Fluorescence confocal microscopy (FCM) is a novel micro-imaging technique providing optical sections of examined tissue. In this study, we compare intraoperative diagnoses from the real-time application of FCM in pre-therapeutic prostate biopsies with the final diagnoses from conventional histology. We found FCM to be an effective tool for the timely assessment of prostate biopsies enabling reliable real-time diagnosis of prostate cancer in patients requiring therapy.Background: Fluorescence confocal microscopy (FCM) is a novel micro-imaging technique providing optical sections of examined tissue. The method has been well established for the diagnosis of tumors in dermatological specimens. Methods: We compare intraoperative diagnoses of the real-time application of FCM in pre-therapeutic prostate biopsies  with the findings of conventional histology. Results: Prostate carcinoma was reliably diagnosed in all patients. Depending on scan quality and experience of the examiner, smaller lesions of well differentiated carcinoma (ISUP1) could not be consistently differentiated from reactive changes. Furthermore, in some cases there was difficulty to distinguish ISUP grade 2 from ISUP grade 1 tumors. ISUP grades 3–5 were reliably detected in FCM. Conclusions: Despite some limitations, FCM seems to be an effective tool for the timely assessment of prostate biopsies enabling reliable diagnosis of prostate cancer in patients requiring therapy. Prostate carcinoma (PCa) is the most common non-skin cancer in men in the western world . The higPCa has long been known to be a heterogeneous disease from a clinical and morphological perspective . The cliEx vivo fluorescence confocal microscopy (FCM) is a digital micro-imaging technique that provides optical sections of unfixed tissue . The imaIn this prospective study, we present our practical experience in the application of FCM for the assessment of prostate biopsies. The primary endpoint of our investigations was the agreement of cancer diagnoses based on FCM and conventional histology. Secondary endpoints were the levels of inter-observer variability and the ability to distinguish between ISUP grade 1 and ISUP grade > 1 cancers based on the FCM scans.MRI-fused prostate punch biopsies from 35 patients between 49 and 79 years (mean 65.7 ± 7.8) were examined. Six patients were under active surveillance with previously known prostate cancer, the other patients were clinically suspected of having prostate cancer. Before the biopsy was taken, the study participants were informed about the examinations and written consent was obtained from all participants. The study was approved by the ethics committee of the Westphalia-Lippe Medical Association (file number 2020-029-f-S) and carried out in accordance with the ethical principles of the Declaration of Helsinki.MRI-fused biopsy cylinders (targeted and random) were taken from each patient. Depending on imaging and clinical presentation, variable numbers of biopsy cylinders were examined in real-time with the confocal microscope in the operating room. The FCM scans were blindly evaluated by two experienced pathologists . Conventional histology was performed by two other experienced pathologists: A third pathologist at our facility performed the routine diagnostics (TH) and a fourth pathologist (ChB) from an external facility blindly re-examined the HE-sections. In case of discrepant histological diagnoses, consensus was established for further analysis. Finally, we statistically compared the findings from FCM to the final results from conventional histology.®, St. Louis, MO, USA). AO is an intercalating fluorochrome for staining nucleic acids, whose DNA -dye complexes have an absorption maximum of 502 nm and an emission maximum of 526 nm [According to the manufacturer’s instructions, the selected native biopsy cylinders were pre-treated with pure alcohol for 10 s (protein precipitation for contrast enhancement). The tissue was then incubated for 30 s with an Acridine Orange solution  with experience in uropathology and diagnoses were established in real-time. Blinded re-evaluation of the FCM-scans (BT) for this study was performed several weeks after the clinical cases were closed.After the scan was made, the tissue was immediately placed into labelled embedding capsules and fixed in 4% PBS-buffered formaldehyde for 24 h. The further histological investigation was carried out immediately for all biopsies following the standard procedure for formalin-fixed and paraffin embedded (FFPE) tissue. The biopsies were not removed from the foam pads during further processing in order not to change their orientation. In this way, the FCM scan could be directly compared with the histological slides of the same tissue. Minor differences in the structures shown in the figures at higher magnifications result from the different penetration depths of the compared methods . TM platform . Diagnoses for routine histology (pathologist TH) were established on the next day according to the current guidelines in order to avoid any delays in patient management. Blinded re-evaluation for this study (pathologist ChB) took place several weeks after conventional histology was completed.Histological diagnoses were established based on eight serial HE-stained sections from each of the paraffin-fixed biopsies. In case of doubt, immunohistological stains for p40 and AMACR  were performed using the Ventana Benchmark® 2500 M-G4 from Mavig, Munich, Germany. Illumination of the specimen was performed by two lasers with wavelengths of 488 nm (ultraviolet) and 785 nm (near-infrared). The short-wave laser represented the cell nuclei marked with fluorescence-dye before the examination. The cytoplasmic and extracellular structures were recorded by the reflected light of the long-wave laser. A built-in algorithm transformed the recorded gray values into an image similar to hematoxylin-eosin (HE) staining, in which the nuclei of the cells were shown in violet, whereas connective tissue fibers and cytoplasm of the cells were shown in pink [The FCM-examinations were carried out with a laser scanning confocal microscope of the type VivaScope in pink . By moduThe presence of prostate parenchyma and periprostatic tissue  was documented in an Excel table. Carcinoma formations, atypical/tumor-suspect glands, enlarged nuclei, prominent nucleoli, basal cell loss, and infiltrative growth patterns were recorded. The concluding ratings were provided in a three-grade variable system . If present, tumor extent (in percentage) as well as GLEASON score/ISUP prognosis group were documented.For the histological diagnoses , tumor manifestations, tumor extent, GLEASON score, ISUP-group and if present perineural, lympho-vascular infiltrates or extra-prostatic manifestations were reported for each biopsy. Inflammatory changes and the presence of pre-neoplastic lesions (Hg-PIN) were also noted.TM. The FCM-ratings  were matched with the diagnoses from conventional histology in error matrices. For the analysis of tumor-diagnoses, FCM-ratings 0 (benign) and 1  were grouped as not diagnostic for malignancy. Sensitivity, specificity, positive, and negative predictive values were calculated for each evaluator and levels of agreement between FCM and conventional histology were analyzed. The inter-observer variability of the FCM ratings was analyzed in the three-part variable system. Biopsy scans with false-negative tumor-diagnoses and differing ratings were re-examined and morphologically compared to the conventional H&E-slide in order to assess the reasons for the differing opinions.All statistical analyses were performed manually using Microsoft ExcelGLEASON/ISUP-gradings based on FCM diagnoses were analyzed with regard to the ability to detect high-grade cancer. Therefore, the diagnoses were grouped in ISUP grade 1 tumors (GLEASON scores ≤ 6) and ISUP grade > 1 tumors (GLEASON scores ≥ 7). The diagnoses were analyzed in error matrices providing sensitivity, specificity, positive and negative predictive values, and levels of agreement for each pathologist. The biopsies were retrospectively grouped according