Functional exercise is crucial for breast cancer patients after surgery, and the use of virtual reality technology to assist patients with postoperative upper limb functional rehabilitation has gradually attracted the attention of researchers. However, the usability of the developed rehabilitation system is still unknown to a large extent. The purpose of this study was to develop a virtual reality upper limb rehabilitation system for patients after breast cancer surgery and to explore its usability.We built a multidisciplinary team based on virtual reality and human-computer interaction technology and designed and developed an upper limb function rehabilitation system for breast cancer patients after surgery. Breast cancer patients were recruited from a grade III-a general hospital in Changchun city for the experiment. We used the System Usability Scale to evaluate the system availability, the Presence Questionnaire scale to measure the immersive virtual reality scene, and the Simulator Sickness Questionnaire subjective measurement scale for simulator sickness symptoms.This upper limb rehabilitation system hardware consisted of Head-mounted Display, a control handle and notebook computers. The software consisted of rehabilitation exercises and game modules. A total of 15 patients were tested on this system, all of whom were female. The mean age was 54.73±7.78 years, and no patients were excluded from the experiment because of adverse reactions such as dizziness and vomiting. The System Usability Scale score was 90.50±5.69, the Presence Questionnaire score was 113.40±9.58, the Simulator Sickness Questionnaire-nausea score was 0.93±1.16, the Simulator Sickness Questionnaire-oculomotor score was 0.80±1.27, the Simulator Sickness Questionnaire-disorientation score was 0.80±1.27, and the Simulator Sickness Questionnaire total score was 2.53±3.40.This study fills in the blanks regarding the upper limb rehabilitation of breast cancer patients based on virtual reality technology system usability research. As the starting point of research in the future, we will improve the system’s function and design strictly randomized controlled trials, using larger samples in the promotion, to evaluate its application in breast cancer patients with upper limbs and other physiological functions and the feasibility and effects of rehabilitation. Breast cancer is the most common malignancy threatening women’s health. The latest report of the International Agency for Research on Cancer shows that in 2018, the number of new cases of breast cancer increased by approximately 2.09 million worldwide, accounting for 24.2% of all cases of female cancer [Surgery is the main treatment for breast cancer –5. PatieVR technology refers to the use of computer systems and sensor technology to generate a three-dimensional environment and to create a new pathway of man-machine communication by mobilizing users’ various senses  to enjoy a more real, immersive feeling. VR is characterized by immersion, imagination and interaction . It has At present, VR technology has been carried out in the field of rehabilitation of breast cancer patients. With the development and diversification of technology, an increasing number of studies are combining it with robots, 3D motion-sensing cameras, machine learning and other technologies to provide process data feedback on the rehabilitation time limit and connotation quality to make the rehabilitation process more accurate and achieve remarkable effects.Usability evaluation is an indispensable link in the process of medical product development and one of the key factors for the successful implementation of telemedicine . The IntThis study aims at VR technology and computer graphics technology, human-computer interaction technology, simulation technology, multimedia technology, sensor technology, network technology to develop a system for exercising upper limb function for breast cancer patients and performs usability study to provide a basis for iterative development and maintenance in the future. In this way, the efficiency and quality of the whole rehabilitation system can be improved, and an effective reference can be provided for data-driven intelligent home rehabilitation.Ten people were involved in a multidisciplinary team, including a breast surgeon (1 person), rehabilitation therapists (2 people), nurses (2 people), postgraduates (3 people) and technical engineers (2 people), on the basis of consulting and summarizing the contents of the guidelines, in accordance with scientific, innovative, practical and feasible principles. Combined with the results of previous research and literature research, the content of the upper limb rehabilitation system was preliminarily conceived, and the functional details were further determined through discussion and brainstorming.Hardware side: The hardware of the system is an HTC VIVE Pro2.0 device , and the helmet is connected to a computer . The handle controller and HMDs use Csharp programming language for secondary development, based on an 850-nm infrared optical orientation method, through the base station to capture the patient’s head and control the handle position and to track the coordinates of the upper limb activity information. The upper limb activity trajectory was calculated and compared with the preset movements of rehabilitation training in the movement database to identify the movements and send the data to the cloud server in real time.Software side: The virtual simulation was developed with the Unity3D game engine and written in C # script language. Component design is completely independent of the hardware and operating system development environment, and the application platform module is relatively independent and convenient for secondary development.https://github.com/2202jasmine1024/breast-cancerWe put the code for the experiment on the github open source site for easy access: A VR-based postoperative rehabilitation system for breast cancer was preliminarily constructed and was mainly presented in the form of web pages and C/S structures, including rehabilitation exercise modules and puzzle game modules.In the rehabilitation exercise module, the rehabilitation exercises were set by the hospital rehabilitation therapists and nurses; they covered making a fist, screw lifting of the wrist, elbow flexion and around the shoulders, touching the ear, climbing a wall, back-handing, and outreach, comprising 10 rehabilitation actions according to the situation of patients with different clinical multidisciplinary teams set in two different mode of rehabilitation  and a nurse (He Wang) from the multidisciplinary team introduced the purpose of the study, the inherent characteristics of the system, module functions and matters needing attention to the participants in the ward, the two researchers demonstrated the login and use methods and informed the participants of the tasks to be completed later. Subjects performed a basic warm-up 10 minutes before the experiment to prevent injuries during the exercise. After the experience, participants rested in the ward for 15 minutes while filling out a questionnaire provided by another researcher. Two postgraduate students (Jiaxin Li and Ze Luan) from the multidisciplinary team, guided the participants in filling out the scale, and conducted unified training for postgraduate students before the survey to make them understand the research objectives and methods During the investigation, two postgraduate students issued questionnaires and guided patients to fill in the scale by themselves with unified guidance language. If there was any doubt about the content of the scale, they explained it in consistent language. Researchers paid close attention to the whole process of the experiment to ensure the patients’ personal safety. We used a general information questionnaire to collect the patients’ age, nationality, marital status, educational level, pathological staging, pathological type, treatment and other information.The SUS is used to evaluate the usability of the system , which iThe SSQ was used to evaluate users’ symptoms of cybersickness . The SSQThe PQ was used to evaluate users’ sense of presence and immersion , which eMicrosoft Excel 2019 and SPSS 24.0 statistical software were used for data analysis. Descriptive statistical methods were used in this study was descriptive analysis: frequency and constituent ratio were used to describe the general information treatment status and other data. Median and quartile spacing were used to describe the SUS score, SSQ score, PQ score.A total of 15 patients were recruited in this study, all female, with an average age of 54.73±7.78 years. See As shown in During the trial, no adverse events were reported, and no patients withdrew for any reason.Using VR technology arm rehabilitation exercise is a new kind of method for the postoperative rehabilitation of breast cancer. When experiencing a new environment, novelty, immediacy and uniqueness can make people more excited. People can give more attention to completing a task or experience using VR devices through multisensory interactive approaches such as seeing, hearing , 41. PatThere are many rehabilitation systems based on VR and its derivative technologies to assist breast cancer patients to recover their upper limb function, quality of life and psychological state. Such as House et al.  developeIn addition, VR and its derivative technologies have been widely used in the diagnosis of complications , palliatSUS was originally designed to evaluate the usability of products and services, including hardware, software, mobile devices, websites, and applications. Due to the combination of certain elements of the rehabilitation system developed in this study, and the increasing number of studies using SUS to test medical devices and products , 52, 53,™ and good software design to build a sense of presence, which allows individuals to focus on the virtual experience. Such participation in immersive tasks is called interactive potential [The VR system uses hardware devices such as HMDs, Cave Automatic Virtual Environment otential , and theotential , 55, 56.Cybersickness is unavoidable in the use of VR. When visual response processing for user input interaction is delayed, the signals from the eyes, the vestibular system of the inner ear and proprioceptive receptors to the brain conflict with each other, and motion sickness is triggered . Halo scThe higher the SSQ score is, the more serious the cybersickness is, and a score over 20 indicates that the user has obvious discomfort , 60. AnaIn addition, some studies have found that presence scores are inversely correlated with cybersickness symptoms. The more a patient feels in a virtual world, the more likely he or she is to experience motion sickness. This study focuses on usability research, with a sample size that is too small and insufficient data collected to support the exploration of the relationship between SSQ and PQ scores. Therefore, the sample size can be increased in future studies to explore the correlation between SSQ and PQ scores.First, in terms of participant recruitment, usability study adopts a convenient sampling method with a relatively small sample size, but this is not uncommon in development iterations , and parThis study is the first step in the new field of VR-based rehabilitation for breast cancer patients to evaluate the usability and feasibility of the newly developed system, to learn from the results of the study and to provide a reference for future research and development projects in this field. The results show that the VR rehabilitation system is available, feasible and easy to learn for breast cancer patients. Through various human-computer interaction methods, such as sight, hearing and touch, participants adapted to the difficulty level according to the selection of rehabilitation mode based on the state of the affected limb and enjoyed the stimulating, diverse and interesting experience of sports games, which presented both physical and psychological cognitive challenges. The system developed in this study is not applied to commercial promotion; in contrast, it is designed to be used as a starting point in other clinical researchers’ potential, immersive VR and interactive technology to explore the potential for the treatment and rehabilitation of breast cancer postoperative rehabilitation purposes. We look forward to the contributions of other researchers in this field.


