Young people, like college students, are at risk of hearing loss from prolonged and excessive exposure to loud sounds. However, behavioral interventional studies on them are inadequate. This study explored the application of a health belief model to the health education intervention on college students for improving hearing health knowledge, health belief, and hearing behaviors. From November 2017 to September 2018, a cluster randomized controlled trial was conducted, enrolling 830 college students, with 419 in the intervention group and 411 in the control group. The intervention group received a 3-month hearing health education, while the control group received no intervention. The information of hearing health knowledge, health belief, and hearing behaviors were collected using hearing health questionnaires before the intervention, after the intervention, and 3 months after the intervention cessation. The intervention significantly improved hearing health knowledge, health belief, perceived severity, and self-efficacy in female students, and effectively reduced the frequency of using headphones per day, duration of using headphones each time, and proportion of using headphones at high volume in female students, and reduced the behaviors of sleeping with headphones listening in females and males. Therefore, this study confirms the effectiveness of health belief model-based intervention for changing hearing loss-related risk behaviors. In the 2015 Global Burden of Disease Study, hearing loss was ranked as the fourth most prevalent chronic disease worldwide [Tung et al. found that 11.9% of 1878 first-year college students in a university in Taiwan had unilateral or bilateral hearing thresholds above 25 dB . BalanaySince there is no effective treatment for hearing loss, it is worth advocating for an active intervention to reduce the risk factors and incidence of hearing loss through health education . KathleeBased on the principles of cognitive theory, the health belief model holds that expectations and beliefs play a key role in persuading people to change harmful behaviors . HearingStudies are still lacking on studies performed on hearing health-related behavioral interventions in college students. This study aimed to examine the effects of a health intervention on hearing health risk behaviors among college students to provide new ideas for the design and application of a hearing health intervention scheme in universities.Classes were cluster-randomized sampled from November 2017 to September 2018, and a total of 830 eligible college students were enrolled from the Department of Medicine, Hangzhou Normal University, with 419 students in the intervention group (IG) and 411 students in the control group (CG). The inclusion criteria were as follows: (1) medical students living on the university campus; (2) signed the informed consent and agreed to participate in the intervention program; and (3) without hearing loss. The hearing status of these college students was tested in our previous research .The intervention group was provided with online and offline hearing health education. The online intervention included releasing hearing health knowledge through the public account “Medical Ear” of WeChat  and establishing WeChat groups for group communication. Offline intervention predominantly comprised the distribution of hearing health leaflets, teaching ear protection exercises, and holding hearing health knowledge competitions. The control group did not receive any intervention .The questionnaire was designed to collect the demographic variables, hearing health knowledge, hearing health beliefs, and hearing loss-related behaviors.The demographic variables include age, grades, ear problems , and the number of people with hearing loss in the family. Tinnitus was divided into two conditions: no tinnitus within the past year, and occasional tinnitus , or frequent tinnitus . Earache and ear tightness were similarly classified.The hearing health knowledge dimension contained 36 items, including hearing risk factors (11 questions), harms of noise to the hearing system (six questions), early symptoms of hearing loss (eight questions), and methods for hearing loss prevention (11 questions). Every correct answer was awarded 1 point, whereas the wrong answers or ‘do not know’ answers were awarded 0 point.The hearing health belief dimension had 45 items, including perceived susceptibility to hearing loss (five questions), perceived severity for hearing loss (nine questions), perceived benefits (seven questions), perceived barriers to changing harmful behavior for hearing-loss (eight questions), cues to action (eight questions), and self-efficacy (eight questions). A 5-point Likert scale rating was used, and a value was assigned from “totally disagree” to “completely agree” with total scores being 45–225 points. The higher the score, the better the level of belief in hearing health. The questionnaire was validated in previous research with 2088 participants involved. Its Cronbach’s coefficient was 0.888, and the KMO value was 0.872. A principal component analysis showed that confirmatory factors accounted for 64.395% of the total variance. We surveyed six types of hearing health-related behaviors: (1) average frequency of using headphones ≥3 times/day; (2) average duration of using headphones ≥30 min each time; (3) probability of increasing headphone volume in a noisy environment ≥50%; (4) volume of the headphones ≥40% of the total volume: (5) using in-ear type headphones; (6) sleeping with headphones for listening to music or radio.Questionnaires were administered to both groups of students before the intervention (T1), at the intervention cessation phase (T2), and 3 months after the intervention cessation . The questionnaire survey was conducted by investigators, including teachers and graduate students who received strictly professional training to ensure data collection quality. They were able to provide accurate interpretation during questionnaire collection by face to face interviews in order to minimize the subjective bias of the participants.n (%). The differences among groups at baseline were analyzed with t-test and chi-square test. The change in scores of health knowledge and health beliefs were calculated as the value obtained at T2 and T3 minus the baseline value, and Repeated Measures ANOVA  were used to analyze the differences in intervention outcomes between the control and intervention groups. If one type of behavior was found to have improved at T2 or T3, compared to T1, the intervention was considered effective (Y = 1), whereas if there was no improvement or the behaviors were found to have worsened, the intervention was considered ineffective (Y = 0). Binary logistic regression analysis was used to estimate the efficiency of health interventions on the hearing of health-related behaviors, and p < 0.05 was considered statistically significant.The database was established using Epidata 3.0, and analyses were performed using SPSS 22.0 for Windows . The demographic data for each student were compiled to produce descriptive statistics. Continuous variables were shown as the mean ± SD (Standard Deviation), and categorical variables showed as n = 241) and females accounted for 71% (n = 589). Sophomores accounted for 50.1% (n = 416) and Juniors 49.9% (n = 414). The proportion of participants with tinnitus, earache, and ear tightness was 51.6% (n = 428), 31.4% (n = 261), and 28.3% (n = 235), respectively. Overall, 61.2% of the students reported a family history of hearing loss. Grade distribution was statistically significantly different between the intervention group and the control group in male students (p < 0.001), while the grade distribution and average age were statistically significantly different in female students (p < 0.001)  .p = 0.045)  .p = 0.007; T3–T1 IG 3.97 ± 8.04 vs. CG 1.95 ± 8.05; p < 0.014). Similarly, the scores of hearing health belief demonstrated significant improvement 3 months after intervention cessation . Among the sub-dimensions of hearing health belief, the scores of perceived severity and perceived self-efficacy showed improvement after intervention respectively   .In male students, the scores of hearing health knowledge and health belief demonstrated no significant improvement at the intervention cessation phase (T2) and 3 months after intervention cessation (T3) between both groups .p < 0.05)  was significantly higher in the intervention group in female students ( < 0.05) .p = 0.013; T3–T1 IG vs. CG 1.612 (1.019–2.550), p = 0.041), the average duration of using headphones ≥30 min each time (T2–T1 IG vs. CG 1.540 (1.014–2.339), p = 0.043; T3–T1 IG vs. CG 1.854 (1.233–2.788), p = 0.003), volume of the headphones ≥40% of the total volume (T2–T1 IG vs. CG 2.416 (1.351–4.320), p = 0.003; T3–T1 IG vs. CG 2.224 (1.275–3.882), p = 0.005) and sleeping with headphones for listening to music or radio (T2–T1 IG vs. CG 1.725 (1.107–2.689), p = 0.016; T3–T1 IG vs. CG 1.991 (1.284–3.088), p = 0.002) , = 0.002) .p = 0.017) , = 0.017) .The health belief model is one of the earliest theories used to explain and predict health behavior , and incIn this study, we found that the 3-month health education intervention effectively improved the level of hearing-health knowledge in female students, with the good effect well-maintained 3 months after the intervention cessation. Similarly, Khan et al. conducted a six-week hearing intervention using computer training, classroom training, and classroom plus application (APP) training on high school students and found that all the three forms increased the average hearing health knowledge score by 20.0%, 14.2%, and 16.3%, respectively .In female students, the effect of the intervention on the total scores of hearing health belief was detected only 3 months after the intervention cessation. Keppler et al. reported that a 6-month hearing education program improved participants’ attitudes and beliefs about hearing protector devices . In contThe ultimate goal of improving the knowledge and belief in hearing protection is to reduce the incidence of hearing health risk behaviors. Common harmful behaviors leading to hearing loss include frequent attendance of concerts, karaoke sessions, high-noise places such as nightclubs, and using of personal music players ,20,21. SThis suggests that intervention programs can play a catalytic role. Liang et al. reported that most teenagers tend to raise the music volume in the presence of background noise in order to hear the music better . SunghwaIn male students, the intervention showed no effectiveness in improving the scores of hearing health knowledge and health beliefs and was less effective in reducing hearing risk behaviors compared with female students. It is probably attributable to that female students have a higher level of health awareness of personal health care than males . TherefoIn the maintenance period, intervention effectiveness still can be seen by the reduced incidence of hearing health risk behaviors, indicating the purpose of cultivating behavioral habits for long-term hearing protection was achieved. It can be a good reference provided for the formulation of health education programs in the future. Marlenga et al. conducted a 16-year follow-up study among 392 students who had participated in a 2–3-year hearing protection intervention and found an increase in the long-term use of hearing protection devices . SimilarThe present study showed several advantages. Firstly, due to the teachers’ participation and students’ cooperation, we found that hearing health education is conducive to ensuring effective participation of students and reducing the attrition rate as much as possible. Secondly, timely communication and feedback between the two sides made it easier for students to obtain hearing health knowledge and encouraged students to develop good hearing habits. There is a proposal to include hearing protection in the students’ curriculum. University organizations are actively exploring how to incorporate the teaching of healthy hearing habits to college students . Klein eThe present study has some limitations. The participants only comprised students from a medical college and may not be representative of students from other colleges and universities. Since medical college students need to communicate with patients and perform auscultation, they need to have good hearing. Additionally, we mainly focused on the behaviors on earphone and cellphone usage, and many other hearing health risk factors like behaviors of seeking medical treatment for ear diseases were not investigated. Follow-up studies should be extended to students of other majors and more hearing health behaviors. Finally, the number of males and females in the intervention and control groups was uneven, and we have controlled for the possible bias to the maximum extent.In this study, a health intervention program based on the health belief model was conducted among college students. It effectively improved hearing health knowledge and hearing health beliefs and effectively reduced the incidence of hearing health risk behaviors in female students but weakly in male students. This study demonstrates the potential benefits of providing hearing health intervention classes to decrease the incidence of hearing health risk behaviors in universities. But specifically, intervention measures targeted to different genders should be carried out.