to the containing ISUP scores in conventional histology and the amount of false ISUP-grades in FCM was evaluated.0) of two estimators is related to the coincidentally expected agreement (pe). The results can vary between κ = 0 for a purely random match  and κ = 1.0 for a perfect match. The interpretation was based on the Landis and Koch categories  [The levels of agreement between FCM and conventional histology were measured using the Cohen’s kappa . This coreement) .A total of 544 biopsies were obtained from 35 patients . The selSince the sample preparation for FCM took a little longer than the biopsy acquisition, not all biopsies could be scanned for organizational reasons in the operating room, especially in the first patients. In these patients, we focused on target biopsies from MRI lesions. In the later cases, all biopsies were scanned. FCM pictures were acquired in 438 biopsies . Tumor manifestations were present in 75/438 (17.1%) FCM images of 17/35 patients that could be compared to conventional histology. No FCM images were obtained of incidental tumor manifestations in 8 random biopsies (9.6%) of 4 patients ; these were diagnosed in final conventional histology only. 31/75  FCM-images contained GLEASON 3 + 3 tumor (ISUP 1). 28/75  images showed GLEASON 3 + 4 patterns (ISUP 2). In 2/75  images GLEASON 4 + 3 pattern (ISUP 3) was present. 9/75  images showed GLEASON 4 + 4 tumor (ISUP 4) and 5/75  scans contained GLEASON 5 + 4/5 + 4 patterns (ISUP 5).FCM scans showed a very good agreement of the tissue structures with histological sections allowing direct correlation of these two presentations. Even small groups of glands of less than 1 mm in diameter could be identified unequivocally in both FCM images and FFPE slides . An impoIn FCM images, luminal secretions are well applicable features for the characterization of glandular structures. Atypical luminal secretions of tumor glands are shown as indistinctly contoured crystalloid signals in reflected light c while cHistoarchitectural criteria of malignancy  could beIntraoperatively (UT) carcinoma was correctly diagnosed in 64/75 biopsies and 11/75 biopsies were diagnosed as false-negative . In the second evaluation (BT), tumor infiltrates were detected in 62/75 biopsies, false-negative diagnoses were made in 13/75 biopsies and 5 tumor-free biopsies were evaluated as false positive . With kappa values of 0.90 and 0.84, the FCM diagnoses showed very good levels of agreement with the gold standard of HE morphology .We found a moderate level of inter-observer agreement  between FCM and conventional histology for the distinction between ISUP1 tumors and ISUP grades > 1 . ISUP grMicro-focal ISUP grade 1 lesions were spuriously upgraded as ISUP grade 2 by the less experienced observer in three biopsies of one patient containing ISUP grade > 1 tumor in other biopsies . ISUP grFCM images were also well suited for the classification of benign and preneoplastic lesions. In two of the biopsies, small foci of high-grade prostatic intraepithelial neoplasia (HgPIN) were detectable using conventional histology and immunohistology. In the FCM scans c,d theseFCM scans also allowed precise statements about inflammatory lesions of the prostate. The extent and the cellular composition of the inflammatory infiltrate can be determined very precisely using the scans. In our scans it was possible to differentiate between acute prostatitis and chronic forms of inflammation  were reliably recognizable in the scans. In contrast, a significant proportion of biopsies containing GLEASON 3 + 4 patterns were sometimes underrated as GLEASON 3 + 3 patterns in the FCM. Recognition of GLEASON 4 patterns are challenging even in conventional histology. Diagnostic criteria for these lesions have repeatedly been redefined in ISUP consensus meetings in order to achieve higher levels of reproducibility ,22,23. TDespite some minor limitations, FCM appeared to be a useful tool for the intraoperative detection of clinically relevant prostate cancers in prostate biopsies. The morphological heterogeneity of prostate cancer was well represented in the scans allowing reliable grading according to the ISUP system in real time. This material-sparing method conserves the biopsies as unfixed material for further histological, immunohistological, and even molecular analysis. In our preliminary examinations, we found that the pre-treatment for FCM did not alter the feasibility of the material for further FISH examinations and extraction of DNA .Further studies are needed to establish FCM in the routine diagnosis. The intraoperative examination of MRI-targeted biopsies from suspicious lesions may lead to a more effective detection of clinically significant cancers . It shouOver the past decade, the understanding of the phenotypic and molecular heterogeneity in prostate cancer has matured indicating changes in future therapeutic and diagnostic strategies . Much ofTo assess the high levels of clonal heterogeneity in future patients, primary tumor samples need to be selected carefully for genomic and epigenetic studies and—if necessary—multi-regional sampling or direct biopsy of metastatic lesions need to be performed. A spectrum of in situ approaches, such as fluorescence in situ hybridization (FISH) and immunohistochemistry have improved the ability to highlight clonally distinct tumor cell populations. FISH-analysis of TMPRSS2-ERG rearrangements and/or immunostaining for PTEN, SPINK1, p53, and RB1 ensure rapid and robust results and have the advantage to be more easily embedded in existing diagnostic workflows  than parTaking this into account, the possibility for real-time examinations of unfixed diagnostic biopsies and surgical specimens makes the ex vivo FCM an interesting mosaic for future multi-disciplinary diagnostic approaches for prostate cancer. FCM represents a step forward toward digitalized pathology, as the specimen preparation is simple and it provides digital images that enable online remote pathological interpretations. Furthermore, large series of digital FCM images might be a basis for building neural networks in the future that contain data from conventional histology, immunohistology, molecular analyzes, MRI findings, and clinical presentations as well as clinical outcome.The main advantage of FCM compared to conventional histology is primarily time. The examinations can be performed in the operating room and provide rapid feedback to the surgeons. The first study demonstrated a good feasibility for the intraoperative assessment of surgical margins during radical prostatectomy as an alternative to frozen sections . In pre-A second advantage of FCM is the preservation of the specimens as unfixed material for downstream immunohistological and molecular examinations. Intraoperative assessment of tumor spread based on mapping biopsies in real-time correlation with MRI-findings, combined with the possibility to perform fast molecular analysis in FISH approaches mentioned above might lead to improved targeting of ablation zones of focal treatments and so could raise their efficacy.Furthermore, FCM might become an important tool in pathology laboratories for bio-banking of PCa samples. Recent data show that only a small part of the relevant mutations of metastatic tumors are represented in the biopsy specimens . FCM enaEx vivo FCM is a feasible tool for the examination of prostate biopsies enabling the diagnosis and grading of prostate cancer in real-time. Rapid feedback to the examiner might lead to intraoperative adaptions of the biopsy strategy and a reduction of biopsies needed in the individual case. Tumors in need of intervention can already be identified intraoperatively, so that the affected patients can be informed about the further procedure immediately after the biopsy has been taken. The efficiency of future focal therapies could be increased through the rapid feedback.The specimens are preserved as unfixed material for downstream immunohistological and molecular examinations. Therefore, FCM might become an important tool in pathology laboratories for bio-banking of PCa samples.