Angelica sinensis Radix (ASR) combinations are used to treat cardiovascular disorders. Astragali Radix (AR) and  This study investigates the protective effects of different compatibility proportions of AR and ASR on cardiac dysfunction in a C57BL/6 mouse model of myocardial infarction (MI). MI mice were induced by ligation of the left coronary artery and divided into six groups: sham, vehicle, 10 mg/kg/d simvastatin and combinations of AR and ASR at different ratios, including 1:1 (AR 2.5 g/kg + ASR 2.5 g/kg), 3:1 (AR 3.75 g/kg + ASR 1.25 g/kg) and 5:1 (AR 4.17 g/kg + ASR 0.83 g/kg). Both AR–ASR combinations and simvastatin were dissolved in saline solution and given daily by gavage. The left ventricle function, infarct size, heart tissue injury, apoptosis of cardiomyocytes, leukocyte infiltrates, capillary density and expression of cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, Bad, IL-1β, IL-6, VEGF, p-Akt and p-eNOS were analysed.p < 0.05, p < 0.01). The combination ratio of 3:1 exerted effect in promoting angiogensis (p < 0.05). In the combination of AR and ASR at 5:1 ratio, angiogenesis was significantly improved (p < 0.01) and the apoptosis was inhibited (p < 0.05). Different combinations of AR and ASR improve cardiac function and reduce infarct size  and tissue injury through different mechanisms. When AR was combined with ASR at ratio of 1:1, the inflammation and cardiomyocyte apoptosis were suppressed ( Our results reflect the regulation of multiple targets and links in herb pairs and provide an important basis for the use of AR and ASR combinations in the treatment of MI. Myocardial infarction (MI) and consequent heart failure are the most common types of cardiovascular disease and the leading causes of morbidity and mortality worldwide  Bge (Fabaceae) known as Huangqi in Chinese, is a major medicinal herb considered to enrich vital energy and treat stagnant blood flow. Angelica sinensis Radix (ASR), the dried radix of Angelica sinensis (Oliv.) Diels (Apiaceae) known as Danggui in Chinese, has been used as a tonic agent to promote blood circulation for several years  herbs are widely used as medicines and daily dietary supplements in Asia on the mode action of ‘multi-component, multi-target, multi-pathway’ . Interleukin 6 (IL-6) and interleukin 1 beta (IL-1β) Mouse ELISA kits were obtained from R&D Systems . Alex Fluor 594 Goat Anti-Rabbit IgG (H + L) was from Life Technologies .The reference compounds of calycosin, formononetin, astragaloside IV (AS-IV), ferulic acid and ligustilide were purchased from Chinese National Institute for Control of Pharmaceutical and Biological Products . Avertin and simvastatin were purchased from Sigma-Aldrich . The anti-CD45, anti-CD31, α-tubulin, VEGF, p-eNOS, eNOS and biotin-conjugated goat anti-rabbit IgG polyclonal antibodies were obtained from Abcam . Caspase-3, cleaved caspase-9, Bcl-2, Bax, Bad, p-Akt, Akt and GAPDH antibodies were bought from Cell Signalling Technology . The All crude herbs were purchased from the specialized market of Chinese herbal medicines  in Anguo, China on 21 March 2017 to 23 March 2017. The two herbs were identified by Dr. Li Tianxiang . Each voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of Traditional Chinese Medicine. The voucher specimen numbers of AR and ASR are no. 20170527 and no. 20170516, respectively.AR, ASR and different ratio combinations of AR–ASR  were prepared. According to different formulations, the appropriate amounts of crude herbs were weighed separately to form a combined weight of 360 g . Before decocting, the medicinal materials were soaked in pure water for 1 h. The mixture was boiled in six volumes of water (v/w) (2160 mL) for 40 min the first time and four volumes of water (1440 mL) for the next two times. All the filtrates were mixed together and condensed to a total volume of 360 mL using a rotary vacuum centrifuge. The final concentration of AR, ASR and the combinations were 1 g/mL. The concentrate was sub-packaged and stored at –4 °C.Calycosin, formononetin and AS-IV act as effective compounds in AR , 22–25 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co. Ltd.  and maintained at the Animal Center of Institute of Biomedical Engineering, Chinese Academy of Medical Sciences . All animals were kept under 22–25 °C and a 12 h light/dark cycle with standard food pellets and free access to tap water. All animal care and experimental procedures were approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (TUTCM20170628) and performed in accordance with the approved guidelines on the use of laboratory animals. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the United States National Institutes of Health  coronary artery, as described before . Mice had no ligation and they were orally administered with saline solution; (2) vehicle group (n = 15). The mice with surgery received saline solution orally; (3) simvastatin group (as a positive control drug) (n = 12). The mice were treated with 10 mg/kg/d simvastatin by gavage; 4–6. AR–ASR combination groups with different ratios  (n = 15 for each group). Before drug treatments, the combinations were diluted with saline solution into 0.5 g/mL. The mice were administered with 10 mL/kg/d plant extracts based on the doses used in clinical practice. Both the AR–ASR combinations and simvastatin were dissolved in normal saline solution and given daily by gavage starting on day 0. Drugs were given for four consecutive weeks. At 7 and 28 days after MI surgery, mice were sacrificed. Hearts were perfused with cold phosphate buffered solution (PBS) and a portion of the LV was either frozen in liquid nitrogen for biochemical analysis or fixed in 4% paraformaldehyde for histological analysis.Post-operatively, the mice were randomly divided into six groups: (1) sham group  and Vevo analysis software at days 7, 14 and 28 post-MI. The investigator was blinded to group assignment. Mice were anaesthetized with 2% isoflurane inhalation and placed in supine position. Heart rates were maintained at 400–500 beats per minute for measurements. Images were obtained from the B-mode parasternal long axis view and M-mode of the parasternal short-axis view to calculate the cardiac diastolic and systolic functions. Left-ventricular end-systolic volumes (LVESVs) and left-ventricular end-diastolic volumes (LVEDVs) were measured through parasternal long-axis scans. The left-ventricular fractional shortening (LVFS) and left-ventricular ejection fraction (LVEF) were calculated in accordance with modified American Society of Echocardiography recommended guidelines.Mice received drug once a day for 4 weeks after surgery. Hearts were harvested after the 28-day echocardiographic analysis. Heart tissue was fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and transversely sectioned into 5 µm pieces. Masson’s trichrome staining was performed to evaluate the degree of fibrosis. Fibrotic tissue was stained to blue-grey and viable myocardium to red. The fibrosis area was calculated as the ratio of the length of fibrotic area to the length of left-ventricular inner circumference with Image J software. The haematoxylin and eosin (H&E) staining was performed to detect tissue recovery after ischaemia injury, as described previously  for 10 min. After washing in PBS 3 times, blocking was done by 5% foetal bovine serum in PBS for 1 h at room temperature.To assess post-MI leukocyte infiltrates, mouse hearts were immunostained for CD45 at seven days post-MI and CD45 positive cells were quantified in the peri-infarct zone. Anti-CD45 antibody  was used for immunohistochemistry of leukocytes. Samples were incubated with primary antibody for 24 h at 4 °C. A biotin-conjugated goat anti-rabbit IgG polyclonal  was used as the secondary antibody.Primary anti-CD31 antibody  was incubated with the sections overnight at 4 °C. Negative controls