgroup ii members appear to be consistently more hydrophobic compared to the members of subgroup i bacteriocins.The amino acid sequences of transporter proteins associated with thirteen subgroup i  and clustered together the most related proteins into groups. To generate the final sequence similarity network, different alignment scores were tested to select an appropriate clustering that grouped most hits with similar gene cluster organization together. This was obtained with a percent identity cut-off of at least 50%. Cytoscape (version 3.9) was then used to visualize the results [A list of the precursor peptides from the RODEO analysis was retrieved and a sequence similarity network was generated using the EFI Enzyme Similarity Tool ,65. This results . The lisGroups from the sequence similarity network that consist of three or more members were subjected to further sequence analysis. The precursor peptide amino acid sequences belonging to each group were aligned using Clustal Omega , and thehttps://mafft.cbrc.jp/alignment/server/) [http://iqtree.cibiv.univie.ac.at/, accessed on 17 October 2021) [http://gravy-calculator.de).Multiple sequence alignment of the putative head-to-tail cyclized precursor peptides was performed using the G-INS-i method implemented in the MAFFT web server (server/) . IQ-TREEer 2021)  was thener 2021) . HydrophIn this study, we performed a new genome mining strategy to search for novel head-to-tail cyclized bacteriocins, specifically by using transporter protein sequences associated with 19 experimentally validated head-to-tail cyclized bacteriocins as query sequences, and the use of a set of bioinformatics tools. A total of 387 strains that potentially encode putative head-to-tail cyclized bacteriocins were identified, 127 of which are unique peptide sequences. Sequence similarity network, sequence analysis, and biosynthetic gene cluster analysis revealed that head-to-tail cyclized bacteriocins are widely distributed and are more diverse than previously thought. Phylogenetic analysis showed that members of group 1 in the sequence similarity network are the most evolutionarily diverse and that the other groups arose from a common ancestor that diverged from group 1. While this work identified putative novel head-to-tail cyclized bacteriocins, it does not predict whether these peptides are easily produced in the laboratory setting and to which pathogens will they be active against. Hence, future work must be directed towards peptide isolation and characterization. It is of particular interest to characterize representative members of groups in the sequence similarity network that do not currently have an experimentally characterized member. Upon the successful production of these bacteriocins, purified peptides can then be used in bioactivity assays against a range of bacterial indicator strains to determine the sensitivity and efficacy of their antimicrobial potential. This work sets the stage for isolation and characterization of diverse head-to-tail cyclized bacteriocins, with likely varying specificities, for their subsequent applications as biopreservatives and antimicrobial agents in various applications in food, animal, and health industries.


The influence of microindentation on the electroluminescence of silicon carbide was studied in forward-biased 4H SiC p-i-n junctions. Four spectral regions at approximately 390, 420, 445 and 500 nm initially observed on virgin samples strongly depend, in regard to magnitude, on the condition of the starting die. These spectral regions may be interpreted as arising from either phonon-assisted band-to-band transitions or from defect-related transitions. The same SiC die were then subjected to mechanical damage brought about by a series of closely spaced microindentations directed approximately perpendicular to the c-axis. The spectra taken after a first set and subsequently a second set of microindentations are distinct from the initial spectra in all cases, and differences are interpreted as being due to the modification of existing defects or additional defects being generated mechanically. The influence of microindentation on the ideality factor is measured and discussed. Measured light flux with respect to a standard light source is also shown at each microindentation stage. Due to the nature of its strong covalent bonding, relatively high thermal conductivity and wide bandgap, SiC is commonly used for high-temperature, high-radiation and high-power applications. It is also known for a low formation energy difference between polytypes, resulting in more than 200 SiC polytypes with distinct stacking sequences . Among aThe recent achievement of high quality 8-inch vapor-grown SiC wafers was motivated by the electric vehicle and sustainable power generation sectors . SiC is The study of SiC p-n or p-i-n junction electroluminescence provides valuable insight into the recombination mechanisms in SiC and can help identify defects . In 4H-SFrom the literature, three additional peaks located at 420, 460 and 480 nm have been detected that provide evidence of recombination at various stacking faults ,11,12. SIn addition to experimental results, modeling by using Density Functional Theory also can help us analyze the change of radiative recombination energy resulting from stacking faults in 4H-SiC . StackinThe identification of such fluorescent defect centers allows further study of defect-induced device degradation or future applications, such as quantum computing and single photon emitters ,20. In tThree 4H-SiC junction transistors purchased from GeneSiC (GA20JT12-263)  were mounted into epoxy pucks  with connections to the base-collector p-i-n junction for convenience of holding and clamping during the surface exposure for optical and electronic testing. A smooth and optically clear die edge surface close to the active junction area was obtained after grinding into the original package by using SiC sandpaper  with grits between 800 and 1200 for the final polish. Samples 1, 2 and 3 were oriented differently during grinding and polishing. Inevitably, samples were subjected to unavoidable heat and mechanical stress due to the soldering, grinding and the polishing process. Light emitted from the exposed die surface of each sample was measured and analyzed.p-type base and n-type emitter intersect with each other periodically with electrode connection on the top of each junction and a n-type collector underneath the interdigitated structure. Samples 1 and 2 were exposed at the direction perpendicular to those interdigitations, whereas Sample 3 was exposed from the direction parallel to such a structure. Specifics of the geometry of the exposed die surface are later described in detail for each sample.Each die consists of an NPN bipolar junction transistor with interdigitated base and emitter electrodes, as shown in EL spectra were obtained by using a grating spectrometer Optometrics Corp. DMC1-03  with an avalanching photodiode Hamamatsu S12053  operated at 158V reverse bias. Data were taken at room-temperature ambient conditions, with 0.2 A current flow across the base-collector junction. Samples were then subjected to indentation by using a micro-hardness tester  to generate a first stage of microindentations to the polished sample edge and near the surface of the sample where the interdigitation is located. After repeating the measurement of spectra, each sample was subjected to a second set of microindentations , a limited plastic deformation driven by the partial dislocation motion is expected [In general, either plastic deformation along the slip direction or a brittle fracture across the cleavage plane results in microscopic defects being generated in SiC when a sufficient external stress is applied . When thexpected . Dislocaexpected . Meanwhiexpected  which asIn In addition to these observations, each as-received SiC sample may possess distinct defect types and structure at the junction interface, and this could be an additional explanation as to the distinctions in their spectra, considering they were not manufactured for LED application. Indications of pre-existing stacking fault formations have been observed as varying from sample to sample in our previous work .Evidence of this hypothesis is from Sample 3. Despite having unstable IV data, the spectrum of Sample 3 shows a dominant 420 nm peak at all microindentation stages. This 420 nm peak is independent of the polish direction. As mentioned before, it is known that stacking faults constitute a prominent defect in SiC and can be active as a quantum well structure with an ideality factor greater than 2 . An inteA camera image of all three samples is shown in Mechanically induced defects by using microindentation were introduced to three 4H-SiC samples. EL spectra were studied at each stage of the microindentation. All three samples had a broadened green emission after each indentation stage, while having distinct intrinsic spectra and intensity data. Sample 3, with a strong 420 nm peak, indicates that stacking-fault-associated recombination dominated the overall radiative recombination. Most of the observed changes to the samples can be explained by the microscopic defects generated during the microindentation process that favor or suppress specific regions of light emission based on the known literature modeling of SiC defects. The diode-ideality-factor results are consistent with a substantial increase in defect-mediated recombination after microindentation.