The precise location of the human female genital representation field in the primary somatosensory cortex (S1) is controversial and its capacity for use-associated structural variation as a function of sexual behavior remains unknown. We used a functional magnetic resonance imaging (fMRI)-compatible sensory-tactile stimulation paradigm to functionally map the location of the female genital representation field in 20 adult women. Neural response to tactile stimulation of the clitoral region (vs right hand) identified individually-diverse focal bilateral activations in dorsolateral areas of S1 (BA1–BA3) in alignment with anatomic location. We next used cortical surface analyses to assess structural thickness across the 10 individually most activated vertices per hemisphere for each woman. We show that frequency of sexual intercourse within 12 months is correlated with structural thickness of the individually-mapped left genital field. Our results provide a precise functional localization of the female genital field and provide support for use-associated structural variation of the human genital cortex.SIGNIFICANCE STATEMENT We provide a precise location of the human female genital field in the somatosensory cortex and, for the first time, provide evidence in support of structural variation of the human genital field in association with frequency of genital contact. Our study represents a significant methodological advance by individually mapping genital fields for structural analyses. On a secondary level, our results suggest that any study investigating changes in the human genital field must map the field individually to achieve sufficient precision. Our results pave the way for future research into the plasticity of the human genital cortex as a function of normal or adverse experience as well as changes in pathologic conditions, i.e., sexual dysfunction, sexual deviation, or sexual risk-taking behavior. The precise location of the female genital representation field in the primary somatosensory cortex (S1) is still a matter of contention . FurtherIn their first presentation of the somatosensory homunculus, The mode of stimulation used in functional mapping studies may contribute to heterogeneous results concerning the location of the genital field in humans. Specifically, electrical stimulation is not equivalent to sensory touch and elicits less focal responses . Self-deCommensurate with the fact that the precise location of the genital field remains controversial, there is no evidence regarding its capacity for structural change in association with use in humans. It is well established that the human brain has substantial capacity for plasticity as a function of experience . Use-depWe here combine the investigation of the location of the female genital field with the question of structural variation of this field as a function of sexual behavior, considering the important issue of individual variability: (1) we provide a precise localization the human female genital representation field by using a focal sensory-tactile nonarousing stimulation paradigm during functional MRI (fMRI) to contrast neural response of stimulation of the clitoral region versus the right hand. (2) We use individually-mapped genital fields based on the 10 most activated vertices per hemisphere for each woman and assess structural thickness in the individually-mapped field using cortical surface analysis. (3) We show that thickness of the individually-mapped genital field varies with the frequency of sexual intercourse in the past 12 months, compatible with use-associated plasticity.We recruited 25 adult healthy women aged 18–45 years. General exclusion criteria applied to select women were lifetime or current psychiatric disorders, exposure to childhood abuse or neglect , neurologic disorders, physical disease, central nervous system or urogenital surgery, psychotropic medication within six months, sexually transmitted disease, sexual disorders , past or current pregnancy, and current menstruation. Exclusionary conditions were assessed using clinician-administered interviews and standard questionnaires . Women wWomen underwent a standardized study visit at the Institute of Medical Psychology and the Berlin Center for Advanced Neuroimaging, both at Charité−Universitätsmedizin Berlin. During the visit, women underwent all study procedures, including interviews and questionnaires for demographics and exclusionary conditions. To localize the genital representation field in S1, women underwent (1) fMRI scanning during sensory-tactile stimulation of the clitoris versus dorsum of the right hand; (2) structural MRI to assess thickness of the individually mapped genital field; and (3) a detailed sexual history to assess frequency of sexual intercourse, i.e., genital sensory touch, in the past year and lifetime for the assessment of use-dependent plasticity of the individually mapped genital field. The study was approved by the institutional ethics committee and was conducted in accordance with the Declaration of Helsinki. Written informed consent of the participants was obtained.3 isotropic T1 anatomic scans were acquired in the sagittal plane using the magnetization-prepared rapid gradient echo sequence . Structural MRI acquisition took 2 × 6:03 min. fMRI scans were obtained using a T2*-weighted echoplanar image (EPI) pulse sequence . The functional imaging paradigm comprised four scanning blocks with a duration of 5:36 min, respectively.Structural MRI was performed using a 3.0 T Siemens Tim Trio MRI scanner  with a standard 12-channel head coil. Two 1-mmWe developed an MRI-compatible sensory-tactile stimulation paradigm that allows for administering a defined focal sensory stimulus to the clitoral region see . The stiThe paradigm was performed in an ABBA versus BAAB block design with stimulation of either the clitoral region (A) or the dorsum of the right hand (B) interspersed with 10-s periods of no stimulation. Each of the four runs started with a period of no stimulation and included a total of eight clitoral and eight dorsum manus stimulation phases. The order of these phases was fixed and counterbalanced between women. Synchronization of the trigger pulses from the MRI scanner and the timing of the stimulation was controlled using Presentation . During the sensory-tactile stimulation, subjects were asked to fixate a cross on a screen. One woman completed only three runs.Pleasantness and sexual arousal during clitoral stimulation were assessed after each run using a seven-point visual analog scale. Subjects were instructed to use a fiber-optic response box, indicating changes in pleasantness and sexual arousal. We then computed combined ratings on overall pleasantness and sexual arousal after the scan. We further inquired on the subjective appropriateness of the location of the clitoral membrane during the experiment as well as on sensations in other body parts during clitoral stimulation. There was no evidence for dislocation of the stimulation membrane in the sample.Statistical parametric mapping 12  was used to perform functional image analysis to localize the genital field in S1. Standard spatial preprocessing of functional images, including realignment and coregistration to T1 image, was separately performed for each of the four scanning blocks. Data were high-pass filtered with a default cutoff period of 128 s to correct for slow drift artifacts. There was no head motion above 3.0 mm and 3.0° of maximal translation and rotation in any direction throughout a scanning block.t tests for contrasts between the two regressors of the design matrix, resulting in individual patterns of neural activation in response to clitoral versus hand stimulation. We identified an activated region in SI for each participant at p < 0.001 without correction or p < 0.05 with family-wise error (FWE) correction for multiple comparisons. Individual neural activations were overlaid onto coregistered anatomic scans and saved as individual regions of interest (ROIs) for the left and right hemisphere, respectively. Individual ROI was multiplied with the t-score map corresponding to the individual contrast image to delineate the most activated vertices within the individually defined ROI. We purposely did not perform spatial normalization to a standard stereotaxic space  or smoothing of the images to allow for subsequent cortical thickness analyses within the individually mapped ROI in native space of the anatomic images, as needed for the use-dependent plasticity analyses. To determine variability of the location of the genital field and hand representation in S1 between women, coordinates of peak neural activation were transformed in MNI space. Barycentre and dispersion across individually mapped fields were computed by averaging individual coordinates in MNI space.After standard spatial preprocessing, fMRI data were analyzed using a general linear model (GLM). The two within-subject conditions of interest  were modeled using a boxcar function convolved with a canonical hemodynamic response function (HRF). Activation maps were calculated with 3. Furthermore, data were spatially smoothed with a 6-mm full-width at half-maximum isotropic Gaussian kernel. Whole-brain group-level analysis with t tests contrasting neural response to the two with-subject factors genital stimulation versus stimulation of the dorsum of the right hand was thresholded at p < 0.05 with FWE-correction for multiple comparisons. Coordinates of the group-based neural activation reflecting the genital field are given in standard MNI space.To additionally localize the genital representation field in S1 on the group level, a random effects GLM was estimated across subjects. For this, individual contrast maps were spatially normalized to a standard MNI template and resampled to an isotropic spatial resolution of 3 × 3 × 3 mmThese statistical analyses and figures were computed using MATLAB Automated image segmentation included (1) spatial registration (affine registration to tissue probability map); (2) initial SPM unified segmentation and skull stripping; (3) local intensity transformation to reduce tissue inhomogeneities  for SPM12 was