Prolonged exposure to negative stressors could be harmful if a subject cannot respond appropriately. Strategies evolved to respond to stress, including repetitive displacement behaviours, are important in maintaining behavioural homoeostasis. In rodents, self-grooming is a frequently observed repetitive behaviour believed to contribute to post-stress de-arousal with adaptive value. Here we identified a rat limbic di-synaptic circuit that regulates stress-induced self-grooming with positive affective valence. This circuit links hippocampal ventral subiculum to ventral lateral septum (LSv) and then lateral hypothalamus tuberal nucleus. Optogenetic activation of this circuit triggers delayed but robust excessive grooming with patterns closely resembling those evoked by emotional stress. Consistently, the neural activity of LSv reaches a peak before emotional stress-induced grooming while inhibition of this circuit significantly suppresses grooming triggered by emotional stress. Our results uncover a previously unknown limbic circuitry involved in regulating stress-induced self-grooming and pinpoint a critical role of LSv in this ethologically important behaviour. Self-grooming is a frequently observed repetitive behaviour in rodents that is believed to contribute to post-stress de-arousal. The authors identified a previously unknown limbic circuit that includes the ventral lateral septum in rats and is involved in regulating stress-induced self-grooming. Generation of adaptive responses to stress involves the evaluation of real or perceived stressors, and the homoeostatic resolution via optimization of emotional and physiological adaptations5. Behavioural adaptations could include increased arousal, attention and vigilance1. On the other hand, maladaptive response to stress has been linked to the aetiology of anxiety, depression and a variety of other neuropsychiatric conditions8. Therefore, strategies evolved to cope with stress are essential for health and survival.Stress in the form of emotional and physiological challenges is ubiquitous in everyday lives. Physical and emotional stressors can upset body homoeostasis that pertains to a steady milieu of physiological parameters as well as states of mind12. These displacement activities are believed to have adaptive values. Indeed, self-grooming is a frequently observed repetitive behaviour in rodents that serve functions more than hygiene maintenance and thermoregulation. This behaviour may represent adaptive response to stress, or restraining force that prevents over-response to stress, such as post-stress de-arousal14. Thus, unravelling the mechanism of stress-induced self-grooming is highly valuable towards understanding the neurobiological basis of stress management.Stress in animals, including human, often results in grooming and other repetitive behaviours such as circling and rocking15, brain stem16 and cerebellum17 that represent the downstream, mechanical motor pathways. On the other hand, some components of the limbic system, including the hypothalamus, amygdala and orbitofrontal cortex, participate in the regulation of grooming. For example, focal activation of specific hypothalamic nuclei could evoke robust grooming20, and control of self-grooming versus social behaviour by distinct amygdala neuronal subpopulations had been demonstrated21. Also, repeated stimulation of the orbitofrontal-striatal pathway could generate compulsive grooming22. Many previous studies that specifically link stress to grooming focused on the neuroendocrine system, namely the hypothalamic–pituitary–adrenocortical axis25, in accordance with the observation that central administration of some stress-related neuropeptides like corticotropin-releasing hormone (CRH), adrenocorticotrophic hormone and melanocyte-stimulating hormone could elicit grooming27, and CRH neurons orchestrate post-stress behaviours including grooming12. More recently, the neural pathways involved in different stress-related responses including grooming have begun to be revealed25. For example, a hypothalamic-septal pathway that mediates the influence of emotional states on grooming, escape and feeding behaviour was identified24. However, the relationship between stress and self-grooming is likely to be complex28, and the neural circuit basis of stress-induced grooming is still unresolved. In particular, the complete neural circuitry that conveys perceived stress, especially those with strong emotional component, that lead to grooming is yet to be uncovered.In the mammalian brain, a number of brain areas have been implicated in the generation of grooming behaviour. These include the basal gangliaIn this study, by identifying and dissecting stress-related neural circuit, we revealed a previously unknown limbic circuit linking the hippocampal ventral subiculum (VS), ventral division of lateral septum (LSv) and lateral hypothalamus that regulates stress-induced self-grooming. Optogenetic activation of this di-synaptic circuitry triggered delayed but robust grooming with patterns closely resembling those evoked by emotional stress. A distinct feature of activation of this circuit is its association with a clear positive valence, unlike many other studies on grooming behaviour. In addition, we found that the neural activity of LSv precedes and is necessary for emotional stress-induced grooming while targeted functional inhibition of this circuitry suppressed grooming caused specifically by stressful paradigms. Our results thus advance our understanding of the neural circuit basis of repetitive behaviour particularly relevant to the adaptation to emotional stress.33, to the animals for an extended period of time, viz 20 min (n = 4). This protocol induced significantly increased time spent in grooming by the animals  488 into LSv resulted in fluorescent signals in a number of brain regions, but a highly discrete and prominent labelling of the VS of the ipsilateral hippocampus was found Fig. . In agre34, we also confirmed in brain slice the GABAergic, monosynaptic connection from LSv to Tu in contributing to grooming , we found significant preference for the animals to stay in the compartment associated with stimulation of the LSv Fig.  or LSv →35 in VS and the retrograde retroAAV-EF1α-DIO-ChR2-mCherry in Tu  rats weighing 300–320 g were used in this study. The animals were bred and maintained by the Laboratory Animal Service Centre of The Chinese University of Hong Kong (CUHK). The animal room was controlled at a temperature of 23 °C on a 12-h light/dark cycle. All animals were handled in strict accordance with the CUHK guidelines and the procedures were approved by the Animal Experimentations and Ethics Committee. All experiments were performed during the light phase (09:00–19:00).12 particles per ml.Adeno-associated viruses (AAV) including AAV5-hSyn-eGFP, AAV9-hSyn-hChR2(H134R)-eYFP, AAV9-CaMKIIα-hChR2(E123A)-mCherry, AAV8-hSyn-hM3Dq-mCherry, RetroAAV-EF1α-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA, AAV9-Ef1α-DIO-eNpHR3.0-eYFP, RetroAAV-hSyn-DIO-hM4Di-mCherry, AAV9-Syn-GCaMP6s-WPRE-SV40 were all purchased from Addgene . All viral titre were >5 × 10SD rats were anesthetized with ketamine  and xylazine , and placed gently in a stereotaxic frame . For micro-injections, a Hamilton syringe (33-gauge) filled with AAV virus or tracer was unilaterally or bilaterally placed into the target area according to the corresponding coordinates: LSv , VS , and Tu  from dura. 0.1–0.5 μl virus or tracer were injected at speed of 10–50 nl/min. The needle was left in place for an additional 10 min before retraction. The scalp incision was sutured, and postinjection analgesics were given for 3 days to aid recovery. The animals were allowed at least 3 weeks to recover and express a virus before optical fibre implantation or behavioural test. For fibre photometry and optogenetics experiments, an optical fibre  or fluid-injection cannula (26-gauge guide cannula possessing a 32-gauge dummy cannula) was implanted directly above. The fibre or guide cannula, together with two stainless steel screws was secured to the skull using dental cement. The rats were allowed at least 1 week to recover before behavioural test. Correct location of implanted fibre/cannula was confirmed postmortem.For ChR2 photostimulation, 473-nm light laser  was delivered via an optic cable  and the stimulation duration varied in different experiments, as described in the relevant results. Laser power was 5 mW measured at the tip of the fibre, which was implanted 0.3 mm above the targeted nucleus. For eNpHR photoinhibition, two optical fibres were attached to the double cannula for constant illumination of the targeted site  of the targeted site. A 589-nm laser  was continuously turned on throughout the post-stress behaviour sessions. For chemogenetic manipulation, clozapine N-oxide (CNO) , or vehicle  was administered via the implanted cannula 40 mins before grooming was assessed. For all behavioural experiments, the animals were videotaped, and the behaviours were evaluated offline.TM 405-conjugated streptavidin–biotin complex (ThermoFisher). The expressions of biocytin and CTB-555 were examined under confocal laser scanning microscope .For retrograde tracing, recombinant cholera toxin-b conjugated to AlexaFluor-488  in PBS was injected to the target site at LSv. At least 7 days were allowed for complete retrograde transport before sacrifice. For anterograde tracing, AAV9-Syn-ChR2-eYFP was injected into LSv and allowed at least 4 weeks for full expression. Brain sections (30 μm) were prepared and examined under confocal laser scanning microscope . To establish the disynaptic circuit of VS → LSv → Tu, AAV9-Syn-ChR2-eYFP was injected into VS for at least 4 weeks for expression. One week prior to sacrifice, recombinant cholera toxin-b conjugated to AlexaFluor-555  in PBS was injected into Tu. Coronal slices of 300 μm were prepared by using a vibrotome (Campden 5100MZ-PLUS Vibrotome) for whole-cell patch recordings. Neurons in LSv that responded to blue light stimulation with EPSC (oSPSC) were filled with biocytin. Biocytin was revealed with Alexa FluorFor all behavioural tests, animals were habituated for 30 min/day consecutively for 3 days before testing. The behaviours of the animals were recorded in a test chamber  by a video camera . Each animal was tested at least three times on different days to verify reproducibility.For spontaneous grooming model, the animal was placed in the test chamber, and the spontaneous activities were recorded for 20 min.32, the animal was restrained in a black tube  for 20 min and then put immediately into the test chamber for 20 min of video recording.For restraint stress-induced grooming37. The animal was exposed to the bright light (60–80 W) from a lamp, 10 cm from above the home cage, for 20 min. Immediately afterward, the animal was placed in the test chamber, and its behaviour was recorded for 20 min.For bright light-induced grooming, animals were transported to the