were without primary antibody. Secondary antibody (Alex Fluor 594 Goat Anti-Rabbit IgG (H + L), diluted 1:200 with 1% foetal bovine serum in PBS) was then incubated with the sections for 1 h in the dark at room temperature. Finally, the sections were viewed and photographed using a Nikon TI-U fluorescence microscope .in situ cell death detection kit at 28 days after treatment with different combinations of AR and ASR. According to manufacturer’s instructions, the dewaxed and rehydrated slides were incubated with 0.1% Triton X-100 for 8 min, rinsed twice with PBS, and then incubated in TUNEL reaction mixture for 60 min at 37 °C. After washed with PBS, samples were analysed by fluorescence microscopy in the range of 520–560 nm and detected in 570–620 nm. The number of TUNEL-positive nuclei was counted at ×200 magnification, 10 fields per section blinded to the study group. TUNEL+ cell density (%) is expressed as the ratio of TUNEL-positive nuclei to the total number of nuclei.For this parameter, mice received drug once a day for 4 weeks after surgery and were sacrificed 28 days after surgery. Apoptosis of cardiomyocytes was detected by TUNEL assay which was performed on paraffin-embedded sections using the g for 10 min to pellet unresolved fragments. Supernatants were collected and the protein concentration was measured by BCA method. After diluting in sample buffer  glycerol, 2% SDS, 0.1% bromophenol blue), the protein samples were separated by 10% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Membranes were blocked with blocking buffer (5% skim milk or 5% BSA in TBS with 0.1% Tween 20) for 1 h. Membranes were then incubated with primary antibodies at 4 °C overnight. Twelve primary antibodies were used in the experiments: caspase-3 , cleaved caspase-9 , Bcl-2 , Bax , Bad , α-tubulin , VEGF , p-Akt , Akt , p-eNOS , eNOS  and GAPDH . After washing, appropriate secondary antibodies were applied to the membranes for 1 h at room temperature. The reactive bands were developed using chemiluminescence according to the manufacturer’s instruction. Images were scanned and band intensities were analysed with photoshop CS5 software .Western blotting analyses were performed to detect the cleaved caspase-3, cleaved caspase-9, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Bcl-2-associated death promoter (Bad), p-Akt, Akt, p-eNOS, eNOS, vascular endothelial growth factor (VEGF) protein expressions at day 28 after surgery. The heart tissues were homogenized in RIPA lysis buffer  with a cocktail of protease and phosphatase inhibitors using tissue homogenizers. The mixture was spun 5000×Levels of IL-1β and IL-6 in heart tissues were detected by mouse ELISA kits. Heart tissues were harvested at seven days after surgery and were frozen in liquid nitrogen. The extracted tissues were homogenized in cold PBS, containing EDTA-free protease inhibitor, phosphatase inhibitor and PMSF mixture. The concentrations of IL-1β and IL-6 in the myocardium were determined by ELISA according to the manufacturer’s instructions.t-test. The results were analysed by one-way analysis of variance (ANOVA) for comparison of each parameter among two or more independent groups. Values of p < 0.05 were considered statistically significant.Statistical analysis was performed using SPSS software . Data were expressed as mean ± standard deviation (S.D.). Differences between two groups were estimated with Student’s p < 0.01). AR–ASR mixtures of 3:1 significantly increased the LVEF at 7 and 14 days (p < 0.05 or p < 0.01). LVEF (p < 0.01).To test the therapeutic effects of different combinations of AR and ASR, a mouse model of acute MI was established via permanent ligation at the middle of the LAD coronary artery, as previously described . LVEF  and LVFS1). LVEF  were alsp < 0.05) (Infarct size is shown in  < 0.05) . These rThe border zone tissue injuries in hearts of mice are shown in p < 0.05). AR–ASR mixed at 3:1 had no significant effect on apoptosis after MI. Consistent with this, AR mixed with ASR at 1:1 ratio decreased activation of caspase-3 and caspase-9 (p < 0.05 or p < 0.01), increased Bcl-2  expression (p < 0.01) and decreased Bad (pro-apoptotic protein) expression (p < 0.01) in response to MI. AR–ASR mixture at 5:1 ratio also decreased activation of caspase-3 and caspase-9 (p < 0.01), and downregulated Bax (pro-apoptotic protein) expression significantly (p < 0.01)  . These rp < 0.05) (p < 0.05). Treatment of AR–ASR mixed at 1:1 significantly reduced the expression levels of IL-1β and IL-6 in the infarcted myocardium compared with those in the vehicle group (p < 0.05)  . Moreove < 0.05) . These r+ vessels. Remarkably, the overall capillary density at the border zone of ischaemic myocardium 4 weeks after MI was significantly higher in the AR–ASR mixtures of 3:1 and 5:1-treated mice compared with the saline-treated animals (p < 0.05 or p < 0.01)  . These fp < 0.01) and increased Akt phosphorylation (p < 0.05). AR mixed with ASR at 5:1 significantly increased Akt and eNOS phosphorylation (p < 0.05). The above results indicated that the acceleration of improved angiogenesis in AR–ASR combinations at 3:1 and 5:1-treated hindlimb ischaemic mice may be explained, at least in part, by the activation of pro-angiogenic factors.To elucidate the molecular mechanism, western blot analyses were performed to detect the VEGF, p-Akt and p-eNOS protein expressions at day 28 after surgery. VEGF is an important cytokine involved in creating new blood vessels in hearts of patients with ischaemic heart disease  deficiency and showed promising effects in improving biochemical and histological changes of heart failure  demonstrated that AR-derived AS-IV, calycosin, formononetin, ASR-derived ferulic acid and total polysaccharides from AR and ASR are higher in combinations of AR–ASR at 5:1 ratios. However, ASR-derived ligustilide is higher in the 1:1 ratio. At 3:1 ratio, the contents of the above active substances are all at the intermediate level. This is consistent with previous reports on chemical assessment of the herbal decoction containing AR and ASR (Dong et al. In the clinical practice of herbal practitioners, the compatibility amongst all the herbs within a decoction will be revised according to the syndrome of the patients. Thus, the investigation of the underlying mechanisms on cardioprotective effects of different AR–ASR combinations is necessary. When AR was combined with ASR at the ratio of 1:1, leukocyte infiltrates were decreased and the inflammatory cytokine secretion was inhibited in the heart tissue compared with the vehicle group after seven-day treatment. At the meantime, the cardiomyocyte apoptosis was suppressed, partially through inhibiting activation of caspase-3 and caspase-9 and decreasing the ratio of Bad to Bcl-2. However, in the combination of AR and ASR at 5:1 ratio, the angiogenesis was significantly improved along with enhanced Akt-eNOS phosphorylation. After MI, a step-by-step myocardial remodelling takes place involving the inflammatory phase and the reparative phase. During inflammatory phase, the milieu in the heart is highly inflammatory. Sequential infiltration of the injured myocardium with neutrophils, monocytes and their descendant macrophages contribute to the initiation of inflammation (Soehnlein and Lindbom In this study, we found that treatments with different combinations of AR and ASR dramatically improved cardiac function, decreased myocardial infarct size and reduced tissue injury in MI mice. Moreover, the myocardial protective effects of AR combined with ASR at 1:1, 3:1 and 5:1 treatment were respectively achieved by inhibiting inflammation and apoptosis, promoting angiogenesis, and regulating angiogensis and apoptosis. These results suggest that the treatment with AR–ASR combinations during different infarct periods might be useful approach to heart protection.