used to perform cortical SBM of the anatomic scans. Image segmentation was conducted using an automated standard procedure. The individually defined ROIs for the clitoris and the dorsum of the right hand were separately mapped onto individual native space cortical surfaces of the left and right hemisphere. After cortical surface registration, mean thickness of the 10 functionally most active vertices within the individually mapped ROIs was separately calculated for each hemisphere in each woman. Cortical thickness at each vertex was calculated as part of central surface estimation , describcorr = 0.025). Using partial correlation analyses, we used age, years since onset of sexual contact, and whole-brain cortical thickness as covariates to control for effects of these variables on genital field cortical thickness. Furthermore, correlations and partial correlations between left-hemispheric cortical thickness of the representation field of the right hand and frequency of sexual intercourse for either time window were calculated to confirm for region-specificity of use-associated variation. These statistical analyses and figures were computed using R Project for Statistical Computing  and IBM SPSS Statistics .We assessed mean frequency of sexual intercourse per week using a standardized biographic questionnaire to quantify sexual intercourse within the past 12 months and in five-year ranges since the onset of the first sexual genital contact. As noted above, we excluded sexual anxiety, discontent or dysfunction as well as dissociation during intercourse using established questionnaires . To assoThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Custom MATLAB Code  for SPM12 and CAT 12 will be provided on request.Demographic and behavioral data are presented in Sensory-tactile stimulation of the clitoral region (relative to right hand) induced significant focal neural activations in S1. Sixteen women exhibited bilateral neural activations in S1. For four women, a significant activation was found in either the right or the left hemisphere only. We next mapped the individual representation of the dorsum of the right hand for use in subsequent cortical thickness analyses. Sensory-tactile stimulation of the dorsum of the right hand  induced significant contralateral focal neural activations in S1. x = −18, y = −34, z = 74; T = 7.72, pFWE-corr = 0.024; right hemisphere: x = 18, y = −40, z = 68; T = 10.26, pFWE-corr < 0.0001). Of note, no other significant neural activations were observed at the group level in response to the stimulation of the clitoral region, suggesting that the stimulation paradigm specifically targeted the genital field and was not overly arousing. When analyzed at the group level across all women, GLMs revealed significant symmetric dorsolateral neural activations in S1 in response to stimulation of the clitoris (relative to hand) in both hemispheres . Similarly, longer-term frequency of sexual intercourse estimated since the onset of sexual contact was significantly correlated with thickness of the individually-mapped left-hemispheric genital field in a partial correlation analysis . Partial correlation analyses between cortical thickness of the right-hemispheric genital field and frequency of sexual intercourse did not reveal any significant effects, suggesting lateralized use-associated structural variation. We mapped individual ROIs for the genital field  onto native cortical surfaces for each subject and estimated cortical thickness of the individual genital representation field  onto native cortical surfaces for each woman and estimated cortical thickness of the individual representation field of the hand (for individual data, see We present novel evidence on the precise location of the female genital representation field and its capacity for use-associated structural variation. Using functional mapping during sensory-tactile stimulation of the clitoral region, we show focal bilateral neural activations within the dorsolateral postcentral gyrus in S1. We show that the individual location of peak neural activations in response to clitoral stimulation varies considerably between women. We applied cortical surface analysis to the individually-mapped ROI to compute structural thickness of the genital field. Correlating the individually-mapped morphologic data with behavioral data on sexual contact, we provide first evidence that thickness of the genital field varies as a function of frequency of genital intercourse in the past 12 months and lifetime, in line with use-associated plasticity.Our results are noteworthy in several ways. To localize the female genital field, we measured neural response in a tactile-sensory stimulation paradigm that delivers a physiologically valid stimulus as opposed to a previous study using electrical stimulation of the clitoris . FurtherOur results suggest profound variability of the individual location of the genital field within the dorsolateral part of S1 with individual peak activations clearly deviating from the group mean. This means that any study looking at structural variation of the genital field as a function of certain conditions, such as sexual behavior, sexual abuse or sexual dysfunction, must necessarily implement individual mapping of the genital field and compute data, i.e., cortical thickness, on an individual level. Clearly, only by using individually-mapped ROIs, such studies yield precise reliable surface-based parameters for association with specific conditions.We computed data on structural thickness of the genital field in individually-mapped ROIs, based on the 10 most activated vertices per hemisphere for each woman. We show that individual thickness of the left genital field associates with frequency of sexual intercourse. The association was stronger for genital intercourse within the past 12 months. While less pronounced, the association was significant for lifetime genital contact. Frequency of genital intercourse was not associated with thickness of the representation field of the right hand nor with thickness of the entire cortical mantle, confirming a specific association between genital touch and genital field thickness. This is compatible with the idea that the female genital field has capacity for structural plasticity depending on its use, commensurate with the general “use-it-or-lose-it” principle of experience-dependent plasticity . While iSeveral mechanisms might contribute to dynamic use-associated structural plasticity of the genital field. Structural thickening of the mature cortex as a function of use most likely reflects formation of new synapses by axonal sprouting, dendritic arborization, and dendritic spine growth rather than induction of new neurons through neurogenesis . There iIt must be noted that use-associated variation of structural thickness of the female genital field in our study was limited to the left hemisphere. This lateralized effect is puzzling given that the neural representation of the clitoris is bilateral. Left-hemispheric dominance of neural plasticity has been reported for learning-dependent structural change after coordination and motor skill training . Such laWhile our localization of the female genital field was experimental in nature, our investigation of the capacity of the genital field for structural variation as a function of genital contact was cross-sectional and relied on retrospective self-report of genital intercourse. Our results align with the general principle of an association between frequency of genital intercourse and structural variation, albeit the direction of effect is a matter of discussion. It is conceivable that thickness of the genital field may drive frequency of sexual intercourse. Results from animal models provide causal that clitoral stimulation drives genital field thickness . Future In conclusion, we provide an unequivocal localization of the female genital field in S1 and support for use-associated plasticity of the human genital field. On a secondary level, our findings support the notion that studies investigating change of the human genital field must map the field individually. Our results pave the way for future research into the plasticity of the human genital field as a function of normal or adverse experience as well as genital field structure, function and plasticity in pathologic conditions, such sexual dysfunction, sexual deviation, or sexual risk-taking behavior.


It is widely believed that cancer is developed due to changes in the genetic codes of our DNA, leading to abnormal growth of cells. In the past few years scientists have discovered a system which is used as an immune mechanism by bacteria in order to cleave the invading viruses, called CRISPR/Cas9. Exploiting this system in humans will allow scientists to attempt to edit genetic errors that lead to cancer. This scientific breakthrough has a lot of potential for treating a variety of diseases, including cancer. It has already been successfully used in treatment of some types of blood cancer. In this article, we discuss the opportunities and limitations of CRISPR/Cas9 in treatment of solid cancer.Cancer is considered by not only multiple genetic but also epigenetic amendments that drive malignant cell propagation and consult chemo-resistance. The ability to correct or ablate such mutations holds enormous promise for battling cancer. Recently, because of its great efficiency and feasibility, the CRISPR-Cas9 advanced genome editing technique has been extensively considered for therapeutic investigations of cancers. Several studies have used the CRISPR-Cas9 technique for editing cancer cell genomic DNA in cells and animal cancer models and have shown therapeutic potential in intensifying anti-cancer protocols. Moreover, CRISPR-Cas9 may be used to correct oncogenic mutations, discover anticancer drugs, and engineer immune cells and oncolytic viruses for immunotherapeutic treatment of cancer. We herein discuss the challenges and opportunities for translating therapeutic methods with CRISPR-Cas9 for clinical use and suggest potential directions of the CRISPR-Cas9 system for future cancer therapy. Our DNA is constantly being damaged by