dimly lit laboratory and left undisturbed for 30 min prior to exposure to the light box on the day of the experimentFor optogenetically induced grooming, the behaviour was monitored for a total of 15 min, following a 5-5-5 min protocol in which blue light pulses  was turned on in the middle 5 min. Grooming and other behaviours were video-taped. To test for social grooming behaviour, two animals were placed in the same chamber, and the same 5-5-5 min protocol was applied.25, the animal was sprayed with water squirts directed to the face, belly, and back (four squirts per area) with a spray bottle pre-filled with sterile water (25 °C) as the “whole-body” water spray model. For “head-only” model, water was directly sprayed to the left and right face (four squirts per side). For “body-only” model, water was sprayed to the belly and back (eight squirts per area). It was then placed in the test chamber for video recording of the behaviour for 10 min. Before testing, the animals were habituated once per day for 3 consecutive days.For water spray-induced grooming38, the animal was placed in an open swimming pool (100 cm × 50 cm and 50 cm high) that was filled with water at 25 °C. Water depth was set at 15 cm to ensure that the rat could stand freely and allowed them to freely swim for 5 min. After at least 3 days of adaptation, animals were allowed to swim for 2 min on the day of grooming testing, and excess water was then removed and allowed to drain away before the animals were placed immediately into the test chamber for 10 min of video recording.For swimming-induced grooming67, the following behaviour tests were performed to evaluate the emotional status associated with different grooming-inducing paradigms.Based on the natural aversion of rodents towards brightly lit areas and at the same time a tendency towards exploratory behaviourFor open-field with a nest test, a dark shelter nest (20 cm × 20 cm and 40 cm high) was put in the corner of an open field (100 cm × 100 cm and 40 cm high). Animals were handled and habituated for 10-min to the open-field 2 days before the test. On the testing day, each rat was allowed to first freely explore the box for 5 min. Then, immediately after going though the induction protocol of a grooming model, the rat was placed in the box at the corner diagonal to the nest. The latency of escape to the nest was recorded as an index of stress-like behaviour. A shorter latency indicates a higher stress level.67, the test box consists of two boxes  and connected by an open door. When testing, the animal was first placed in light box and the total time spent in light box was recorded for a period of 5 min. The less time spent in the light box indicates a higher stress level.For light–dark box test67. The maze was consisted of a central square (15 cm × 15 cm), two open arms without walls (15 cm × 40 cm) and two closed arms with walls (15 cm × 40 cm and 40 cm high). Each rat was placed in the centre of the maze, after which the rat behaviour was recorded for a period of 5 min. The time spent in the open arms was used as an index of stress-like behaviour.For EPM test, it was used to measure anxiety-related behaviour in rodents37.Behavioural analysis: The grooming and other behaviours were quantified manually by three observers with the aid of a video editing software . The observers were blind to the experimental conditions. Social grooming was assessed when a rat licked and chewed the fur of the conspecific, while placing its forepaws on the back or the neck of the other rat. Arousal behaviours including rearing and heading were also examined. For gross analysis of self-grooming, the number of grooming bouts, the duration of individual bout and therefore the total grooming time in the test period, were evaluated. Self-grooming was defined as when the animal licked, or used the forelimb to stroke, its own body parts including the paws, nose, eyes, head, body, legs, tail and genital. An interruption of 6 s or more separated two individual bouts37. In addition, no grooming was defined as phase 0. To facilitate the identification of these patterns by the observers who were blind to the treatment, the recorded videos were replayed at 1/4 of the actual speed. By noting the time points of the start and end of these different phases, the exact sequences of the different phases within a grooming bout was determined. A grooming bout was considered interrupted if at least one pause in action (<6 s) was recorded within its transitions of phases. In addition, the transitions between different phases of grooming pattern were used to evaluate the ‘correct’ transitions based on the conventional cephalocaudal progression stereotypy: (0 → 1), (1 → 2), (2 → 3), (3 → 4), (4 → 5), and (5 → 0) and the otherwise ‘incorrect’ transitions represented by other patterns. From these data, the total number of transitions and the percentages of incorrect transitions were calculated.To analyse the patterns and microstructures of grooming behaviour under different conditions, different phases, or stages, of grooming activities were defined, including paw licking (phase 1), nose/face/head grooming (phase 2), body grooming (phase 3), leg grooming (phase 4), tail and genital grooming (phase 5), according to conventional protocolTo analyse the microstructures, all grooming bouts under each of the six experimental conditions were collected and pooled. Then, the transition probabilities from one phase to another phase were calculated, and a transition probability matrix was obtained. As an example, in spontaneous grooming, the animals together spent a total of 112 times in phase 1 (paw-licking). The transitions from paw-licking to other phases took place at the following frequencies: 9 (1 → 0), 96 (1 → 2), 4 (1 → 3), 2 (1 → 4) and 1 (1 → 5). The transition probabilities were therefore 8.04%, 85.71%, 3.57%, 1.79% and 0.89%, respectively. The overall patterns of phase transitions could be visually captured by the transposed transition matrix and connectivity graphs shown in Fig. D) and Pearson’s correlation coefficient (CC) for each pair of matrices, whereasTo be able to compare quantitatively the similarity among the transition probabilities matrices, we computed two indices, namely the Euclidean distance . The percentages of time that the animal spent on the stimulated and unstimulated sides of the chamber in a period of 15 min were quantified.The animal was put in a box , which was divided into two equal chambers without any contextual cues69. The procedure consisted of three stages: preconditioning (baseline), conditioning, and testing phases. On the first day, the sliding door was retracted, and rats could explore the entire apparatus freely for 15 min (3 × 5 min). Animals that spent >70% of time in either of the compartments were excluded from further analysis. Immediately following the preconditioning phase, the rats underwent conditioning in both sides, respectively. During conditioning, one of the two chambers was paired with a photo-stimulation  for 2 × 3-min/day for 3 days. During the test phase, the animals did not receive any treatment and had free access to both compartments for a total time of 10 min. Animal movements in each of the chambers were recorded, and the time spent in each chamber was analysed by the Anymaze software.The animals were habituated to handling for 3 days prior to the beginning of the procedure. Experiments were conducted in two interconnected chambers (60 × 40 and 40 cm high each) that could be separated by a sliding door. The two chambers were decorated with different forms of stripes on the wall. Animal movements in each chamber were recorded and analysed with the Anymaze software71. The rats having completed in vivo optogenetic stimulation experiments were deeply anesthetized by isoflurane, and then perfused transcardially with 100 ml room temperature NMDG artificial cerebrospinal fluid (aCSF) containing (in mM): 92 NaCl, 2.5 KCl, 1.25 NaH2PO4, 20 NaHCO3, 10 HEPES, 25 Glucose, 5 Na-ascorbate, 2 thiourea, 3 Na-pyruvate, 10 MgSO4, 0.5 CaCl2, 12 N-acetyl-l-cysteine, which was saturated with carbogen (95% O2/5% CO2) prior to use. After perfusion the rats were decapitated and the brains were gently extracted from the skull and placed into the cutting solution (NMDG aCSF) for an additional 30 s. Coronal sections at 300 μm were cut with a vibrotome (Campden 5100MZ-PLUS Vibrotome) and transferred into a holding chamber containing 34 °C NMDG aCSF to recover for 10 min. After the initial recovery, the slices were transferred into a new holding chamber containing HEPES holding aCSF containing (in mM): 92 NaCl, 20 NaHCO3, 25 Glucose, 2.5 KCl, 1.25 NaH2PO4, 10 HEPES, 5 Na-ascorbate, 2 thiourea, 3 Na-pyruvate, 2 CaCl2, 2 MgSO4, 12 N-acetyl-l-cysteine. After another hour of recover, the slices could then be transferred to the recording chamber with normal aCSF containing (in mM): 125 NaCl, 2.5 KCl, 11 Glucose, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, and 2 MgCl2.Adult rat brain slices were prepared as following procedures2, 2 mM Na2-ATP, 0.4 mM Na3-GTP. For optically evoked inhibitory postsynaptic currents (oIPSCs), the internal solution was (in mM): 50 mM KCl, 10 mM HEPES, 1 EGTA, 2 mM MgCl2, 2 mM Na2-ATP, 2 mM Tris GTP. Light pulses at 470 nm were delivered through a patterned light stimulator  to activate ChR2 while recorded neurons were voltage-clamped at −70 mV. Access resistance (<20 MΩ) was not compensated and monitored continually throughout each experiment. Recordings were terminated whenever the input resistance increased >30% or access resistance exceeded 20 MΩ. Signals were acquired using a MultiClamp 700B amplifier controlled by Clampex 10.4 software via a Digidata 1550 interface (Molecular Devices). Responses were filtered at 3 kHz, digitized at 10 kHz, and analyzed using Clampfit 10.7 (Molecular Devices). In a subset of experiments, biocytin was included in the internal solution. After recording, slices were fixed overnight in 4% paraformaldehyde. Filled neurons were identified by staining using streptavidin-conjugated Alex 405 (ThermoFisher).For recording of optically evoked action potential, excitatory postsynaptic currents (oEPSCs) and spontaneous firing, we used internal solution containing (in mM): 130 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 1 EGTA, 2 mM MgClTo monitor the neuronal activity in LSv, the rats were injected with 100 nl of AAV9-Syn-GCaMP6s-WPRE-SV40 or AAV5-hSyn-eGFP into the right LSv. Two weeks after the virus injection, an optical fibre  was implanted to target the right LSv. With at least 1-week recovery, the optical fibre was connected to FibreOptoMeter (Plexon’s Multi-Fibre Photometry) through an optical fibre patch cord  for photometry recording. To record fluorescence signals, a beam from a 470 LED was reflected with a dichroic mirror, focused with a lens coupled to a PMT. The LED power at the tip of the patch cord was <50 μW. Fluorescent signals were collected and synchronized with behaviour