Listeria monocytogenes in the environment. L. monocytogenes outbreaks have been linked to fruits and vegetables; thus, a better understanding of the factors influencing its ability to colonize plants is important. We tested how environmental factors and other soil- and plant-associated bacteria influenced L. monocytogenes’ ability to colonize plant roots using Arabidopsis thaliana seedlings in a hydroponic growth system. We determined that the successful root colonization of L. monocytogenes 10403S was modestly but significantly enhanced by the bacterium being pregrown at higher temperatures, and this effect was independent of the biofilm and virulence regulator PrfA. We tested 14 rhizosphere-derived bacteria for their impact on L. monocytogenes 10403S, identifying one that enhanced and 10 that inhibited the association of 10403S with plant roots. We also characterized the outcomes of these interactions under both coinoculation and invasion conditions. We characterized the physical requirements of five of these rhizobacteria to impact the association of L. monocytogenes 10403S with roots, visualizing one of these interactions by microscopy. Furthermore, we determined that two rhizobacteria  were able to similarly impact 10 different L. monocytogenes strains, indicating that the effects of these rhizobacteria on L. monocytogenes are not strain specific. Taken together, our results advance our understanding of the parameters that affect L. monocytogenes plant root colonization, knowledge that may enable us to deter its association with and, thus, downstream contamination of, food crops.In spite of its relevance as a foodborne pathogen, we have limited knowledge about IMPORTANCEListeria monocytogenes is ubiquitous in the environment, being found in or on soil, water, plants, and wildlife. However, little is known about the requirements for L. monocytogenes’ existence in these settings. Recent L. monocytogenes outbreaks have been associated with contaminated produce; thus, we used a plant colonization model to investigate factors that alter L. monocytogenes’ ability to colonize plant roots. We show that L. monocytogenes colonization of roots was enhanced when grown at higher temperatures prior to inoculation but did not require a known regulator of virulence and biofilm formation. Additionally, we identified several rhizobacteria that altered the ability of 11 different strains of L. monocytogenes to colonize plant roots. Understanding the factors that impact L. monocytogenes physiology and growth will be crucial for finding mechanisms  that enable its removal from plant surfaces to reduce L. monocytogenes contamination of produce and eliminate foodborne illness. Listeria monocytogenes is a foodborne pathogen that predominantly infects immunocompromised individuals and causes the disease listeriosis  influences plant colonization of L. monocytogenes in a bacterial strain- and plant-dependent manner  harbor a diverse microbial community  with A. thaliana seedling roots. This assay uses floating mesh to keep the leaves of A. thaliana seedlings above the liquid (which decreases plant stress) while allowing the roots to free-float in the liquid medium below  (1× and 0.1×) and Murashige and Skoog (0.5× MS). After a 24-h incubation, 10403S was similarly abundant on A. thaliana seedlings in all media (∼104 to 105 CFU/plant) (7 CFU/ml) compared to the LB media (∼108 to 109 CFU/ml), indicating that 10403S more specifically associated with the roots in 0.5× MS. Because of this, we elected to use 0.5× MS in all subsequent assays.To establish hydroponic assay conditions, we compared the ability of U/plant) . HoweverA. thaliana roots in 0.5× MS. To test persistence, we first preinoculated A. thaliana roots with 10403S to an attachment level of ∼106 CFU/plant  (L. monocytogenes 10403S can persist on plant roots for at least 3 days under these conditions.We next wanted to investigate if 10403S was able to persist over time on  to 72 h . Additio CFU/ml) . This suL. monocytogenes can grow under a range of temperatures, 0 to 45°C, and growth temperature can significantly alter its transcriptional, translational, and metabolic state (600)-normalized dilutions to inoculate our hydroponic assay, after which the seedlings were incubated at RT and number of CFU/plant was assessed after 24 h.ic state , 42, 43.3 CFU/plant) compared to bacteria pregrown at 4°C or RT (∼3 × 102 CFU/plant) (4 CFU/ml) (10403S pregrown at 30°C and 37°C had modest but significantly increased number of CFU/plant (∼10U/plant) , with no CFU/ml) . As a coprfA gene or constitutively expressing prfA (prfA*). All strains were grown at 37°C (preinoculation), and after 24 h, we compared the plant-associated and planktonic CFU numbers between the three L. monocytogenes strains. There were no significant differences in either number of CFU/plant   or number of CFU/ml planktonic  . To further validate these findings, we performed an alternative root colonization assay that used a modified hydroponic approach involving higher inoculation levels but reduced colonization time. Even with these altered conditions, we did not observe any significant differences in number of CFU/plant between the ΔprfA, prfA*, and WT strains  . We also tested  whether flaA and actA impacted L. monocytogenes’ ability to colonize roots at elevated temperatures and saw colonization levels similar to those of the parental strain. These data support the conclusion that L. monocytogenes 10403S root colonization is not regulated by PrfA, flaA, or actA.Based on the response of 10403S to growth temperature, we speculated that the biofilm and virulence regulator PrfA are involved in root colonization of 10403S. To determine this, we compared the root colonization of the 10403S parental strain to strains either lacking the L. monocytogenes’ ability to colonize A. thaliana roots. Plant roots in native soil harbor diverse microbial communities , Curtobacterium oceanosedimentum (ES1096), Microbacterium oleivorans (ES1039), and Pseudomonas simiae WCS417r (ES620)  of 10403S and its coinoculation partner within the same liquid well and then assayed for 10403S colonization after 24 h using streptomycin to select for 10403S. To verify that the rhizobacteria were still colonizing the root and present in the liquid media, we also assessed the number of CFU/plant  and number of CFU/ml of the rhizobacteria in monoculture with the plant and when coinoculated with 10403S .Next, we investigated whether we could identify other environmental factors that influenced munities ; we ther (ES620) . We test5 CFU/plant) (P < 0.05) when it was coinoculated with ES620 (to ∼106 CFU/plant) (P < 0.05) numbers of CFU/ml than when grown alone  (M. oleivorans) does not appear to affect the growth of 10403S in liquid or on roots, while ES1024 (A. nicotinovorans) and ES1096 (C. oceanosedimentum) appeared to stimulate 10403S planktonic growth without altering its levels on roots.We discovered that when 10430S was coinoculated with ES1014, ES1096, or ES1039, 10403S had a similar number of CFU/plant as when inoculated in monoculture (∼10U/plant) . HoweverU/plant) . We alsoctively) . Howeverctively) . Thus, EL. monocytogenes 10403S. To address this, we collected cell-free, conditioned medium (CM) from ES620 and 10403S when grown alone. We then preinoculated seedlings with 10403S and added these seedlings to wells containing CM from either 10403S or ES620 (1 part fresh 0.5× MS to 1 part CM). After 24 h, we did not detect a significant difference in root-associated 10403S number of CFU/plant  when grown in CM from itself versus from ES620 (both ∼105 CFU/plant). However, we did detect a significant increase (P < 0.05 with ∼2-log increase) in the number of CFU/ml of 10403S planktonic cells when grown in ES620 CM compared to 10403S CM . These data indicate that the enhanced root association of 10403S observed when coinoculated with ES620 requires the physical presence of ES620.We then wanted to determine if the physical presence of ES620 was essential for enhancing the root association of P. simiae) altered colonization patterns or localization of 10403S on the root. To do this, we conjugated Escherichia coli SM10 carrying the constitutively fluorescent plasmid, pHPL3-mCherry (Cmr), with 10403S to generate a strain we called 10403S(pHPL3). 10403S(pHPL3) was cultured in the hydroponic assay in monoculture or in coculture with ES620. ES620 did not autofluoresce in the mCherry channel . We targeted three main areas of the seedling root for imaging: the crown, middle, and root tip  appears to colonize all three regions we assessed, with the most colonization appearing in the middle . Four strains  had significantly reduced numbers of CFU/plant, and two strains (RM3169 and RM3000) had significantly reduced planktonic numbers of CFU/ml in monoculture compared to 10403S (P < 0.05) . Overall, these data demonstrate that all 10 of these additional L. monocytogenes strains could colonize A. thaliana to substantial levels.We next wanted to determine whether ES620 could affect the root association of other  strains  represenL. monocytogenes strains with ES620 (P. simiae), all had significantly (P < 0.05) enhanced root colonization compared to when inoculated alone  were coinoculated with ES620  or next to one of the 125 bacterial isolates (in coculture) onto agar plates. We identified 18 bacterial isolates, mostly represented by Pseudomonas and Burkholderia species, that demonstrated noticeable colony-level antagonism toward L. monocytogenes 10403S based on visual increases in translucence of L. monocytogenes 10403S colonies or a reduction in overall biomass .We spotted cultures of either A. thaliana roots both when inoculated on their own or during coinoculation with L. monocytogenes 10403S ; they all also had detectable planktonic numbers of CFU/ml in the wells . All 10 strains significantly reduced L. monocytogenes 10403S’ ability to colonize roots (P < 0.001)  to ∼103 CFU/ml (coculture)  . ES1010,culation . These sculture) . This inP < 0.05) 10403S growth in the liquid  all significantly inhibited 10403S root colonization  but did e liquid  and C. TL. monocytogenes CFU/plant to determine whether we could identify bacteria able to inhibit L. monocytogenes even after it was established on roots. We modified the assay to mimic an invasion scenario, where 10403S was preinoculated on roots (to ∼105 CFU/ml) before being moved to wells containing individual rhizobacteria (rather than inoculating the two strains simultaneously). After 24 h, we compared the number of 10403S CFU/plant under rhizobacterial invasion to its number of CFU/plant in monoculture without invasion. The rhizobacterial strains able to reduce 10403S CFU both on and off the root during coinoculation  also significantly reduced (P < 0.05) the number of 10403S CFU/plant when added as invaders (5 to 104 CFU/plant) (P < 0.05) the number of 10403S CFU/ml in the media (from ∼105 CFU/ml when 10403S was grown without an invader to <103 CFU/ml [below the level of detection])  . Notablyection]) . In contection])  and incrhe wells .5 CFU/plant or greater even as invaders and were present in the media at levels near 108 CFU/ml . Collectively, these data highlight the importance of investigating the sequential colonization of bacterial consortium members: several bacteria  that were antagonistic when added concurrently with 10403S during colonization were unable to impact 10403S cell numbers on the root if it had already become established there.To determine whether the inability of these rhizobacteria to invade 10403S on plant roots was because 10403S simply excluded them from accessing the root, we quantified the number of CFU of the invading bacteria both on the plant and in the liquid culture medium. We found