internal and external sources. It is widely accepted that genomic instability and accumulation of driver aberrations lead to clonal cancer evolution. Mammalian cells have developed systems to repair and maintain the integrity of their genome through employing various DNA damage response (DDR) mechanisms. DNA double strand breaks (DSB) are considered one of the most lethal insults to cells. Following induction of DSB, the DNA repair machinery is activated to repair the DSB. There are two main pathways for repairing a DSBs, non-homologous end joining (NHEJ) or homologous recombination repair (HRR). The NHEJ repair system simply processes the broken DNA ends, joins the two ends of DSB and ligates the two damaged ends together. Although an efficient repair mechanism, it gives rise to mutations, deletions, or insertions (Indels). In contrast, HRR is an accurate repair mechanism that uses the homologous sister chromatid as a template to precisely repair the DSB. This system requires a complex and coordinated machinery of proteins and enzymes to first resect the damaged ends of DNA, identify and localize the homologous sister chromatid at the site of the damage and facilitate the invasion of 3′ single-stranded tails of the sister homologous chromatid into the damaged DNA to act as a template to restore the damage to its original sequence using DNA polymerases .Gene editing has a long history and technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have previously been described . Such plClustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-9 (Cas9) is a gene editing tool that comprised of a single guided RNA (sgRNA) and a DNA endonuclease Cas9 protein. The sgRNA identifies the matched complimentary sequence of the genome and guides the Cas9 to the site of target DNA . FollowiMultiple sgRNA sequences can, in principle, allow gene editing at multiple locations . This abCRISPR/Cas9 technology offers significant potential in laboratory cancer research, including the ability to generate better disease models for cancer, drug targets and discovery and study of treatment resistance. It is a flexible, relatively simple-to-use technology with a high degree of efficacy which is affordable and easy to use in laboratory research [CRISPR/Cas9 has been successfully used to inactivate several tumor suppressor genes in order to create cancer disease models. For example, CRISPR-induced deletion of P53, Nf1, Pten and Ptch1 in a mouse brain resulted in forming medulloblastoma and glioblastoma . As discDrug resistance is a significant issue in oncology. CRISPR/Cas9 technology has provided an opportunity to study the mechanism of resistance to anti-cancer drugs by correcting activating mutations that confer resistance to certain therapies. Examples of using CRISPR/Cas9 include models of EGFR exon 20 mutations to study the efficacy of the EGFR tyrosine kinase inhibitor (TKI) osimertinib , or ablaEnhanced toxicities from combining anticancer drugs are a major factor in anticancer drug development. For instance, despite the synergistic effect of platinum and PARP inhibition, such concurrent combination strategies have largely failed, mainly due to high grade myelosuppressive toxicities. It has been demonstrated that CRISPR-induced inhibition of PARP1 synergizes cisplatin cytotoxicity in ovarian cancer , which mIn the last decade, the field of oncology has witnessed a revolution in the way we treat patients with cancer. Immunotherapy remains at the forefront of this therapeutic evolution. Immune checkpoint blockade, CAR-T cells, and adaptive T cell therapy (ACT) are among the treatments that have successfully entered the clinical setting.Chimeric antigen receptor (CAR) T cells (CAR-T) are harvested T cells that have been genetically engineered in order to express receptor to recognize neoantigens that are expressed on the cancer cells surface . Such tuWhile CAR-T cells have been very successful in the treatment of hematological malignancies, particularly B cell malignancies ,30, theiAnother obstacle is T cell exhaustion, which is the result of chronic exposure to antigens . SeveralSuch limitations, as well as the high rate of toxicities, including cytokine release syndrome (CRS) and the risk of graft versus host disease (GVHD), have hindered the development of CAR-T cell therapies in treatment of solid cancers . The CRIAdaptive cell therapy (ACT) has the potential to overcome the reliance on tumor infiltrating lymphocytes (TILs) that are not always found in all tumors. The T cell receptor (TCR) complex is at the core of these treatment modalities and is responsible for “foreign antigens”. In ACT, patients’ T cells are engineered to express particular TCRs that are able to detect antigens expressed by tumor cells, and direct T cell killing of the tumor cells. Such transgenic TCRs specifically engineered to recognize NY-ESO1 antigens in certain tumors provide an opportunity for targeted killing of tumor cells presenting those immunogenic antigens. A potential problem with ACT is the mispairing of α and β chains of the therapeutic and endogenous TCR complex resulting in reduced efficacy for engaging TCR with the target antigen. Additionally, the negative regulatory effect of PD-1 expression on T cells reduces the antigen response and therefore the efficacy of T-cell-directed tumor killing. Preclinical mouse model studies indicate that a combination of PD-1 blockade and transgenic TCR T cells against NY-ESO1 antigen presenting tumor cells is associated with improved efficacy . This haOne patient with advanced and treatment refractory metastatic liposarcoma showed mixed response to treatment with a large abdominal lesion showed 50% reduction in the size of the disease with the duration of response lasting for 4 months, while other metastatic lesions progressed . The besThe second clinical trial using CRISPR/Cas9 PD-L1 edited T cells was conducted in advanced treatment of refractory non-small-cell lung cancer. Of twenty-two patients enrolled in the trial, twelve patients received the edited T cell infusion. The investigators reported that the treatment was safe and that all treatments related to adverse events were low grade. The editing efficiency in the infused cells was low, which could be due to the delivery method rather than the gene editing issue. This resulted in short term persistence of the edited T cells in patients. The off-target mutation frequency was low , evaluated by next generation sequencing at 18 potential off-target loci. None of the patients showed objective response to treatment, although one patient with higher PD-1 editing efficiency showed stable disease for 76 weeks, indicating that better editing efficiency may lead to greater antitumor activity .These two studies have addressed the safety concerns associated with the use of CRISPR/Cas9 technology to some extent, as well as providing the proof of principle for the feasibility of CRISPR/Cas9 as a therapeutic option for solid and hematological malignancies. Multiple clinical trials are currently underway to assess safety and antitumor activity of CRISPR/Cas9 edited T cells in multiple cancers .While CRISPR/Cas9-directed ex vivo gene modification has been very promising, in vivo delivery of CRISPR/Cas9 into solid malignancies is possibly the biggest challenge ahead. A few delivery methods have recently been used, such as viral vectors like adeno-associated virus and lentivirus, or non-viral delivery methods like lipid- or polymer-based nanoparticles ,50,51. ITM are in development to monitor the off-target mutations more accurately [P53 is that CRISPR/Cas9 gene modification may not be optimal due to the tumor suppressor effect of P53 [TP53 mutated cells [TP53 may lead to mutations and DNA rearrangements in proto-oncogenes which may subsequently increase the chance of malignancy. These concerns regarding the safety of the CRISPR/Cas9 system are the subject of extensive research to increase the site specificity and reducing off target nuclease activities. Among the strategies currently being researched are more advanced editing techniques such as base and prime editors that reduce the chance of off-target mutations [Another major challenge with CRISPR/Cas9 technology is off-target gene editing. Off-target DNA strand cleavages leading to off-target mutations and larger chromosomal rearrangements, such as inversions and translocations, are observed in all gene editing platforms ,54. In Ccurately . One obst of P53 . Two stued cells ,59. Thisutations . Base edutations . One advutations .The CRISPR/Cas9 gene editing platform holds great research and clinical promise in cancer therapeutics. This simple and versatile system has the ability to help us understand the mechanisms of cancer predisposition and mechanism of metastasis, as well as predicting response to treatment and drug resistance. In the clinical context, it is set to revolutionize the use of CAR-T and adaptive cell therapies by overcoming some hurdles such as GVHD and T cell exhaustion. There are considerable challenges ahead of developing CRISPR/Cas9 technology as a routine treatment of solid cancer. Manufacturing timeline, high cost of manufacturing, off-tumor effects, CAR-T delivery, trafficking and tumor infiltration, and associated safety and toxicities are a few examples of the major challenges ahead. Inherent clonal selection and expansion of cancer which lies at the center of therapeutic failure for the majority of anticancer therapies, including chemotherapy and targeted therapy, is another major obstacle. Even with effective and safe CRISPR/Cas9 targeting, tumor heterogeneity and presence of sub clones which harbor different driver mutations may lead to the emergence of resistant clones. Identification of multiple sub clones before treatment and multiplex CRISPR/Cas9 sgRNA may be one way to address this problem to some extent. Another significant challenge is how CRISPR/Cas9 might be useful in treating cancers caused by structural events or copy number aberrations. Nonetheless, more clinical trials are underway to address these points which we expect to set the gene editing technology as an integral part of cancer treatment in the future.