of the animal via the CineLyzer system .F/F values obtained from CineLyzer system were exported to Matlab for further analysis. The data were represented by heatmaps or averaged response (shaded area indicate SEM) in a time window between 9 s before and 3 s after grooming onset. Smoothed ΔF/F values were calculated through three different steps: To begin with, the raw fluorescence was averaged to get FAVG (n) over a sliding time window (0.75 s) in a symmetrical pattern. The baseline for a particular frame (FBASELINE (n)) was the minimum of the FAVG (n) in a time window (3 s) preceding this frame. Next, ΔF was calculated by subtracting FBASELINE (n) from FRAW (n) and then dividing by FBASELINE (n). Finally, ΔF was smoothed with an exponentially weighted moving average. The exponential weighting was described by a time constant For analysis of photometry data, smoothed ΔRats were anaesthetized and perfused with PBS followed by 4% PFA in PBS. The brain was fixed in 4% PFA at 4 °C overnight. Fixed samples were sectioned into 30-μm coronal sections using a cryostat (ThermoFisher). For c-Fos staining, the brain samples were prepared 60 min after behavioural test followed by fixation. The brain sections were blocked in 10% normal goat serum in PBS with 0.3% Triton X-100 in PBS for 2 h and then incubated with anti-c-Fos antibody  at 4 °C overnight. After thorough rinsing in PBS, the slices were incubated with goat anti-rabbit lgG secondary antibody  in block solution. The sections were rinsed in PBS and counterstained with DAPI and mounted. Images were taken under a confocal laser scanning microscope .Patch pipettes were pulled and filled with 3.0–5.0 μl of nominally RNAse-free internal solution using a microloader. The tip diameter of the pipette was around 1/3 of the size of cell body. Under visual inspection, the cell was harvested into the patch pipette by applying a negative pressure. The cell mixture was put into a tube on wet ice for at least 1 min. The tube was frozen in liquid nitrogen and stored at −80 °C until the RT reaction was carried out. Synthesis of first-strand cDNA from 3 μl (for qPCR) of total RNA (in 10 μl) was carried out with the Prime Script RT reagent Kit . qPCR was run on iCycler (Bio-Rad) by using SYBR Green Supermix (Bio-Rad). A multiplex two-round single-cell qPCR was carried out for simultaneous detection of vesicular glutamate transporters (vGluT2), glutamic acid decarboxylase 67 (GAD67). The first PCR conditions included an initial denaturation for 5 min at 94 °C, 30 cycles with 45 s denaturation at 94 °C, 45 s annealing at 61 °C and 70 s extension at 72 °C, final elongation 7 min at 72 °C. An anliquot (5 μl) of first round PCR product used as a template for the second round PCR (40 cycles) using nested primers. The conditions were the same as described for the first round. Products were identified with gel electrophoresis with GelRed nucleic acid gel stain (Biotium). The oligonucleotide primers used for single-cell RT-PCR are listed in Supplementary Table Experiments were repeated independently with similar results at least three times. Micrographic images presented in figures are representative ones from experiments repeated independently: Fig. t-test, paired two-tailed t-test, one-way ANOVA with Tukey post-hoc test, two-way ANOVA unless otherwise indicated. Values are reported as mean ± standard error of the mean (SEM). The cutoff value of significance was P = 0.05. Electrophysiology data was analyzed using Clampfit 10.7 (Molecular Devices). All figures were prepared with Photoshop CC 2017, Adobe Illustrator CS6 (version 16), Microsoft Excel 365.Statistical analysis was performed using GraphPad Prism 7.02, using unpaired two-tailed Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Reporting Summary


Conventional protocols utilize core needle biopsy (CNB) or fine needle aspiration (FNA) to produce cell suspension for flow cytometry (FCM) is a diagnostic challenge for lymphoid malignancies. We aim to develop an alternative CNB rinsing technique (RT) to produce cell suspension for FCM during this mini‐invasive procedure of CNB for lymphoma diagnosis.FNA and CNB specimens from the same lesion of 93 patients with suspected lymphoma were collected under the guidance of B‐ultrasound simultaneously. The fresh CNB samples were prepared to cell suspension by RT for FCM immunophenotyping analysis (Group CNB‐RT). Then, the CNB tissues after performing the RT process and the fresh FNA tissues were processed by conventional tissue cell suspension (TCS) technique to obtain the cell suspensions (Groups of CNB‐TCS & FNA‐TCS), respectively, as comparison. The diagnostic efficacies, as well as the concordances of the FCM results with reference to the morphologic diagnoses were compared in these three groups.p = 0.819). The diagnostic sensitivity and specificity of CNB‐RT  was not inferior to that of CNB‐TCS  and FNA‐TCS .RT could yield sufficient cells for FCM immunophenotyping analysis, though a lower cell numbers compared to TCS technique. The diagnostic concordance was comparable in group CNB‐RT (91.1%) to the group CNB‐TCS (88.9%) and group FNA‐TCS (88.4%)  rinsing technique (RT) to produce cell suspension for flow cytometry (FCM) during this mini‐invasive procedure of CNB for lymphoma diagnosis. We found that the CNB‐RT presented non‐inferior diagnostic concordance and efficacy as compared to the conventional tissue cell suspension (TCS) technique. CNB‐RT has the potential to produce cell suspension for FCM immunophenotyping while preserving tissue for lymphoma diagnosis and research. It has been reported that bone marrow core biopsy specimens were prepared to cell suspension by "vortex" method for FCM immunophenotyping detection.22.1About 93 consecutive patients with suspected lymphoma were recruited in this prospective study from May 3, 2017 to February 8, 2018. We collected the FNA and CNB specimens from the same lesion simultaneously during Ultrasound guided CNB for these patients. Each patient was numbered from case 1 to case 93 according to the sampling sequence. The lesion sites were classified into superficial versus deep‐seated, and lymph node versus extranodal lesions. The superficial lesions included the cervical, clavicular region, parotid region, lateral lobe of thyroid, axilla, breast, groin, thigh muscle layer, and testis. The deep‐seated lesions included the thorax, abdomen, retroperitoneum, and pelvis regions.The primarily collected CNB tissues (at least two cores) were sent to pathological department for the histopathological evaluation which was regarded as the standard reference. Additional one core of CNB tissue was subsequently collected for preparation of the cell suspension by RT (Group CNB‐RT), which was processed as follows: One core of fresh CNB was submerged and stirred gently for three to four times in phosphate buffer (PBS) with PH7.4 using sterile forceps at room temperature immediately after they were collected. The cell suspension in PBS medium which was obtained by RT, devoid of tissue, was sent for FCM immunophenotyping analysis immediately. Then, the CNB tissues after performing the RT process and the fresh FNA tissues were processed by TCS to obtain the cell suspensions for FCM detection respectively (Groups of CNB‐TCS and FNA‐TCS). The conventional TCS technique was performed using the traditional protocol in which a single or a portion of representative tissue core was disaggregated mechanically with knives and needles to produce a cell suspension for FCM analysis.The diagnostic concordances between the FCM immunophenotyping results and the morphologic diagnoses as well as the diagnostic efficacy of each group were compared in these three groups of consistently matched data. The FCM data of all patients in the study were analyzed by the same pathologist（Dr. Chi) without blinding to the technique type. The pathologic reports of CNB specimen biopsies were issued by the others pathologists on duty. It was double blind for that the pathologists of FCM immunophenotyping and biopsy detection did not know each other's results before the reports were issued. Dr. Chi judged whether the results of FCM immunophenotyping and CNB biopsy were concordant according to the following rules first. Then, a review group composed of Dr. Chi, Dr. Huang and Prof. ZM. Li would review all the reports of FCM and biopsies of each patient, and make a final judgment on the concordance of the results. For those discordant cases, Dr. Huang was responsible for reanalyzing the FCM data and rereading the biopsy slides. If the analysis results of Dr. Huang are consistent with initial reports, respectively, the case would still be classified as non‐concordance. If not, two or more other pathologists would be recruited to reanalyze the FCM data and CNB biopsy. The consensus diagnoses of the discordant cases were made by these pathologists. The concordant judgment standard of each disease entity was as follows: For cases of B‐cell and T‐cell lymphoma which were diagnosed by morphology, concordances were made if FCM analysis detected a monotypic B‐cell population with a restricted light chain expression or a dominant T‐cell population with aberrant phenotypes. Negative results or misdiagnosis to the other diseases by FCM would be considered discordant. For reactive and atypical cases, concordances were considered if the FCM results were negative, whereas discordances were made if diagnosed as lymphoma by FCM. Concordant judgments would be made if a borderline light‐chain excess or a small subset of monotypic B‐cells or abnormal T ‐cells were detected by FCM in suspicious cases diagnosed by morphology, otherwise it was considered discordant. Finally, for classical Hodgkin lymphoma (CHL) and non‐hematopoietic system diseases, a negative FCM finding for these two disease categories was considered concordant to morphologic diagnosis. Because the panels with B‐ and T‐cell markers we used for FCM analyses usually were limited without CD30, CD15, or other specific markers for CHL and non‐hematopoietic system diseases since the insufficient cells; Furthermore, FCM did not play an important role in the diagnosis of CHL and non‐hematopoietic system diseases due to the lack of a clonal population or specific antibodies. In our study, a total of 28 FCM specimens from three groups of CNB‐RT, CNB‐TCS, and FNA‐TCS were not concordant with the biopsy results, which were from 16 CNB tissue specimens. After reanalyzing by Dr. Huang, all these 28 FCM specimens and 16 CNB biopsy specimens were consistent with initial reports respectively and were still classified to the discordant cases. Thus, No more pathologists were arranged to review the data and slides.Sensitivity and specificity were used to evaluate the diagnostic efficacies of these three cell suspension methods. According to the definition of sensitivity (true positive rate), the concordant rate between FCM immunophenotyping and biopsy is consistent with the value of sensitivity.Prior to the use of