that all rhizobacterial strains could colonize the plants to ∼10A. thaliana roots. We placed seedlings containing 10403S (∼105 CFU/plant) in wells containing 1:1 fresh 0.5× MS-CM and assessed 10403S number of CFU/plant and number of CFU/ml liquid after 24 h. 10403S grown in CM of ES558, ES1010, ES1032, or ES1035 had significantly reduced (P < 0.05) number of CFU/plant compared to when it was grown in 10403S-CM . The CM from ES558 led to the greatest decrease in root-associated 10403S (dropping colonization from 105 to 104 CFU/plant), while the other isolates reduced 10403S colonization by a half log . The CM of these four strains had divergent impacts on 10403S growth in the hydroponic liquid: number of 10403S CFU/ml was significantly enhanced (P < 0.05) by the CM from ES1010, reduced by the CM from ES558 and ES1035 (P < 0.05), and unaffected by the CM of ES1032 . This suggested that the antagonistic effects of these strains toward L. monocytogenes 10403S root association is at least partially mediated by secreted chemical cues.Having found that physical contact was required for the enhanced root colonization observed when 10403S was coinoculated with ES620, we next wondered whether physical contact was also necessary for the negative impact that ES1010, ES558, ES1032, and ES1035 had on root colonization by 10403S. To test this, we obtained CM from these strains grown in monoculture with L. monocytogenes 10403S with roots, we next wanted to determine whether the CM from these rhizobacteria were also antagonistic toward other L. monocytogenes strains. Visual indicators of antagonism during agar-based cocultures demonstrated that all four rhizobacteria antagonized the additional L. monocytogenes strains, with ES558 (P. protegens) appearing to be the strongest antagonizer . We therefore tested whether the CM from ES558 could inhibit the other L. monocytogenes strains during plant inoculation. When we compared the 10 L. monocytogenes strains grown in either CM from themselves (−) or from ES558 (+), we observed a significant decrease (P < 0.05) in root-associated L. monocytogenes CFU number . Additionally, we did not see any colonization defects when we assessed the flaA and actA mutants . This indicates that there are still-unidentified factors regulating the surface association of L. monocytogenes and that some of these pathways are affected by temperature. Other possible gene candidates that may be influencing these outcomes are the alternative sigma factor B (sigB) or the response regulator AgrA, both of which are influential in environmental survival and adaptation in L. monocytogenes, as well as playing roles in biofilm/plant colonization  as well as enable the identification of genes specifically responsive to temperature. This idea is particularly intriguing given the evidence that L. monocytogenes does not form robust biofilms on stainless steel coupons or other abiotic surfaces typically used during in vitro assays . Altogether, these data suggest that L. monocytogenes forms more robust biofilms on biotic surfaces than on abiotic surfaces and indicate that the genetic factors governing the association of L. monocytogenes with roots remain to be discovered.Exposure to stressors, such as temperature, pH, limited nutrients, and other physiochemical stressors, has been found to enhance nditions , 51–53. surfaces  as well surfaces . In addisurfaces , 26, 41. studies , 56, we nization . Future o assays . Such poL. monocytogenes’ ability to colonize food-processing-facility-like surfaces and dairy food items . Furthermore, when colonized with P. simiae WCS417r (ES620), 11 different L. monocytogenes strains, representing a range of serotypes and lineages, all exhibited enhanced root colonization compared to when inoculated on roots in monoculture. This particular interaction was dependent on the physical presence of ES620, since conditioned medium from this strain could not enhance L. monocytogenes 10403S colonization. These synergistic observations are consistent with studies describing how Pseudomonas fluorescens enhances L. monocytogenes attachment to glass coverslips and stainless steel  typically contain multiple species of bacteria , 60 that61–od items , 65. In od items , 67 or aod items , 65 L. mn plants . In our ss steel . In addifectants , 71. It L. monocytogenes 10403S’ ability to coexist with our initial panel of four rhizobacteria was intriguing, as much of the literature often highlights antagonistic outcomes  enhanced L. monocytogenes’ root attachment. Thus, even within bacterial genera with strong impacts on L. monocytogenes, a wide range of potential outcomes are possible. Interestingly, we saw strong relationships between the outcomes observed during agar coculture and those from the hydroponic root assay. Of the 10 pseudomonad isolates interrogated on roots, the strains exhibiting the strongest inhibition of L. monocytogenes during agar coculture inhibited L. monocytogenes both on and off the plant root, while those pseudomonads that only moderately inhibited L. monocytogenes during agar coculture were more likely to demonstrate root colonization-specific effects in our hydroponic assay. Thus, in spite of extensive strain-level variation, we observed some phenotypic outcomes that were consistent across assay formats, indicating that monitoring colony-level antagonism using agar-based assays is a useful tool to identify bacterial coculture partners able to impede L. monocytogenes plant root colonization.outcomes , 73; howoutcomes . Of the cilities , 74 and,possible . Indeed,L. monocytogenes’ ability to associate with plant roots, we also wanted to understand the ecologically important question  was able to reduce the CFU/plant numbers of 11 different preestablished L. monocytogenes strains in a contact-independent manner. From the existing data, it is unclear whether this reduction is due to cell lysis, growth inhibition, or dissociation with the root surface. Regardless, the ability of Pf-5 to broadly impact L. monocytogenes strains highlights the potential use of this strain (or the bioactive compounds it secretes) to reduce L. monocytogenes colonization on crops or in food-processing facilities. P. protegens Pf-5 dedicates ∼6% of its genome to secondary metabolites, many of which are genes predicted to generate antibiotic and antifungal compounds (We did observe that the four strongest antagonists (from both the agar and coinoculation assays) were able to reduce 10403S burden on the plant root even when ompounds . Additioompounds , making L. monocytogenes is prevalent in the environment; however, compared to the knowledge we have about L. monocytogenes’ role as a human pathogen, we know very little about the requirements for L. monocytogenes’ existence in natural settings. Overall, our study provides new data regarding how exogenous factors  impact L. monocytogenes-plant-root interactions and demonstrate the need for continued research into these associations. As our global population and, thus, food crop production and consumption increase, disease-causing L. monocytogenes outbreaks from contaminated produce are likely to increase as well. Thus, understanding the influence of specific bacteria or communities of microbes on L. monocytogenes’ physiology and growth will be crucial for identifying mechanisms that can reduce the carriage of L. monocytogenes in pastures and on plant surfaces to reduce the possibility of foodborne illnesses.Escherichia coli, on Miller-lysogeny broth  agar plates made with 1.5% (wt/vol) Bacto-Agar (BD Biosciences) and were grown overnight at 37°C. To begin liquid inoculations, several isolated bacterial colonies were suspended in LB to an OD600 of 0.5 and inoculated into the assay wells at a final concentration of OD600 of 0.02. All references to LB indicate the use of the lower-salt Lennox composition of LB. In instances where L. monocytogenes was grown at alternative temperatures prior to inoculation in the hydroponic assay, 10403S was grown at 4°C (for 3 weeks), RT (for 3 days), 30°C (2 days), and 37°C (24 h).All bacterial isolates and strains  were stoE. coli SM10  seedlings were sterilized by chlorine gas exposure. Briefly, 3 ml of concentrated HCl was added to 100 ml of concentrated Clorox bleach in an enclosed container for 3 h inside a chemical fume hood. After sterilization, seeds were stored in Eppendorf’s in the dark at 4°C until use.2O and 7.45 g of Na2·EDTA) at 50 mg/liter (RPI M70300-5.0); this is referred to as 0.5× MS throughout the paper. There was no sucrose added to plates or liquid cultures for assays using 0.5× MS + MES. Individual sterile seeds were placed on the mesh disks, and the plate was sealed using a gas-permeable tape  and placed in a Conviron incubator for long-day conditions  for 7 to 9 days.Prepunched, 0.5-cm-diameter mesh circles  were autoclaved and placed onto agar plates containing 0.5× Murashige-Skoog (MS) salts plus MES buffer  at RT for 24 h unless otherwise stated. After 24 h, seedlings were removed from the mesh with sterilized forceps and placed into 1.5-ml Eppendorf tubes for sonication and quantification of root associated cells (number of CFU/plant). Sonicated samples were serially diluted and plated on 1× LB and 1× LB plates supplemented with 200 μg/ml streptomycin (to select for L. monocytogenes 10403S). For quantification of planktonic number of CFU/ml, medium was serially diluted from each well and plated to count CFU on 1× LB and 1× LB with 200 μg/ml streptomycin (to select for L. monocytogenes 10403S).After 7 to 9 days, germinated seedlings and their mesh were placed into 24-well Corning plates containing 1.6 ml of the indicated liquid medium and inoculated with bacteria at an OD600 of 0.5 and inoculated into a single well (L. monocytogenes plus one other bacterium per well) each at an OD600 of 0.02 and incubated at RT under static conditions for 24 h.Experiments are performed as stated in “Hydroponic experiments,” above. Bacteria were individually suspended to an ODL. monocytogenes colonies were resuspended to an OD600 of 0.5 in 1× LB, and seedlings were placed into this inoculum for 3 h, static, at RT. The L. monocytogenes-inoculated roots were then placed into wells where the invading bacteria were added at an OD600 of 0.02, incubated at RT, and kept static. Wells were treated as stated in “Hydroponic experiments,” above.L. monocytogenes strains were grown overnight in brain heart infusion  at 30°C in a Cel-Gro tissue culture rotator, speed 8 . L. monocytogenes strains were centrifuged and resuspended in 10 mM MgCl2, where the number of CFU was adjusted to ∼1 × 109 CFU/ml. Seedlings were transferred to 24-well plates containing the bacterial suspensions. Plates were covered with a gas-permeable membrane and shaken at 150 rpm at room temperature for 3 h. Following incubation, seedlings were removed from the wells and transferred to a new 24-well plate containing 1 ml of fresh 10 mM MgCl2 for 10 min. After this incubation, seedlings were removed from the mesh using an aseptic technique and transferred to a new 24-well plate containing 1 ml of 10 mM MgCl2, and plates were sealed and sonicated. Homogenate was serially diluted and plated on 1× LB to determine the number of CFU/seedling.Seeds were sterilized and grown as stated above, except that for this assay two seeds were placed on a single mesh disk. Liquid cultures of L. monocytogenes or onto LB plates to quantify the other rhizobacteria.Colonized seedlings were removed from the mesh and placed into an Eppendorf tube with 500 μl of 0.5× MS and sonicated (Qsonica sonicator q700) at 15 A using a microtip for 12, 1-s pulses, with 1 s off between pulses. The sonicator tip was thoroughly cleaned between samples using 70% ethanol. Sonicated samples were serially diluted