BMC Pregnancy and Childbirth, aiming to summarize the complex interaction between SARS-Cov-2 infection, pregnancy and diabetes. This commentary will summarize and discuss the main findings of this article and its implications for future research.SARS-Cov-2 (Severe Acute Respiratory Coronavirus 2) infection confers a non-negligible risk for younger pregnant women with diabetes, which is still less well investigated. This topic was recently addressed by a systematic scoping review in  BMC Pregnancy and Childbirth, aiming to summarize the complex interaction between SARS-Cov-2 infection, pregnancy and diabetes. Thereby the authors identified three major topics, with need of further research [Diabetes mellitus is a major risk factor for a severe course of COVID-19 (Corona Virus Disease 2019) with higher risk for manifesting a viral pneumonia-induced acute respiratory distress syndrome (ARDS) and leading to increased mortality rates , SARS-Coresearch .As comprehensively summarized by the authors, the prognosis of COVID-19 is related to several factors including platelet activation and increased proinflammatory cytokine release, which is associated with worsening metabolic control, poor disease progression and consequently poor pregnancy outcomes . NoteworIt is challenging to uphold GDM screening and diagnosis standards while there is a purpose to limit as possible in-person contact in order to reduce the risk of exposure and dissemination of SARS-Cov-2. Consequently, several health care authorities modified their recommendations for GDM screening and testing during the COVID-19 pandemic. Fasting plasma glucose (FPG), glycated haemoglobin A1c and random plasma glucose were discussed as alternative screening strategies to universal testing by use of oral glucose tolerance tests (OGTT) . In thisEberle et al. clearly summarized the potential advantages of “digital care” in the treatment of pregnancies with diabetes . For surHowever, despite our achievements in the pathophysiological understanding of the disease as well as in the development of novel diagnostic strategies and telemetric approaches the effective treatment of diabetic pregnancies during the COVID-19 pandemic is still a great challenge for obstetrics and feto-maternal medicine. Regular appointments with obstetricians are required to assess possible fetal malformations and growth trajectories in fetal ultrasound. Leaving out advanced fetal sonography even at early gestation  can potentially cause severe complications in later pregnancy – for example due malformations which could be otherwise detected already at the first trimester . Researc


Background: Recent studies have utilized data-based dynamic modeling to establish strong association between dysregulation of cerebral perfusion and Mild Cognitive Impairment (MCI), expressed in terms of impaired CO2 dynamic vasomotor reactivity in the cerebral vasculature. This raises the question of whether this is due to dysregulation of central mechanisms (baroreflex and chemoreflex) or mechanisms of cortical tissue oxygenation (CTO) in MCI patients. We seek to answer this question using data-based input-output predictive dynamic models.Objective: To use subject-specific data-based multivariate input-output dynamic models to quantify the effects of systemic hemodynamic and blood CO2 changes upon CTO and to examine possible differences in CTO regulation in MCI patients versus age-matched controls, after the dynamic effects of central regulatory mechanisms have been accounted for by using cerebral flow measurements as another input.Methods: The employed model-based approach utilized the general dynamic modeling methodology of Laguerre expansions of kernels to analyze spontaneous time-series data in order to quantify the dynamic effects upon CTO (an index of relative capillary hemoglobin saturation distribution measured via near-infrared spectroscopy) of contemporaneous changes in end-tidal CO2 , arterial blood pressure and cerebral blood flow velocity in the middle cerebral arteries . Model-based indices (physio-markers) were computed for these distinct dynamic relationships.Results: The obtained model-based indices revealed significant statistical differences of CO2 dynamic vasomotor reactivity in cortical tissue, combined with “perfusivity” that quantifies the dynamic relationship between flow velocity in cerebral arteries and CTO in MCI patients versus age-matched controls (p = 0.006). Significant difference between MCI patients and age-matched controls was also found in the respective model-prediction accuracy (p = 0.0001). Combination of these model-based indices via the Fisher Discriminant achieved even smaller p-value (p = 5 × 10–5) when comparing MCI patients with controls. The differences in dynamics of CTO in MCI patients are in lower frequencies (<0.05 Hz), suggesting impairment in endocrine/metabolic  mechanisms.Conclusion: The presented model-based approach elucidates the multivariate dynamic connectivity in the regulation of cerebral perfusion and yields model-based indices that may serve as physio-markers of possible dysregulation of CTO during transient CO2 changes in MCI patients. CO2 vasomotor reactivity, whereby an increase in blood CO2 tension causes a decrease of cerebrovascular impedance (through vasodilation) and, consequently, an increase of cerebral blood flow. The latter enables the gradual reduction of the original surge in blood CO2 tension in order to achieve homeostasis (from hypercapnia to normocapnia). This mechanism also acts in the reverse manner in the event of a decrease in blood CO2 tension to achieve normocapnia from a state of hypocapnia  of cerebral vasculature and the cortical oxygenation response (COR) to changes in blood CO2 tension, discovering significant impairment of both DVR and COR in patients with amnestic MCI relative to age-matched controls  the controls . These f-holding . Notable-holding .CO2  under resting spontaneous conditions  and significant impairment in the chemoreflex gain was found in MCI patients that correlated strongly with their respective deficit in DVR at middle cerebral arteries—but not with the deficit in cortical tissue oxygenation (CTO) at the lateral prefrontal cortex  is actually an index of relative hemoglobin saturation distribution in the arterial/venous compartments. This relative distribution can be altered by capillary recruitment caused by changes in cerebral blood flow, thus providing a “directional hypothesis” to the present study.In this broad effort, our work has sought to contribute quantitative measures of cerebral flow regulation (and possible dysfunction) in the form of data-based DVR indices computed from predictive dynamic models of how beat-to-beat cerebral flow velocity or cortical tissue oxygenation (CTO) are influenced by changes in arterial blood pressure and end-tidal nditions , 2019. Tl cortex . This inCO2) with output being the CTO measurements at the lateral prefrontal cortex. This data-based model allows the quantification of the dynamic effects of transient systemic CO2 changes upon localized CTO at the lateral prefrontal cortex—separately from blood CO2 effects on cerebral arteries (vasomotor reactivity). This may prove useful for quantifying CTO regulation/dysregulation in cerebrovascular or neurological disease at the level of small/micro cortical vessels. Furthermore, proper scientific interpretation of the obtained predictive models may provide valuable insights into the physiological mechanisms that affect the regulation of cortical perfusion and oxygenation in healthy subjects and patients.To this purpose, we include in this study the measurements of blood flow velocity at the middle cerebral arteries as another “input” of our predictive dynamic model  as indicated in Equation (1). A complete characterization of the linear dynamics of this system is achieved when the three “kernels” of the convolutional terms in Equation (1) are estimated from input-output data. These kernels are characteristic of each system and can be viewed as “the patterns of weighing the input epochs in order to construct the present output value.” The kernels can be estimated robustly when the Laguerre expansion technique is used . The obtained subject-specific models offer novel insights into the dynamics of this system and can advance our scientific understanding when properly interpreted. They can also generate model-based “physio-markers” regarding various aspects of the regulation of cerebral perfusion or oxygenation and its possible association with MCI. For example, the findings indicate significant reduction in the CTO response of MCI patients, relative to age-matched controls, in response to a step change in blood CO2. This statistical differentiation increases when the CTO response to a step change in cerebral blood flow is also taken into account. The full potential scientific and diagnostic utility of this approach remains an important goal for future studies as more relevant knowledge