the tissues, written informed consent was obtained from each of the patients. This study was approved by the ethics committees of Sun Yat‐sen University Cancer Center   and was conducted in accordance with the ethical standards of the World Medical Association Declaration of Helsinki. The authenticity of this article had been validated by uploading the key raw data onto the Research Data Deposit public platform (www. researchdata.org.cn), with the approval RDD number as RDDA2020001528.2.2The cases of CNB with adequate material were evaluated by hematopathologists based on the WHO classification of tumors of hematopoietic and lymphoid tissues 2016.2.3Before the FCM detection, an accurate dilution of cells in trypan blue vital stain counted on a hemacytometer chamber which gave an accurate volume allowed assessment of the viability as well as the number of cells in each suspension derived from three groups of CNB‐RT, CNB‐TCS, and FNA‐TCS.2.4U Test. Fisher's exact test was used to analyze the difference in diagnostic concordance among three groups. The data with a normal distribution were expressed as mean ±standard deviation, while the data with nonnormal distribution was expressed as median (range). All tests were two‐tailed, and p value <0.05 was considered significant.All statistical analyses were performed using SPSS 19.0 statistical software package . The comparison of the cell number was analyzed by Mann–Whitney 33.1A total of 93 patients with 279 cell suspension samples collected from FNA and CNB specimens in the same lesion simultaneously were enrolled. The mean age of the patients ± the standard deviation was 49.1 ± 17.2 years  and included 50 men and 43 women. There were 49 cases of superficial lesion and 44 cases of deep lesion. The nodal and extranodal lesions were 45 and 48 cases, respectively.p = 0.094) (Table 6 (0.002‐2.080), 1.060 × 106 (0.004‐6.338), and 3.296 × 106 (0.003‐59.803), respectively. Group FNA‐TCS had more cell number than the other two groups (p < 0.001).A total of 13 cases yielded an insufficient cell number for FCM within the cohorts. Both 3 cases  in groups CNB‐RT and CNB‐TCS, respectively; seven cases  in group FNA‐TCS. FCM was performed on the rest of quantity sufficient cases, including both 90 cases  in the groups CNB‐RT and CNB‐TCS, respectively; 86 cases  in the group FNA‐TCS. No significant difference was found in the cases of insufficient cell number for FCM analysis among these three groups (4) Table . The via3.2p = 0.819). More specifically, the concordant cases between the FCM results and morphologic diagnosis in three groups according to the categories of morphological diagnosis are listed in Table The concordant and discordant cases with sufficient cells for FCM detection in three groups are listed in Table For classical Hodgkin lymphoma (CHL), reactive and atypical /suspicious lymphadenopathies, FCM and morphologic evaluation also showed 100% concordance rate among three groups, respectively.Twenty cases (58 samples) of non‐hematopoietic system diseases were detected by FCM including one case of gastrointestinal stromal tumor, low‐differentiated squamous cell carcinoma, fibroma, lympho papillary cystadenoma, and neurinoma, respectively; two neuroendocrine neoplasm; three cases of low‐differentiated adenocarcinoma, small cell carcinoma and thymoma, respectively; and four lymphoepithelioma‐like carcinoma. One case with three samples that the histologic diagnosis was small cell carcinoma, a significant CD45‐negative population of intermediate sized cells was identified by FCM in three groups. We added the cytokeratin marker to confirm the epithelial origin. The other 19 cases (55 samples) of non‐hematopoietic system diseases were negative by FCM studies. In these non‐hematopoietic system diseases, the concordance rates were 100% in both groups of CNB‐RT and CNB‐TCS but with a lower rate of 94.4% (17/18) in group FNA‐TCS. The discordant case in group FNA‐TCS was abnormal T‐cell by FCM analysis, whereas the histologic diagnosis was thymoma.For DLBCL cases, 22 of 26 samples (84.6%) in group CNB‐RT, 20 of 26 samples (76.9%) in group CNB‐TCS and 21 of 26 samples (80.8%) in group FNA‐TCS were successfully identified by FCM studies. Overall, there were in total 15 of 78 DLBCL samples (19.2%) had a negative FCM diagnosis, probably due to those large neoplastic cells did not survive the harsh of tissue sampling and FCM processing.Discordance between FCM and morphologic diagnosis was mainly identified in unclassified B‐cell lymphoma (B‐UCL) and T‐cell Lymphoma among three groups. Two cases  of B‐UCL were both diagnosed as B‐cell lymphoma, unclassifiable, with features intermediate between DLBCL and CHL by morphology whereas the FCM results were all negative in three groups. This directly led to the concordant rate of B‐UCL in all three groups being zero. For T‐cell lymphoma, there was only one out of three samples  was concordant in groups CNB‐RT and CNB‐TCS, respectively; while one of two samples  was concordant in group FNA‐TCS. The concordant samples in three groups were derived from the same patient that was diagnosed as T‐cell prolymphocytic leukemia by morphology. The other two discordant samples were diagnosed as angioimmunoblastic T‐cell lymphoma (AITL) and NK/T‐cell lymphoma by morphology while the FCM results were all negative.Herein, we presented a case of DLBCL, germinal centre B‐cell (GCB) subtype detected by morphology and FCM using three cell suspension preparation methods in Figure 3.3The values of sensitivity and concordant rate of each disease category were the same according to the definition of sensitivity. There was no significant difference in the diagnostic sensitivity of the three cell suspension preparation methods, although the value of group CNB‐RT was slightly higher. The specificities of groups CNB‐RT, CNB‐TCS, and FNA‐TCS were very high, reached 100%, 100%, and 98.8%, respectively Table . There w4p < 0.001), it could yield sufficient cells for FCM immunophenotyping analysis. Furthermore, the concordant rate of which was gratifying and was not inferior to that of groups CNB‐TCS and FNA‐TCS (p = 0.819). FNA specimen usually mixed with blood due to the aspiration movement during sampling, furthermore, groups CNB‐RT and CNB‐TCS shared the same CNB tissue core. These two reasons could well explain why the groups CNB‐RT and CNB‐TCS had fewer cells than group FNA‐TCS. In addition, the number of QNS cases in the CNB‐RT was the same as that in the group CNB‐TCS (three cases), and less than that in the group FNA‐TCS (seven cases). In six of seven QNS cases in group FNA‐TCS, the corresponding cases in groups CNB‐RT and CNB‐TCS were able to obtain enough cells for FCM study  using CNB cores to prepare the cell suspension for FCM analysis. We found that though the cell number of suspension in group CNB‐RT was the fewest among three groups and there was a significant difference  and specificity of group CNB‐RT were both 100%, which were better than or equal to that of groups CNB‐TCS and FNA‐TCS. In these types of diseases, not only the techniques of CNB‐TCS and FNA‐TCS, but also CNB‐RT strongly supported the final diagnosis.For DLBCL, the sensitivity (concordant rate) of group CNB‐RT (84.6%) was lower than the above five diseases entities (100%). However, the specificity of group CNB‐RT was also 100%, indicating a high true negative rate. Some scholars conclude that in cases of DLBCL, the malignant morphologic features are typically so apparent that FCM analysis is often not necessary for a confident morphological diagnosis of lymphoma.B‐UCL and T‐cell lymphoma were the two disease categories with low sensitivity (concordant rate). Two B‐UCL cases  could not be detected by all the three methods which resulted in a concordant rate of 0%. However, the other seven disease categories had not been misdiagnosed as B‐UCL, thus, the specificities of all three groups in B‐UCL disease was 100%. Since grey zone lymphoma usually did not express surface immunoglobulins, if these large cells did not die during processing, we could see some large B‐cells with negative for both light chains, which were abnormal. Nevertheless, we could not observe these FCM results. We thought either the large cells died during the processing or unsuccessful sampling resulted in the discordance. Extensive necrosis or much less tissue was seen under the microscope in these two B‐UCL cases. For T‐cell lymphoma, the sensitivity (concordant rate) of group CNB‐RT  was not superior to group CNB‐TCS  and group FNA‐TCS . The specificity of CNB‐RT was 100%, which was equal to that of group CNB‐TCS and slightly better than that of group FNA‐TCS (98.8%). Although FCM analysis was helpful in identifying an abnormal T‐cell population, not all T‐cell lymphomas had distinct immunophenotypes. The inherent limitations and the sampling error could add the difficulty of FCM analysis for T‐cell lymphoma.Whether the sampling is successful or not as well as the status of specimen directly affects the FCM results, either the RT method or the TCS method. We noted that almost all the QNS samples  for FCM analysis in three groups although the pathological examination gave a definite diagnosis; there were more necrosis, fibers, fat, or connective tissue in the background under the microscope. Therefore, it was comprehensible that the cell numbers of suspensions sampled from the same lesion site for FCM study would be insufficient. Furthermore, 24 of 28 (85.7%) discordant samples could also be observed with extensive necrosis and more fiber by the histologic detection, hence, the sampling errors could not be excluded in the discordant cases either.In general, our results indicated that RT had a high sensitivity and specificity on assisting diagnosis for the cases with suspicious lymphoma, especially for the low‐grade B‐cell lymphoma, followed by DLBCL. The specificity of RT was superior to the sensitivity, which was the same as those reported by TCS method.5RT is a simple, rapid, and effective way to preserve the maximum amount of tissue for lymphoma diagnosis and research while providing the suitable quantity and quality cell suspension for FCM detection as compared to the traditional TCS technique. RT can replace TCS technology, especially in cases with very few tissues. Therefore, we advocate the application of RT in the primary diagnostic procedure for patients with suspected lymphoma.The authors confirm that there are no conflicts of interest.Pei‐Dong Chi: Data analysis of FCM immunophenotyping, methodology, and writing‐original draft, data curation, and formal analysis. Yi‐Jun Liu: Treatment of tissue specimens and FCM immunophenotyping detection. Yu‐Hua Huang: Pathological evaluation of lymphoma. Ming‐Jie Mao: Statistics of data. Yu Wang: Resources. Zhi‐Ming Li: Resources, supervision, and writing‐review and editing. Jian Li: The sampling of tissue specimens, conceptualization, supervision, and writing‐review and editing.Table S1Click here for additional data file.