and plated onto LB plates supplemented with 200 μg/ml streptomycin to select for 600 of 0.5 and 1 μl spotted on LB-agar plates with 0.5 cm in the center of each colony. Initial screen plates were grown at 37°C and imaged at 48 h; plates in subsequent experiments were grown at 30°C and imaged at 48 h.Cells were scraped from overnight LB-agar plates and suspended to an OD600 of 0.5 and added to the hydroponic assay wells at an OD600 of 0.02. The strains were grown with seedlings present in 0.5× MS + MES for 24 h. After 24 h, the liquid culture medium was collected and centrifuged for 3 min to pellet the bacteria, and the supernatant was filtered through a 0.22-μm filter to remove any cells. This sterile, cell-free conditioned medium (CM) was then added to wells as 1 part conditioned medium and 1 part fresh 0.5× MS + MES.Several colonies from overnight streak plates were suspended to an ODL. monocytogenes 10403S(pHPL3-mCherry) was colonized, with and without ES620, as previously stated for the hydroponic experiment. Seedlings were removed from culture after 24 h and placed on a slide. To prevent the seedlings from being crushed, frame seals (Bio-Rad SLF0601) were used to create a space between slide and coverslip and filled with 0.5× MS to allow for better imaging. Images were taken with a Nikon Eclipse 80i compound fluorescence microscope.For microscopy imaging, t tests. When only two groups were compared, we only used Mann-Whitney t tests. Asterisk denotes P values .Statistical comparisons were performed using GraphPad Prism, version 10.0. Each dot represents a biological replicate (single seedling), and data were obtained from at least three independent experiments for all experiments. Error bars represent standard errors of the means. Statistics were first performed by comparing all groups using Kruskal-Wallis analysis of variance (ANOVA) when applicable. If the Kruskal-Wallis ANOVA demonstrated significance, then individual groups were compared using Mann-Whitney


Although visual inspection is the preferred route in everyday clinical practise for detecting early caries lesions, novel technologies like light fluorescence-based devices (Vista Proof iX HD smart) have been developed to enhance early caries detection.Occlusal surface of 45 molar and 49 premolar teeth from 34 adult participants who fulfilled the eligibility criteria were examined by two observers using three diagnostic methods. Examination was performed visually using the International Caries Detection and Assessment System (ICDAS-II) followed by Vista Proof. Fissurotomy was applied for histological validation. Intra- and inter-observer agreement were measured for ICDAS-II and light-induced fluorescence camera using Kappa test. The overall diagnostic accuracy parameters, area under the receiver operating characteristic curve (AUC-ROC) and 95% confidence interval (95% CI) of the (AUC) for caries detection by Vista Poof were evaluated.For ICDAS-II and Vista Proof methods, there was almost perfect intra- and inter-observer agreement. Based on ICDAS-II as a reference standard 1, Vista Proof showed a low level of agreement in enamel carious lesion detection with low sensitivity value of 48%, high specificity of 100%, and AUC was 0.112, while a high level of agreement in dentin carious lesion detection with high sensitivity value of 100%, low specificity of 48% and AUC was 0.888. Based on fissurotomy as reference standard 2, Vista Proof showed a high level of agreement in dentin carious lesion detection with high sensitivity value of 95% and 0% specificity and AUC was 0.814. Quantitative light-induced fluorescence camera with reference to ICDAS-II is considered as an accurate diagnostic modality for detection of early occlusal caries. Histological findings validate the diagnostic accuracy of the camera in dentin. Key words:Histological validation, Initial caries, ICDAS, Light induced fluorescence, Vista Cam. First and foremost, dental caries is a disease that affects 60-90 % of the total of school-aged children and adults worldwide . To adeqClinically, caries diagnosis is commonly performed by multiple methods as visual tactile methods, assessments of translucency, color, and dental hardness, as well as by means of radiographic imaging. International caries detection and assessment system (ICDAS) based on standard method for visual diagnosis of dental caries had been widely used as gold standard for clinical diagnosis of dental caries . The visAs a result, occlusal caries detection technologies should not only be capable of detecting and monitoring lesions at all phases of caries process, but also be very reliable. Because detecting early occlusal caries is challenging, novel approaches for detecting early caries have been developed. Some of these technologies use light-based fluorescence devices to utilize fluorescence properties of hard tissues . The ligThe current study’s protocol was registered in protocol registration and results system  database under identification number NCT03940170. All procedures involving human subjects in this study were carried out in accordance with ethical requirements of Research Ethics Committee of Faculty of Dentistry, Cairo University . The outpatient clinic of Conservative Dentistry department, Faculty of Dentistry, Cairo, Egypt, hosted this diagnostic clinical trial and each participant that included in this study signed an informed consent form after describing research procedure in-depth.et al. 2017  assessed 60 extracted teeth for calibration. Each examiner diagnosed the teeth and recorded their results. They compared the results and returned the discrepancy cases until they reached 100% consistency . True blRegarding quantitative analysis of lesion activity using Vista Proof Camera, teeth were examined using fluorescence-induced interchangeable head of the camera (Vista Proof) following manufacturer’s instructions. The head of the camera was positioned perpendicular to the occlusal surface of teeth. This scenario was conducted under isolation with cotton rolls and suction tip and after tooth drying with a triplex air syringe for 15 seconds. By pressing focus button, the camera focused sharply on the tooth followed by pressing the trigger button for capturing the image. Image was analyzed by special software (DBSWIN) version 5.15.1. The software produced a digital image that showed lesions in different colors with numerical values between 0 and 3 predicting depth and extent of the lesion Figure  10)..10).The true histological extent of carious lesions was determined by fissurotomy which was considered as a confirmatory test. Clinical fissure evaluation was decided according to the reading obtained from Vista Cam, ICDAS-II, in addition to lesion activity and caries risk assessment. When Vista Cam reading ≥ 2 and ICDAS-II as score 3, fissurotomy was carried out. In case of ICDAS-II score 2, fissurotomy was only carried out when Vista Cam reading was ≥2, and the lesion appeared to be active on visual (matt appearance) and tactile (soft) examination and the participant was categorized as high caries risk . It is wP ≤ 0.05. Intra- and inter-observer agreement regarding ICDAS-II and Vista Proof modalities were evaluated using Cohen’s Kappa test. Diagnostic validity of Vista Proof device was determined, including sensitivity, specificity, overall accuracy, positive and negative predictive values, and ROC curve analysis, in detection of caries compared with traditional ICDAS-II and detection of enamel and dentin lesions compared with fissurotomy and ICDAS-II.Statistical analysis was performed using IBM SPSS Statistics Version 20 for Windows. Significance level was set at There was nearly perfect intra-observer agreement for ICDAS-II (Kappa= 0.943 for observer 1 and Kappa= 0.891 for observer 2), and Vista Proof methods (Kappa = 0.841 for observer 1 and Kappa= 0.810 for observer 2). Inter-observer agreement was nearly perfect for both ICDAS-II and Vista Proof methods . Vista Proof showed a low level of agreement with ICDAS-II in enamel carious lesion detection with low sensitivity value of 48%, high specificity of 100% to achieve an overall accuracy of 67% . Positivex-vivo studies or can only be performed in clinical studies with a relatively small number of teeth that can be extracted and having laboratory examination    was taken as reference standard for detection of diagnostic accuracy of fluorescence camera (Vista Proof iX HD Smart) in addition to use of fissurotomy technique not histopathological cross section for a histological validation of results. The main challenge for using histopathology as reference standard is that it is limited to the mination . In addimination . Kappa v. (2008) . While R. (2008)  discoveret al. (2012)   . This fi. (2012) , who dis12 . ThisFlorescence camera (Vista proof HD smart) showed a low level of agreement with ICDAS-II in enamel carious lesion detection with low sensitivity high specificity, an overall accuracy of 67% with a very poor AUC .These results could be justified by the fact that the device is very sensitive to any changes in the carious lesion with great sensitivity to any bacterial biproducts which made its scores exceeding the scores of ICDAS II. On the other hand, other studies revealed the inability of the device to quantify the scattered fluorescence light in the early demineralized enamel areas as it could not measure the intrinsic changes in enamel structure, so it has a lower performance for early enamel lesions. In addition, the type of enamel affects the ways of scattering of the device . MoreoveFlorescence camera (Vista Proof HD smart) showed a high level of agreement with ICDAS-II in dentin carious lesion detection with high sensitivity, low specificity , an overall accuracy of 67% and a very good AUC. This high sensitivity can be explained as the red fluorescence monitored by the device as caries, was related to the microbial metabolic products which were expected to be present in larger amounts in such dentin lesions  . Also, The level of agreement between Vista Proof HD smart and fissurotomy in detection of dentin lesions showed strong correlation between the two modalities. However, in this study validation of enamel lesions efficiently could not be achieved due to ethical issues. Where ICDAS II scores 0 and 1 were assessed with no further validation by fissurotomy, as they were indicated for remineralization and not pits and fissures sealing. ICDAS-II score 2 was validated depending on individual caries risk assessment, lesion activity and when Vista Proof HD smart score was ≥ 2. Follow-up appointments for re-assessment of teeth with ICDAS-II score 0-2 was conducted to monitor lesions progression.et al. (2015) and Melo et al. (2017)  and Jablonski-Momeni et al. (2014) , which led to 0% specificity and 100% sensitivity. This finding was in accordance with Melo . (2017) ,29, whilet al. 20 and Jabl. (2014) . This coFollowing the analysis of study results, the null hypothesis of this study was partially accepted as light-induced fluorescence camera was equivalent to ICDAS-II modality in distinguishing carious and non-carious teeth. However, it exceeded ICDAS-II in distinguishing between enamel and dentin caries. Histological validation agreed with Vista Cam scores in dentin carious lesions.Under the conditions of the present study, the following conclusions might be drawn:1- Quantitative analysis of carious lesions is crucial in achieving preventive dentistry rather than using a subjective visual assessment.2- Quantitative light-induced fluorescence camera with reference to ICDAS-II is considered as an accurate diagnostic modality for detection of early occlusal caries.3- Histological findings validate the diagnostic accuracy of the camera in dentin carious lesions.