is accumulated.The present paper employs this quantitative methodology to extract predictive models of the dynamic regulation of cortical oxygenation (viewed as an “output” variable) as a function of concurrent spontaneous changes of blood We collected the following time-series datasets over 5 min in 36 patients with amnestic MCI and 12 age-matched cognitively normal controls:(1)mean arterial blood pressure (ABP) over each heart-beat, measured continuously with finger photo-plethysmography (Finapres); the heart-beat interval was also determined from Finapres;(2)CO2 (CO2) measured via nasal cannula using capnography (Criticare Systems);end-tidal (3)mean cerebral blood flow velocity (CFV) over each heart-beat, measured continuously in the middle cerebral arteries using a transcranial Doppler (TCD) probe  placed over the temporal window and fixed at constant angle with a custom-made holder;(4)via Near Infrared Spectroscopy (NIRS) (Hamamatsu). As noted in Introduction, this index is not quantifying directly “tissue oxygenation” but rather the relative hemoglobin saturation distribution in the arterial/venous compartments (which we assume to be related to tissue oxygenation).cortical tissue oxygenation (CTO) as tissue oxygenation index (TOI), defined as the ratio of oxyhemoglobin to total hemoglobin multiplied by 100, measured 1 Healthy older adults (control subjects) were cognitively normal and did not have MCI or dementia. Both healthy controls and patients with MCI were excluded if they had major neurologic, vascular, or psychiatric diseases, uncontrolled hypertension, clinically diagnosed or self-reported diabetes mellitus, clinical histories of stroke, unstable heart diseases, sleep apnea, body mass index (BMI) ≥ 35 kg/m2, and current or past history of smoking. Participants were instructed to abstain from caffeinated beverages, alcohol, and vigorous exercise over the 24 h prior to the clinical visit. All hemodynamic data were collected in a quiet, environmentally controlled laboratory with an ambient temperature of 22 degrees Celsius, under resting seated conditions.All patients/subjects participated voluntarily in this study and signed the Informed Consent Form that has been approved by the Institutional Review Board of the UT Southwestern Medical Center and Texas Health Presbyterian Hospital Dallas. Participants were recruited from the University of Texas Southwestern Medical Center Alzheimer’s Disease Center, senior centers in the Dallas-Fort Worth metropolitan area, and local newspaper advertisements. The diagnosis of amnestic MCI was based on modified Petersen criteria . The scoAll measurements were non-invasive, safe and comfortable for the subjects. After 20 min of rest, 5-min recordings were made at an initial sampling rate of 1 KHz and subsequently pre-processed prior to analysis as described below. The gender composition, the age (mean and standard deviation) and the neuropsychological scores (MMSE and DLMR) of the patients and controls are given in via cubic-spline interpolation. Breath-to-breath end-tidal CO2 values were placed at the mid-point of each breath (unevenly sampled) and were re-sampled every 0.25 s via cubic-spline interpolation to make the data-samples of all physiological variables contemporaneous. All time-series data were high-pass filtered (via subtraction of a 2-min moving-average Hanning window) to remove the constant baseline and very low frequency content below 0.01 Hz, and low-pass filtered using an 8-s Hanning window in order to alleviate the effects of respiratory sinus arrhythmia and concentrate on dynamic effects below 0.2 Hz where hemodynamic regulation is thought to take place  and were re-sampled every 0.25 s ke place . Figure via finger photo-plethysmography; (2) beat-to-beat data of mean cerebral flow velocity (CFV) measured via transcranial Doppler at the middle cerebral arteries; (3) breath-to-breath data of end-tidal CO2 (CO2) measured via capnography through a nasal cannula; and the putative “output” of beat-to-beat data of mean CTO measured via Near Infrared Spectroscopy as Tissue Oxygenation Index (TOI) at the lateral prefrontal cortex. The estimated model for each of 36 MCI patients (MP) and 12 age-matched control subjects (Controls) is then used to compute indices quantifying certain physiological functions of interest via the proper simulations. For instance, a model-based index of CTO/TOI response to CFV is computed as the time-average over 50 s of the model-predicted TOI response to a unit-step (1 cm/s) change of CFV, as described later. These input-output predictive dynamic models are estimated using the novel methodology of Laguerre kernel expansions and Principal Dynamic Modes that was pioneered by our lab and has been shown to yield accurate model estimates from noisy and relatively short datasets  and the output (CTO/TOI):In order to obtain the requisite predictive dynamic models, we employ the input-output modeling methodology that has been pioneered by our lab , 2019 andatasets . The keyy(n) denotes the n-th CTO/TOI output sample; the discrete time-series data p(n), x(n) and z(n) denote the three inputs ABP, CO2 and CFV respectively; while kp, kx and kz denote the three “kernels” of the model with respect to the inputs p(t), x(t), and z(t), respectively. The summations represent discrete convolution operations, which constitute a general mathematical/computational model form for all linear dynamic systems with invariant characteristics over time. This general modeling approach has also been extended to the analysis of nonlinear dynamic systems using multi-dimensional convolutions  for any given set of input waveforms p(n), x(n), and z(n). An outline of the kernel estimation procedure from the input-output time-series data is provided in where: olutions , 2014—al2 and CFV. The average kernels and their standard deviation (SD) bounds are shown in 2 input . For the CFV input , as time-averages of the respective model-predicted TOI response of each MCI patients or Controls over a specified time-horizon. 2 has the greatest effect on the TOI model-predicted response, and the effect of a unit-step ABP change is the smallest. Likewise, the difference of unit-step responses between MCI patients and Controls is greatest for the CO2 input and smallest for the ABP input.The statistical significance of these average kernel differences between MCI patients and Controls can be examined in the context of the respective estimation variances by simulating the predictive models (defined by the respective kernels) for each unit-step input, while the other two inputs are set to zero/baseline. We can then compute According to the aforementioned logic of computing model-based indices by time-averaging of the TOI response to each of the three unit-step inputs, we can define the following indices for three specific aspects of CTO (assumed to be related to the TOI measure of relative capillary hemoglobin saturation distribution) by selecting the appropriate input-output pair and time-averaging of the TOI response over 50 s to obtain the following indices:(1)2 ReactivityCortical CO (CCR): time-average of model-predicted TOI response to unit-step CO2 input; Mean (SD): 0.144 (0.248) for Controls vs.–0.059 (0.244) for MCI patients, p = 0.022.(2)Cortical Tissue Perfusivity (CTP): time-average of model-predicted TOI response to unit-step CFV input; Mean (SD): 0.184(0.187) for Controls vs. 0.085(0.138) for MCI patients, p = 0.115.(3)Cortical Autoregulation to Pressure (CAP): time-average of model-predicted TOI response to unit-step ABP input; Mean (SD):–0.022 (0.117) for Controls vs.–0.029 (0.131) for MCI patients, p = 0.861.p-values are based on t-test of the difference of the means  under the Gaussian assumption. It is evident that only the CCR index is significantly different between MCI patients and Controls (p < 0.05) and reverses sign on the average (from positive in Controls to negative in MCI patients). This result suggests a significant impairment of cortical CO2 vasomotor reactivity in MCI patients. A similar result, but with a smaller p = 0.006, was found in our previous work , the mean value for MCI patients is about half the mean for Controls, suggesting the potential utility of a Composite Index defined as a linear combination of CCR and CTP according to the Fisher Discriminant , which influence the dynamic regulation of CTO and are not accounted by the employed three-input model. For example, it has been reported that the function of the neurovascular unit . Using the Fisher Discriminant  of the estimated model prediction was significantly smaller in Controls vs. MCI patients: Mean (SD): 22.17 (8.06) vs. 37.95 (18.15), corresponding to lar unit  and the lar unit  may be alar unit , to achip = 0.025) for the Composite index [CCR–1.77 × NMSE] but marginal (p = 0.072) for the CCR index alone.In order to examine whether these model-based indices correlate with the cognitive performance of the subjects/patients as quantified by the scores