Background: Placental chorioangioma is the most common benign non-trophoblastic neoplasm of the placenta. Its clinical relevance lies in the size of the tumor since larger masses cause pregnancy complications, including an unfavorable neonatal outcome. Case presentation: We report the case of a 34-year-old second gravida and nullipara at the 35th week of gestation, admitted to the gynecological department for antibiotic-resistant fever. The cardiotocography performed during hospitalization showed an abnormal fetal pattern. A 2250 g newborn was delivered by cesarean section. No complications were observed during childbirth and postpartum was insignificant. On gross inspection a white fleshy intraparenchymal mass blooming on the maternal surface was noted; routinely stained sections revealed features consistent with chorioangioma with vascular channels lined by inconspicuous endothelial cells immunoreactive for CD31 and CD133. Focal expression of CD133 was also observed in placental villi. Discussion: CD133 expression indicated the presence of stem cells in chorioangioma, suggesting their possible role in the development of mesenchymal lesions including chorioangioma. Placental chorioangioma is a fetal-stromal vascular lesion, being part of a wider subgroup of lesions, called villous capillary lesions, which includes the following variants characterized by excessive angiogenesis: chorioangiosis , chorioangiomatosis , chorioangioma (similar to chorioangiomatosis for stem and immature intermediate villi involvement but different for the formation of a nodular expansile lesion) and multiple chorioangioma syndrome, an infrequent variant with several concomitant chorioangiomas occupying up to 80% of the total volume of placental parenchyma ,3,4. ChoThe first vascular tumors were described by Clarke in 1798 ,7. In teThe main purpose of our report was to demonstrate the expression of CD133 in placental chorioangiomas which have never been described before. We believe that the widespread positivity of CD133 in chorioangioma may be suggestive of stem cell involvement in its formation.secundigravida nullipara, was admitted to our Obstetrics and Gynecology Unit of the Policlinico Hospital of Modena at the 35th week of gestation because of an antibiotic-resistant fever. A rapid swab test for SARS coronavirus 2 was negative. She was hemodynamically stable. Pertinent laboratory examinations resulted within the normal limits, and other infections were ruled out. Ultrasound examinations of placental plate and placental annexes were unremarkable and fetal biometry was within the ranges. However, a pathological cardiotocography tracing was observed and subsequent hospitalization was required to perform an urgent cesarean section. A vital 2250 g newborn (Apgar score 8 at 1 min and 9 at 5 min) was delivered.A 34-year-old woman, Placenta, membranes and umbilical cord were sent for histological examination. On gross inspection, the fixed placenta specimen measured 17 × 14 cm and weighed 406 g, showing a 6 cm white fleshy intraparenchymal mass blooming on the maternal surface A,B. HistEndothelial cells were diffusely immunoreactive for CD31   A. PlacenPlacental chorioangioma is the most common non-trophoblastic benign neoplasm of the placenta. Chorioangiomas occur in about 1% of all pregnancies ,8,9,10 aSmall chorioangiomas <5 cm) are usually asymptomatic and are usually not diagnosed or incidentally found at histological examination , while l cm are uChorioangioma can sometimes be confused at ultrasound examinations with placental teratoma, degenerated myoma and blood clot , but hisWe present an unusual case of giant chorioangioma in a second gravida nulliparous woman at the 35th week of pregnancy. Clinically, most of the large or giant chorioangiomas are symptomatic and they can lead to pregnancy disorders, ranging from vaginal bleeding to poor fetal outcome and severe intrapartum complications in the most serious cases. The case in examination was asymptomatic, being one of the few clinical exceptions to what is reported in the literature ,32, and reservoir for the blood supply favoring the symbiotic growth of the fetus and the lesion, without any manifestation of the latter. The fever that the patient had at the beginning was probably caused by a possible sub-clinical infection, then spontaneously regressed.In terms of clinical implications, a review of 2010 confirmed the high rate of fetal complications in large or giant chorioangiomas . Differe+ precursor cells may be the source of endothelial cells underlying the congenital vascular malformation [Histologically, the mass was defined as a cellular chorioangioma because it showed numerous small vessels within a relatively scant stroma with the presence of coarse calcifications. The neoplasm does not seem to depend on the maternal estro-progestin hormone state, as opposed to other gynecological vascular lesions that express estrogen. However, interestingly the tumor cells in the vessels showed widespread positivity to CD133, a typical marker of cancer stem cells ,35. The ormation . On the ormation . The preIn this paper we would like to highlight the characteristics of a clinical unexpected manifestation of a giant chorioangioma and to report the observed widespread positivity of CD133, suggesting a pivotal involvement of stem cells in its formation. The identification of these cells, likely responsible for the growth of vascular structures presumably through a switch from a trophoblastic-mesenchymal phenotype to a vascular phenotype, may allow the identification of new possible future applications of CD133 as a histological marker of chorioangiomatous lesions as well as new therapeutic targets in order to decrease pregnancy complications. Nevertheless, the prenatal screening of placental vascularization may help to select the population of pregnant women who need a strict follow-up in order to identify earlier fetal impairment.


Pelodiscus sinensis using transmission electron microscope (TEM), immunohistochemistry (IHC), and immunofluorescence (IF) techniques. TCs surrounded the acini and ducts of the connective tissue of the exocrine pancreas and between lobules and gland cells. The cells were located preferably close to the blood vessels, interlobular ducts, and nerve fibers. Ultrastructurally, TCs exhibited small and large bodies with thick and thin portions, podoms, and podomers, and prolongations that form dichotomous branching with hetero-cellular and homo-cellular junctions. The podom (thick) portions showed caveolae, mitochondria, rough endoplasmic reticulum, and vesicles. The nucleus carries heterochromatin and is irregular in shape. The shape of TCs depends on the number of telopodes (Tps) bearing long, short, spindle, triangular, and “beads on a string” shapes with twisted, tortuous prolongations and ramifications. Shed extracellular vesicles and exosomes were found frequently released from projections and Tps within connective tissue in the vicinity of the acini and collagen fibers. IHC and IF results showed CD34+, α-SMA+, and vimentin+, long and triangle-shaped TCs, consistent with the TEM findings. The presence of shaded vesicles from TCs might implicate their possible role in immune surveillance, tissue regeneration as well as regulatory functions in the reptilian pancreas.The existence of telocytes (TCs) has not yet been established in the pancreases of aquatic reptiles. Here, we report TCs in the exocrine pancreas of  Telocytes (TCs) are interstitial cells of mesenchymal origin, first reported by Popescu and Faussone-Pellegrini in 2005. They were named interstitial Cajal-like cells (ICLCs) and interstitial cells of cajal (ICCs) based on their morphology, ultrastructural features, immunophenotype, and site of existence. Later on, ICLCs were renamed TCs ,2,3,4. TThe distinct morphological features of TCs entail the unique physiological roles in the tissues. More roles of TCs could be expected in the cellular organization of the tissues during development and in the postnatal life. In heart, TCs guide and maintain the tissue organization and complex three-dimensional morphology during development , and forFurthermore, TCs secrete extracellular vesicles that play an important role in supporting tissue repair/regeneration and nurse function, intercellular signaling, cell differentiation , homeostPelodiscus sinensis) are a fresh water species that belongs to the family Trionychidae. The turtle are ectothermic animals, and these animals are, commercially, the most important species of aquaculture in Asian countries. P. sinensis have high nutritional and pharmacological values [Chinese soft-shelled turtles  via transmission electron microscope (TEM), immunohistochemistry (IHC), and immunofluorescence (IF). Close associations between TCs and other neighboring cells are reported in blood vessels (BVs), smooth muscle cells (SMCs), nerve fibers (NFs), nerve tracts, inter lobular ducts, gland cells, and collagen fibers (CFs). Extracellular / shed vesicles from podoms and Tps in the vicinity of BVs, CFs, and ducts were observed. This will help for understanding the role of the pancreas in innate immune functions, tissue regeneration in the field of cell biology, and ultrastructural insights into the possible functions of TCs in reptiles. Our present study confirms the presence of TCs within the connective tissue in the lobules and gland cells within the pancreas of Chinese soft-shelled turtles , along with collagen fibers  A,B. Tps 1 and Tp2 (podoms to podomers), adjacent to the acinus (1), in close contact with podoms to podomers (Tp2)  C. Tps cors (Tp2) D. It is Tps surround the interlobular duct long and short convoluted processes with many twists, with overlapping of the podoms and podomers between the acinus, where the macrophage is shown inside the duct surrounded by Tps A,B. The 1), and Tp2 mitochondria and vesicles  and podomers (thin). Furthermore, Tps are long, convoluted, and moniliform. The podomic “triad” (dilated segment) consisting of caveolae, mitochondria, and endoplasmic reticulum is involved in Caovements . Our resovements ,19,30,43ovements . Some stovements . There hovements ,8,68. Coovements , in the ovements  and testovements . Using dDevelopmental studies on lungs in rabbit have established the proportion between TCs during gestational and postnatal stages. There was a significant reduction of population and cell diameters of TCs, while similar increment in the length of Tps during the advancement during postnatal life. The study concluded that the TCs contributed to the angiogenesis, establishment of blood-air barrier and organization of the lung parenchyma during the development . TCs andAmphibian telocytes have a classical similarity in ultrastructure to mammals. The difference in TCs between amphibians and mammals is that Tps of amphibian TCs are not convoluted like mammalian