In advanced prostate cancer, access to recent diagnostic tissue samples is restricted and this affects the analysis of the association of evolving biomarkers such as AR-V7 with metastatic castrate resistance. Liquid biopsies are emerging as alternative analytes. To clarify clinical value of AR-V7 detection from liquid biopsies, here we performed a meta-analysis on the prognostic and predictive value of androgen receptor variant 7 (AR-V7) detected from liquid biopsy for patients with prostate cancer (PC), three databases, the Embase, Medline, and Scopus were searched up to September 2021. A total of 37 studies were included. The effects of liquid biopsy AR-V7 status on overall survival (OS), radiographic progression-free survival (PFS), and prostate-specific antigen (PSA)-PFS were calculated with RevMan 5.3 software. AR-V7 positivity detected in liquid biopsy significantly associates with worse OS, PFS, and PSA-PFS (P <0.00001). A subgroup analysis of patients treated with androgen receptor signaling inhibitors  showed a significant association of AR-V7 positivity with poorer OS, PFS, and PSA-PFS. A statistically significant association with OS was also found in taxane-treated patients (P = 0.04), but not for PFS (P = 0.21) or PSA-PFS (P = 0.93). For AR-V7 positive patients, taxane treatment has better OS outcomes than ARSi (P = 0.01). Study quality, publication bias and sensitivity analysis were integrated in the assessment. Our data show that liquid biopsy AR-V7 is a clinically useful biomarker that is associated with poor outcomes of ARSi-treated castrate resistant PC (CRPC) patients and thus has the potential to guide patient management and also to stratify patients for clinical trials. More studies on chemotherapy-treated patients are warranted.PROSPERO, CRD42021239353. Prostate cancer (PC) is one of the most common male cancers. The androgen receptor (AR) pathway is critical in maintaining normal prostate tissue homeostasis, cancer development and progression . TherapiBiomarkers detected in liquid biopsy (such as circulating tumor cells and cell-free tumor DNA) demonstrate good concordance with biomarkers detected in conventional tissue biopsy, especially for metastatic CRPC . Liquid To clarify the clinical utility of AR-V7 detection from liquid biopsies, we undertook a comprehensive systematic review and meta-analysis to evaluate the available data from the clinical studies published up to September 2021. Prognostic and predictive value of liquid biopsy derived AR-V7 data in PC patients were evaluated from 37 studies that met the inclusion criteria.https://www.rayyan.ai/) for independent systematic review according to selection criteria. Two independent, blinded observers (TK and YM) reviewed all candidate articles. Any discrepancies in the article selections were resolved by discussion.This study was conducted according to preferred reporting items for systematic reviews and meta-analysis (PRISMA) . The proPre-set exclusion criteria of this study were: (1) publication type: review articles, letters, comments, questionnaires, conference papers, corrections, reply to editor, case reports, book chapters, abstracts only, research highlights, summaries; (2) non-human studies ; (3) non-prostate cancer; (4) AR-V7 data are not derived from human; (5) survival data not related to AR-V7 or with insufficient data to calculate the hazard ratios (HRs) and their 95% CIs, or the Kaplan–Meier (K–M) curve unable to calculate HRs and 95% CI parameters. Finally, studies were only included when they met the following criteria: (1) AR-V7 assayed in liquid biopsies ; (2) A reported relationship between AR-V7 and prognostic/predictive indicators, namely, OS, PFS, and PSA-PFS; (3) patient cohorts with n >25, and (4) English language only.This study focuses on the prognostic value of AR-V7 detected from liquid biopsy and its predictive value for ARSi and chemotherapy. According to a pre-designed table, the items of data extraction included the last name of the first author, publication year, study country, number of patients included, age of patient, sample resource (processing method) and AR-V7 detection method, type of therapies, endpoints of oncological outcomes, HRs and 95% CIs , follow-up durations and definitions of OS, PFS, and PSA-PFS . Data waThe adapted Newcastle–Ottawa Scale (NOS) scales for cohort study  were use2 statistical methods were applied to evaluate the heterogeneity among included studies and a random effects model was used for data consolidation. If the heterogeneity was very high, only a descriptive score was given. Further subgroup analysis based on patient treatment was also conducted. The inverted funnel plots with Egger’s test were used to analyze potential publication bias with R software. A sensitivity analysis was carried out to assess the influence of each individual study on the pooled results by sequentially excluding each study. A two-tailed p-value <0.05 was regarded as statistically significant.Pooled HR and 95% CI were used to evaluate the prognostic and predictive value of AR-V7 presence or high expression  on the patient survival parameters  in Review Manager 5.3 software . The Cochran Q and I® (Qiagen) (n = 13), Oncoquick® (Greiner Bio-One GmbH) (n = 1), red blood cell (RBC) lysis (n = 3), and immunomagnetic beads-based methods  (n = 9). The method of AR-V7 detection was primarily by PCR . Endpoint of patient outcomes include OS (n = 30), PFS (n = 28) and PSA-PFS (n = 10) (The flowchart outlining the results of the literature search and application of the strategic inclusion and exclusion criteria is presented in (n = 10) Table 1.2 ≥80%), random effects model was applied to calculate HR value and 95% CI for all survival parameters.Thirty studies including 976 AR-V7 positive  and 2,056 AR-V7 negative (or low level) patients were used for OS comparison, while 28 studies including 697 AR-V7 positive and 1,553 AR-V7 negative patients were used for PFS analysis and 10 studies including 216 AR-V7 positive and 425 AR-V7 negative patients for PSA-PFS analysis. Most patients in the cohort of studies were treated with ARSi  or taxane-based chemotherapy. Some reports included miscellaneous treatments [such as Bipolar Androgen-based therapy ]. Overal–AR-V7 positive patients treated with ARSi  had significant poorer OS , PFS  and PSA-PFS  compared with AR-V7 negative patients –4. WhenIn the subgroup analysis of the patients treated with taxane-based chemotherapy, the association of AR-V7 positivity with worse OS was observed  Figure 2For the studies in which the authors did not clarify treatments and were unable to be classified as either ARSi or taxane chemotherapy, AR-V7 presence is associated with worse OS  and PFS  3.Four studies compared treatment response in AR-V7 positive or negative patients. Taxane treatment is linked to superior OS  in patients positive for AR-V7, compared to ARSi  and in vivo (PC tissue)  for OS data and lack adequate power for PFS (P = 0.21) or PSA-PFS (P = 0.93). However, there are relatively fewer publications in this subgroup, so these conclusions are based on weaker datasets compared to the ARSi treated subgroup; for instance, the omitting one publication changes the P-value and AR-V7 impact on OS would no longer be significant ( tissue) , 48. On Three other meta-analyses on AR-V7 prognostication , 49, 50 In conclusion, ARSi and taxane-based chemotherapy are approved treatment options for CPRC patients and are used globally. Use of emerging methodologies, such as liquid biopsy-determined AR-V7, to optimize utility of a known predictive biomarker could help to guide the optimal treatment sequencing pathway for each patient in a personalised manner and is therefore of clinical importance. Standardization of liquid biopsy AR-V7 detection would underpin utility in clinical practice. Avoiding ineffective therapies or early switching to more effective approaches should ensure better outcomes for patients. However, further studies on chemotherapy-treated patient cohort and direct comparison of chemotherapy vs. ARSi are warranted.The original contributions presented in the study are included in the article/Project development, methodology, data collection and analysis: TK and YM. Conceptualization: YM and TK. Project development: TB, KS, PDS and WC. Statistics: JD, TK and YM. Manuscript writing, editing, and reviewing: all authors. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.TK received an Ingham Institute/Narellan Rotary Club Men’s health grant 2018 and a WSU School of Medicine Androgen Receptor Research scholarship.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.