telopodes, perhaps because they do have dichotomous branching . PreviouIn this study, we demonstrated the presence of TCs and Tps within the connective tissue in between lobules and gland cells. Within the connective tissue, there are a lot of TCs, and their long elongated Tps surround the large blood vessels and the interlobular duct. In between the gland cells, a small amount of Tps and a large quantity of small blood capillaries exist. Moreover, the shed/extracellular vesicles can also be found from the podoms within the connective tissue in between the gland cells, collagen fibers, large and small blood vessels, Tps, and interlobular duct. On the basis of this evidence, it is suggested that TCs and Tps within the connective tissue in between lobules and gland cells in the pancreas of Chinese-soft shelled turtles, along with large and small blood vessels, interlobular ducts, nerve fibers, and collagen fibers, may be functionally related. Accordingly, telocytes may play a functional role in the immune reactions and secretion in the pancreas, the endocrine, as well as the exocrine system.P. sinensis (3–4 years), were purchased from a wild breeding base in Jiangsu province of China. Each animal weighed 650–750 g. The animals were anesthetized by sodium pentobarbital (20 mg/kg body weight), administered intraperitoneally, and were then killed by cervical dislocation. The pancreas samples were collected for transmission electron microscopy. The experimental procedure was so designed as to minimize the suffering of the animals. The slaughtering and sampling procedures were approved by the Nanjing Agricultural Veterinary Experimental Animal Ethics Committee and Science and Technology Agency of Jiangsu Province (SYXK (SU) 2010-0005, approved on 1 January 2010).Ten adult (of either sex) Chinese soft-shelled turtles, 3 in size were fixed in 2.5% glutaraldehyde in 0.1 MBPS solution (pH 7.4) at 4 °C overnight. Post-fixation of the turtle pancreas was done in 1% osmium tetroxide  for 1 h. The specimen washing was done in distilled water and stained en bloc with 2% aqueous uranyl acetate, then washed again in distilled water (3 × 5 min). Dehydration was done in ethanol, and specimens were embedded in Epon 812 R . Ultrathin 50 nm-thick sections were mounted on copper grids and contrasted with uranyl acetate and lead acetate. Specimens were observed under the Hitachi TEM system .Blocks of turtle pancreas approximately 1 mmThe turtle pancreas samples used 10% formalin for tissue fixation. Tissue samples were first embedded and made into paraffin wax blocks, and then tissue sections were cut into 6 µm before being deparaffinized and rehydrated. Ethyl alcohol  was then used in decreasing concentrations. Samples were incubated in 3% hydrogen peroxide to block endogenous peroxidase activity for retrieval of antigen. Bovine serum albumin (BSA) 5% was used for blocked specimens, which were placed for 30 min at room temperature, followed by incubation with rabbit anti-CD34  and mouse anti-αSMA  antibodies for 24 h at 4 °C. Sections were washed and incubated using biotinylated anti-rabbit/anti-mouse IgG , then placed for 1 h at room temperature. The sections were incubated with avidin-biotinylated peroxidase complex. Next, the activity of peroxidase was determined by staining with diaminobenzidine , and hematoxylin stain was used for the nucleus.The paraffin sections of the pancreas tissue were deparaffinized, followed by antigen unmasking in sodium citrate buffer. Then, for block non-specific antibody binding, the tissue sections were incubated with 1% BSA for 30 min at room temperature. Following this, the samples were incubated overnight at 4 °C, with primary antibody pairs: mouse anti-vimentin , rabbit anti-CD34 , and mouse anti-αSMA . Then, after washing with 0.1M phosphate buffered saline , the samples were incubated with Alexa Fluor-488-conjugated goat anti-rabbit IgG  and Tritc-conjugated goat anti-mouse IgG , Tritic-conjugated goat anti-rabbit IgG , and Alexa Fluor-488-conjugated goat anti-Mouse IgG  secondary bodies for 2 h at 37 °C. The nuclei were then counterstained with 4’,6-diamidini-2- phenylindole (DAPI) , after which glycerin was used for mounting. Using a BX53 Olympus microscope, sections were observed, and a digital color camera, namely, an Olympus DP73, was used for capturing fluorescent images.Our results confirm the existence of TCs and long process Tps between lobules and gland cells within the connective tissue in the pancreas of Chinese soft-shelled turtles. TCs establish homo-cellular and hetero-cellular junctions that are involved in intercellular communications. TCs reduce the work load by releasing shading/secretary vesicles or exosomes for long and short distances. These conveys information to the damaged tissue for repair. Exosomes perform multifunction, specific immune reactions with macrophages or lymphocytes, peristaltic contraction in smooth muscles, chemical reactions in the case of damaged blood vessels, and repair. This information will help us to better understand the role of immune systems and different immunological disorders in the pancreas of reptiles.


Despite its high prevalence, the impact of hearing impairment on completion of cognitive tests, many of which rely on auditory input to access test material, has not been described. We investigated if hearing impairment is associated with missing scores in 3602 adults . Cognition was measured using 10 neurocognitive tests. Pure tone better-ear hearing thresholds (0.5-4 kHz) were averaged and categorized. ≥Moderate hearing impairment (versus none) was associated with greater missingness on two auditory tests: Logical Memory  and Digits Backwards ; and the non-auditory Trail Making Test Part B . Compared to models using complete cognitive data, models that imputed missing scores showed stronger associations of hearing impairment with poor cognitive performance. Older adults with HI are less likely to complete cognitive testing, resulting in biased estimates of the hearing impairment-cognitive performance relationship.


Beauveria bassiana and Beauveria brongniartii produced structurally different congeners of the cyclodepsipeptide beauveriolides, whereas the rather divergent species B. brongniartii and Cordyceps militaris biosynthesized similar analogs under the same growth condition. The conserved biosynthetic gene cluster (BGC) containing four genes present in each species is responsible for beauveriolide production. In contrast to the compound formation profiles, the phylogenies of biosynthetic enzymes or enzymatic domains show associations with fungal speciation. Dependent on the insect species, production of beauveriolides may contribute to fungal virulence against the susceptible insect hosts. The findings in this study augment the diversity of fungal secondary metabolisms.Fungal chemotaxonomy is an approach to classify fungi based on the fungal production profile of metabolites, especially the secondary metabolites. We found an atypical example that could question the reliability of fungal chemical classifications in this study, i.e., the more closely related entomopathogenic species  Beauveria bassiana and Beauveria brongniartii produced structurally distinct analogs of BVDs, whereas the less-close relatives B. brongniartii and Cordyceps militaris biosynthesized structurally similar congeners under the same growth condition. It was verified that a conserved BGC containing four genes is responsible for BVD biosynthesis in three fungi, including a polyketide synthase (PKS) for the production of 3-hydroxy fatty acids (FAs) with chain length variations. In contrast to BVD production patterns, phylogenetic analysis of the BGC enzymes or enzyme domains largely resulted in the congruence relationship with fungal speciation. Feeding assays demonstrated that an FA with a chain length of eight carbon atoms was preferentially utilized, whereas an FA with a chain longer than 10 carbon atoms could not be used as a substrate for BVD biosynthesis. Insect survival assays suggested that the contribution of BVDs to fungal virulence might be associated with the susceptibility of insect species. The results of this study enrich the knowledge of fungal secondary metabolic diversity that can question the reliability of fungal chemotaxonomy.Fungal chemodiversity is well known in part due to the production of diverse analogous compounds by a single biosynthetic gene cluster (BGC). Usually, similar or the same metabolites are produced by closely related fungal species under a given condition, the foundation of fungal chemotaxonomy. Here, we report a rare case of the production of the cyclodepsipeptide beauveriolides (BVDs) in three insect-pathogenic fungi. We found that the more closely related fungi IMPORTANCE Fungal chemotaxonomy is an approach to classify fungi based on the fungal production profile of metabolites, especially the secondary metabolites. We found an atypical example that could question the reliability of fungal chemical classifications in this study, i.e., the more closely related entomopathogenic species Beauveria bassiana and Beauveria brongniartii produced structurally different congeners of the cyclodepsipeptide beauveriolides, whereas the rather divergent species B. brongniartii and Cordyceps militaris biosynthesized similar analogs under the same growth condition. The conserved biosynthetic gene cluster (BGC) containing four genes present in each species is responsible for beauveriolide production. In contrast to the compound formation profiles, the phylogenies of biosynthetic enzymes or enzymatic domains show associations with fungal speciation. Dependent on the insect species, production of beauveriolides may contribute to fungal virulence against the susceptible insect hosts. The findings in this study augment the diversity of fungal secondary metabolisms. Tolypocladium inflatum and other fungi   and two l-type and one d-type amino acid residues  , 10. Howeria sp. . For simresidues . Current10.1128/mSphere.00667-20.2TABLE S1Table S1, PDF file, 0.5 MB.Compositions of BVDs identified before and in this study. Download Copyright © 2020 Yin et al.2020Yin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B. bassiana infection  larvae with beauverolide L could not kill insects in a dosage of less than 30 μg per larvae but induced immune responses in insects  for those in B. bassiana, which is conserved in both gene order and orientation to those of B. brongniartii and C. militaris (B. bassiana are mostly similar to those of B. brongniartii (84% to 95% at the amino acid level) followed by those of C. militaris (69% to 81% similar) and A. nidulans (40% to 54% similar) (Considering that the cyclodepsipeptide BVDs contain a 3-hydroxy fatty acid (FA) as one of the building blocks like emericellamides produced by nidulans , the put to easD , similar to easD , a conseilitaris  and C. Tilitaris . A recipsimilar) , which isimilar) , 24.A. nidulans diverged first and then the Cordycipitaceae fungi in the order of C. militaris, B. brongniartii, and B. bassiana  domain within the module 4 that is absent in EasA . The PKSbassiana . Howeverbassiana , B. bronC. militaris and B. brongniartii, which were different from the similar patterns produced by two strains of B. bassiana  analysis. Intriguingly, the data revealed that the chromatographic profiles were similar between bassiana , i.e., tB. bassiana, only the strain ARSEF 2860 was used for further analysis), multiple peaks disappeared in the mutant samples  and metabolite purifications. The individually purified compounds were subject to nuclear magnetic resonance (NMR) analysis. As a result  and two HMDA-type BVDs, La and II  and beauverolides  identified in this study from three Cordycipitaceae fungi. HMDA, 3-hydroxy-4-methyldecanoic acid; HMOA, 3-hydroxy-4-methyloctanoic acid. Download Copyright © 2020 Yin et al.2020Yin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00667-20.6FIG S213C, 125 MHz; 1H, 500 MHz) were collected in pyridine-d5. HSQC, heteronuclear singular quantum correlation. HMBC, heteronuclear multiple bond correlation; COSY, correlation spectroscopy. Download FIG S2, TIF file, 0.9 MB.1D and 2D NMR analysis of the compound beauveriolide X. NMR data  Phenotype of fruiting bodies formed on caterpillar pupa (left) and rice medium. The silk moth pupae were injected with spore suspensions or the rice medium were inoculated for 50 days. (B) HPLC profiling of metabolite production or nonproduction by different samples of C. militaris. SDB represents the extraction from the mycelial sample harvested from SDB. Download FIG S3, TIF file, 1.3 MB.Fruiting body (FB) induction and metabolite profiling of Copyright © 2020 Yin et al.2020Yin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the besB, acyltransferase besC, and CoA-ligase besD were also individually deleted in B. bassiana strain ARSEF 2860. Similar to that for the null mutant of the NRPS gene, ΔbesB and ΔbesD mutants lost their abilities to produce BVDs Ba, Ka, and Ca compared with that by the wild type (WT) strain. However, relative to the WT, a reduced amount of BVDs was still produced by the ΔbesC mutant , PKS for oosporein biosynthesis in B. bassiana; AfoG (AN1026), PKS for asperfuranone biosynthesis in A. nidulans; TRI101 (AAD19745) trichothecene 3-O-acetyltransferase of Fusarium sporotrichioides; InpC (AN3490) acyl-CoA ligase for the biosynthesis of fellutamide B in A. nidulans. Download FIG S4, TIF file, 0.4 MB.Phylogenetic analysis of the PKS domains and enzymes involved in BVD biosynthesis in different fungi. Different domains are as follows: KS, ketosynthase; AT, acyl transferase; KR, ketoreductase; ER, enoyl reductase. The domains of other PKS or other enzymes included to root the tree are as follows: OpS1  compared with the null mutant  HPLC profiling of the PKS gene besB deletion mutant after feeding with different demethyl-hydroxy-FAs. Each FA was added at a final concentration of 100 μg/ml. 3-HDDA, 3-hydroxydodecanoic acid; 3-HAD, 3-hydroxydecanoic acid; 3-HOA, 3-hydroxyoctanoic acid. (B) HPLC profiling of B. bassiana after feeding with different concentrations of d-Val and d-Leu. The spores of the B. bassiana WT strain ARSEF 2860 were inoculated in SDB for four days, and then the d-type amino acids were added at the final concentrations as indicated for another four days. Mock control was incubated in SDB without the addition of any d-type amino acid. Download FIG S5, TIF file, 0.5 MB.Substrate feeding assays of Copyright © 2020 Yin et al.2020Yin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B. bassiana also differ from those of B. brongniartii and C. militaris in that d-allo-Ile is used by B. bassiana, whereas the residues d-Leu and d-Val can be additionally integrated at position 4 by the latter fungi  and B. brongniartii, C. militaris barely infected and killed the female adults of fruit flies (Drosophila melanogaster) . The results indicated that, relative to the corresponding WT strain, BVD-nonproducing mutants of ΔbesB  and ΔBrbesB  had significantly reduced virulence against fruit flies (P > 0.05) was observed between the WT and respective mutants against the wax moth larvae  . Thus, tit flies . Howeverh larvae . The resMetarhizium fungi  produce C-type fumonisins (with a 19-carbon atom chain), whereas the recently diverged species  predominantly produce B-type fumonisins (with a 20-carbon atom chain)  . We repoeps spp. , differeilitaris , 30. Theilitaris . In addiP. fumosorosea (syn. I. fumosorosea)  and BGC losses in different species such as C. confragosa and C. cicadae. Additional support of an HGT event from A. nidulans to C. militaris is the presence of the conserved BGC for the joint production of cordycepin and its protection molecule pentostatin  . Howeverae fungi , 30. Likilitaris . Consideilitaris , high cotostatin . Neverthl-/d-amino acids. Even without being detected in this study, a previous report has indicated that B. bassiana strains also produce beauverolides D, E, Ea, F, and Fa that all contain a 3-hydroxy FA of HMOA  and ARSEF 8028 (Bb2), B. brongniartii RCEF 3172 (Br), and C. militaris Cm01 (Cm) were used in this study for metabolite production and gene deletions. Both the WT and mutants were maintained on potato dextrose agar . For BVD production, the WT and mutants were inoculated in SDB (BD Difco) and incubated in a rotatory shaker at 25°C and 180 rpm for 10 days.The WT strains of A. nidulans  for transformation of the WT strain ARSEF 2860 of B. bassiana. In addition, BrbesA and BrbesB were individually deleted in B. brongniartii, and CmbesA and CmbesB were disrupted in C. militaris. The drug-resistant colonies were verified by PCR, and at least two independent null mutants of each gene were obtained by single-spore isolation for BVD production profiling. After preliminary analysis, one null mutant of each gene was used for further experiments.Whole-genome analysis of fungal secondary metabolic gene clusters was performed using the program antiSMASH ver. 5.0 . A PKS anidulans . To detebassiana . To gene10.1128/mSphere.00667-20.4TABLE S3Table S3, PDF file, 0.4 MB.PCR primers used in this study. Download Copyright © 2020 Yin et al.2020Yin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the besD mutant of B. bassiana was complemented with the CmbesD gene from C. militaris. Thus, the full-length open reading frame of CmbesD was amplified using both the genomic DNA and cDNA of C. militaris as the templates by fusion PCRs using the ClonExpress II one-step cloning kit . The gene was under the control of the constitutive gpdB gene (BBA_05480) promoter (sur gene for conferring sulfonylurea resistance)   for tranB.bassiana and EasA and EasB of A. nidulans was performed using the program antiSMASH  in SDB medium (100 ml in each 250-ml flask) and incubated at 25°C and 180 rpm in a rotatory shaker for 10 days. The mycelia of each sample were then harvested by filtration, washed twice with sterile water, and extracted twice with 50 ml methanol for 30 min under sonication. The extracted samples were concentrated under vacuum, and the crude extract was dissolved in 1.5 ml methanol. There were three replicates for each strain.The conidia of the WT and different mutants of C. militaris are mainly consumed as health-beneficial mushrooms (Antheraea pernyi) pupae and on rice medium  equipped with an SPD-20A UV-visible (UV-Vis) detector and a C18 reverse-phase column . Sample aliquots (15 μl each) were eluted with deionized water (solution A) and acetonitrile  at a flow rate of 1 ml/min and monitored at 194 nm. The column temperature was set at 40°C.Considering that the fruiting bodies of ushrooms , the frue medium . After iB. bassiana, mycelia were harvested from 12 liters of SDB broth and extracted with methanol to generate approximately 10 g of crude extract. The crude sample was first fractionated using an Inertsil ODS (octadecyl silica) column with a gradient elution of deionized water (solution A) and methanol . BVDs were concentrated in the 80% methanol fraction, which was further purified using an LC-20AD HPLC system with a C18 reverse-phase column . Samples were eluted with deionized water (solution A) and acetonitrile  at a flow rate of 4 ml/min.For purification of BVDs from B. brongniartii, mycelia were collected from 2 liters of SDB culture broth and extracted with methanol to generate crude extract for purification with an LC-20AD HPLC system. Three fractions enriched with BVDs were collected. Beauveriolides V, VI, and VII were concentrated in fraction 1. Beauveriolides I and III were concentrated in fraction 2. Beauveriolides II, IX, and X and beauverolide La were concentrated in fraction 3. These three fractions were further purified with the LC-20AD HPLC system to obtain individual BVDs. The purified compounds were individually subject to one-dimensional (1D) NMR analysis in pyridine-d5 to collect the 1H and 13C spectra using a Bruker Avance III-500 system equipped with a 5-mm PABBO BB-1H/D probe. Standard pulse parameters were used for all NMR experiments. For the new compound beauveriolide X, 2D NMR spectra were also collected to obtain the data of heteronuclear singular quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), and correlation spectroscopy (COSY). All spectrum data were processed using the inbuilt program MestReNova .For purification of BVDs produced by besB mutant of B. bassiana and the ΔCmbesB mutant of C. militaris. The cultures were grown in SDB supplemented with each FA at a final concentration of 100 μg/ml at 5 days postinoculation and kept for cultivation for an additional 5 days. After incubation, mycelial samples were harvested for metabolite extraction with methanol as described above. Relative to the ΔCmbesB sample, additional peaks obtained in individual FA feeding experiments were collected and subjected to LC-MS analysis. Compound structures were deduced based on the obtained mass data. To determine the effect of d-type amino acid additions on BVD production, d-Leu and d-Val were used to feed the WT strain ARSEF 2860 of B. bassiana. The spores were inoculated in 250-ml flasks containing 100 ml SDB for 4 days, and d-Leu and d-Val were individually added at the final concentrations of 100 μg/ml and 500 μg/ml, respectively. The samples were incubated for another 4 days, and mycelia were harvested and extracted with methanol for HPLC analysis.To determine the substrate specificity, three 3-hydroxy FAs with chain length variations, i.e., 3-HDDA, 3-HAD, and 3-HAD, were ordered  and used to feed both the ΔB. bassiana (ARSEF 2860), B. brongniartii, and C. militaris were first examined using the female adults of Drosophila melanogaster by topical infection with spore suspensions (1 × 107 conidia/ml)  . After vd before . Insect d before .