Many people with neurological conditions experience challenges with movement. Although rehabilitation is often provided acutely and sub-acutely following the onset of a condition, motor deficits commonly persist in the long-term and are exacerbated by disuse and inactivity. Notably, motor rehabilitation approaches that incorporate exercise and physical activity can support gains in motor function even in the chronic stages of many neurological conditions. However, delivering motor rehabilitation on a long-term basis to people with chronic neurological conditions is a challenge within health care systems, and the onus is often placed on patients to find and pay for services. While neurological motor rehabilitation is largely the domain of physical and occupational therapists, kinesiologists may be able to complement existing care and support delivery of long-term neurological motor rehabilitation, specifically through provision of supported exercise and physical activity programs. In this perspective style review article, we discuss potential contributions of kinesiologists to advancing the field through exercise programming, focusing on community-based interventions that increase physical activity levels. We conclude with recommendations on how kinesiologists’ role might be further optimized towards improving long-term outcomes for people with chronic neurological conditions, considering issues related to professional regulation and models of care. An estimated 1 billion people live with neurological conditions, ranging from stroke and traumatic brain injury to neurodegenerative diseases . For sudPersistent and progressive losses in motor function commonly propel people with neurological conditions into a cycle of ever-declining health. Motor deficits interfere with performance of activities of daily living –10 and pThe primary aim of the article is to initiate conversation on whether long-term motor rehabilitation for adults with chronic neurological conditions might be advanced through greater and more deliberate integration of kinesiologists into the field. We begin with a brief overview of motor rehabilitation for people with chronic neurological conditions, discussing its premise, its relationship to exercise and physical activity, and barriers to participation. Next, we describe the scope of practice of a kinesiologist with consideration of where kinesiologists might fit within the traditional neurological motor rehabilitation team. Finally, we provide recommendations on how kinesiologists might be better utilized to complement existing motor rehabilitation services for people with chronic neurological conditions. We consider limitations of kinesiologists in working in the neurological rehabilitation field and acknowledge alternative approaches to improving access to long-term neurological motor rehabilitation. The discussion is framed within our research and clinical experiences with neurological rehabilitation in Canada and the United States but are relevant to other countries with similar health care resources.Most recovery of motor function after sudden neurological damage occurs in the initial 3 to 6 months post-injury during in- and out-patient rehabilitation . While tGiven that chronic neurological motor rehabilitation benefits are partly dependent on physical conditioning, there is a degree of overlap with what might be considered “exercise” or “physical activity” services. A distinction could be made that “rehabilitation” is delivered to improve function or reduce disability associated with a specific underlying condition or impairment , while “Generally, the neurological motor rehabilitation team includes an inter-disciplinary group of physiatrists, physical therapists, and occupational therapists, with supporting contributions from other health care providers such as recreational therapists, psychologists, and nurses , 4, 36. Kinesiology broadly refers to the scientific study of human movement . It is aThe broad scope of practice of kinesiologists could be perceived as a strength in terms of versatility of the profession; however, it also contributes to confusion about the distinct role of a kinesiologist relative to other health care professions , 44, parTo our knowledge, the practical contributions of kinesiologists to the neurological motor rehabilitation process have traditionally been minimal. With increased evidence that exercise and physical activity benefits most chronic health conditions including neurological conditions , there iA valuable and appropriate contribution that kinesiologists can make through a role in chronic neurological motor rehabilitation is in the delivery of community-based exercise programs. The relatively recent development of community-based exercise programs for people with chronic neurological conditions represents a positive shift in the field towards addressing the need for long-term, affordable motor rehabilitation services. Of note, some existing community-based programs, mainly targeted to people with chronic stroke, already utilize instructors with skillsets consistent with kinesiologist training –52. HereTIME™ is a community-based group exercise program that serves people who are ambulatory but experience balance and mobility challenges, including people with neurological conditions . In the Use of kinesiologists in community settings could move beyond general exercise programming to also include programs focused on re-acquisition of motor skills through task-oriented motor skill practice. Researchers in kinesiology-related departments across the world have advanced understanding of the neural control of movement and skill acquisition, and influenced the field of neurological rehabilitation e.g., –61. LikeOutside of community-based group program delivery, kinesiologists commonly work alongside physical and occupational therapists in private practice . In clinAlthough on a generally small scale that is inconsistent across locales, the examples we provided demonstrate that kinesiologists and people with related skillsets  are already being used to support exercise programming for people with chronic neurological conditions. Given the contributions that kinesiologists can, and to some extent already do, make to chronic neurological motor rehabilitation, development of established roles for kinesiologists could be a constructive step for the field toward enhancing long-term functional outcomes for people with neurological conditions. Specifically, the examples provided indicate that kinesiologists could play a crucial role in the expansion of community care for people with chronic neurological conditions. In this role, kinesiologists could complement the traditional neurological rehabilitation team towards providing increased opportunity for people to participate in motor rehabilitation activities on a long-term basis.A major challenge for the kinesiology profession in working with clinical populations in general is the lack of regulatory bodies that protect the public , such asAlthough more widespread regulation of kinesiologists is a critical step for the recognition of the profession, kinesiologists’ formal adoption into the neurological motor rehabilitation team may also depend on the establishment of neurological motor rehabilitation as a sub-field within kinesiology. Many undergraduate kinesiology programs offer concentrations of study, with some common concentrations including content relevant to chronic neurological motor rehabilitation . Likewise, related academic programs in “kinesiotherapy” that focus on using exercise as rehabilitation for people with functional limitations have a rich history in the United States . DevelopGiven the long-term nature of efforts to create regulatory bodies and establish concentrations of study, it is our current recommendation that kinesiologists working within a neurological motor rehabilitation team should have, at a minimum, an undergraduate degree in kinesiology from an accredited institution and be registered with a local professional association that requires participation in a professional liability insurance program . With tUltimately, the need for ongoing and often life-long exercise and motor rehabilitation services for people with neurological conditions , 5 suggeIn advance of this formal model of care, Fig. The use of remote technologies to deliver rehabilitation services could provide an efficient means of delivering in-home motor rehabilitation services to people with mobility and transportation barriers, or those who are geographically isolated from direct services . Beyond It could be argued that long-term neurological motor rehabilitation needs would be better met by expanding access to regulated health professionals, such as physical and occupational therapists, than by increasing the use of kinesiologists. The more advanced training of these therapists is critical for management of complex clinical issues and use of various relevant treatment techniques and modalities. Potential approaches to expanding access to physical and occupational therapists could involve increasing educational funding, development of programs that do not repeat training acquired by prior kinesiology program graduates, or modification of restrictive reimbursement patterns for therapeutic services. In our view, these are also necessary steps for the field of neurological motor rehabilitation but should not be considered exclusive alternatives to advancing the kinesiologist role. Instead, our view is that a more established role for kinesiologists that respects their proficiencies and limitations in working with this population will support increased access to, and more efficient use of, therapists’ skills and expertise.Long-term exercise and physical activity supports for motor rehabilitation for people with chronic neurological conditions are needed. Kinesiologists are movement practitioners with a skillset well-suited to contribute to such strategies, particularly through community-based exercise and physical activity programming. These programs supported by kinesiologists and targeted to people with neurological conditions benefit motor function but are not yet widely offered. Development of relationships among the traditional neurological rehabilitation team and kinesiologists through clinic-to-community models of care could be a step toward advancing kinesiologists’ role in improving long-term outcomes of people with chronic neurological conditions. Yet, lack of professional regulation in most jurisdictions remains a key challenge. Overall, this viewpoint is meant to serve as a starting point for further discussion as we work towards improving long-term motor rehabilitation outcomes for individuals with chronic neurological conditions.