of the Delayed Logical Memory Recall (DLMR) neuropsychological test, we present in (new) CO2 tension and the key hemodynamic variables of arterial blood pressure and cerebral blood flow in the middle cerebral arteries (viewed as three “inputs”). These subject-specific models are used to generate model-based “physio-markers” of the regulation of cerebral perfusion in order to gain quantitative insight into the possible association between the regulation of CTO dynamics and MCI. These physio-markers cannot be evaluated for diagnostic purposes at this time, because we do not currently know what percentage of MCI patients actually have the impairment captured by these model-based indices (lack of “ground truth”). However, the potential diagnostic utility of this approach remains an important goal for future studies as more relevant knowledge is accumulated.This paper presents the results of the application of a quantitative methodology that extracts predictive models of the dynamic regulation of cortical oxygenation (viewed as an “output” variable) as a function of concurrent spontaneous changes of blood Dynamic Vasomotor Reactivity (DVR) in the middle cerebral arteries, finding that these model-based DVR indices are significantly different between MCI patients and age-matched Controls  that is obtained via NIRS and actually measures the relative hemoglobin saturation distribution in the cerebral capillary arterial/venous compartments). The main findings of this study are:The present paper utilizes the same methodological approach to examine the effects of key hemodynamic variables and blood COTissue Oxygenation Index (TOI) to a unit-step change of blood CO2 (1 mmHg) was significantly different for MCI patients vs. Controls  index (p = 0.022), indicating impaired CTO response to transient changes of blood CO2 in MCI patients.(1) The response of cortical Cortical Tissue Perfusivity (CTP) index was not significantly different between MCI patients and Controls (p = 0.115). Nonetheless, this reduction of CTP index in MCI patients may be combined with the reduction of the CCR index in MCI patients (see #1 above) to achieve smaller p-value between the two groups (p = 0.006) via a Composite Index that is defined by the respective Fisher Discriminant (see left panel of (2) The response of TOI to a unit-step change of CFV (1 cm/s) at the middle cerebral arteries was about half (on the average) in MCI patients relative to Controls  had significantly smaller Normalized Mean-Square Error (NMSE) for Controls (p = 0.0001). This finding suggests that CTO is influenced in MCI patients by more factors (relative to Controls) that confound its prediction by the three inputs of the model  via a Composite Index defined by the respective Fisher Discriminant (see right panel of (3) The model prediction of TOI from spontaneous time-series data of the three inputs  of CO2 and/or CFV.An important issue that was elucidated by the Transfer Functions obtained in this study regards the frequency-response characteristics of CTO to concurrent changes of blood ents see . The mag2 changes upon the cortical TOI .In the time-domain analysis, the average kernels shown in The counterintuitive result of CCR polarity reversal in MCI has some experimental corroboration in the neurovascular coupling literature , where iafter the dynamic effects of cerebral blood flow, blood CO2 and arterial blood pressure have been taken into account. In other words, these NMSE differences may pertain to changes in the cerebral microvascular environment and the neurovascular unit occurring in MCI. If this hypothesis can be validated in future studies, then this NMSE measure may offer quantitative insight into the state of the cerebral microvascular environment for diagnostic purposes.This discussion points to the confounding effects of possible dysfunction of the neurovascular unit , since this would explain the increased normalized mean-square error (NMSE) in the model prediction for MCI patients canonical linear dynamic models that are distinct from the data-based classification methods of “machine learning,” which may utilize any type of data/features in connection with ad hoc computational structures/models  to separate two or more classes via iterative computational schemes. In machine learning, the validation of the results is based on comparisons between “training” and “testing” datasets—but their interpretation (if any) has to be made with reference to the employed computational structure/model and tends to be difficult due to the nonlinear elements  contained therein. The results obtained via such machine learning methods are not unique on account of the employed iterative computational schemes (that may converge to different answers at different runs) and their dependence on the employed ad hoc computational structure/model. By contrast, the presented approach of linear dynamic models using the canonical  convolutional representation provides a unique answer for the given input-output data of each subject, which has universal validity (within the assumption of dynamic linearity) and can be interpreted readily with the well-developed concepts of Transfer Functions or Impulse Response Functions (kernels).It is useful to note that the presented methodology utilizes linear dynamic models and only two inputs, although this system is likely to exhibit some nonlinearities and be influenced by more than two physiological “input” variables. Our group studied previously dynamic nonlinearities in this system  and do not perform dynamic analysis oftime-series data of the type presented herein. Thus, there is no basis for direct comparison of the results obtained by these two distinct approaches. This underlines the novelty of the proposed approach.A limitation of this study is the relatively small size of the cohort that prevents proper evaluation of the model-based diagnostic physio-markers and the lack of balance in terms of the number or gender of MCI patients and control subjects in the cohort. Therefore, larger and balanced cohorts of MCI patients and Controls must be analyzed before any conclusions can be drawn regarding the potential clinical utility of the presented model-based physio-markers. In this regard, we note the confounding effects of potential vasomotor cross-sectional changes occurring in the middle cerebral arteries upon the obtained kernel estimates of subject-specific models. This is bound to increase the intragroup variability of the obtained models. Another limitation pertains to the use of s system , 2013 an outputs , provide outputs  have perCO2 dynamic reactivity in cortical tissue combined with perfusivity from cerebral arteries to CTO in MCI patients vs. age-matched Controls (p = 0.006). The observed change in the dynamics of CTO in MCI patients is primarily in lower frequencies (<0.05 Hz), suggesting impairment in endocrine/metabolic  mechanisms and neurovascular coupling. These model-based indices may serve as physio-markers of possible dysregulation of CTO during transient CO2 changes in MCI patients.In conclusion, the presented approach generates model-based indices that reveal significant statistical differences of The original contribution presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.This study has been approved by the Institutional Review Board of the UT Southwestern Medical Center and Texas Health Presbyterian Hospital Dallas. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.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.


RFP) gene to mark the transgenic seed (maintainer). The transgenic plant produces 1:1 NMS seeds and maintainer seeds that can be distinguished by the RFP signal. However, the RFP signals in the partially filled or pathogen-infected maintainer seeds are often too weak to be detected by RFP-based seed sorting machine, resulting in intermingling of the maintainer seeds with NMS seeds.The third-generation hybrid rice technology can be constructed by transforming a recessive nuclear male sterile (NMS) mutant with a transgenic cassette containing three functional modules: the wild type male fertility gene to restore the fertility of the mutant, the pollen killer gene that specifically kills the pollen grains carrying the transgene, and the red fluorescence protein (amiRNAs). In this system, the NMS seeds have normal endosperm and are heavy, but the maintainer seeds have shrunken endosperms and are light-weighted. The maintainer seeds can be easily and accurately sorted out from the NMS seeds by weight-sorting machines, so pure and fully filled NMS seeds are available.Here we constructed a weight-based seed sorting system for the third-generation hybrid rice technology by silencing the genes encoding ADP-glucose pyrophosphorylase (AGP) essential for endosperm starch biosynthesis via endosperm-specific expression of artificial microRNAs (The weight-based seed sorting system shows obvious advantages over the RFP-based seed sorting system in accuracy, efficiency, and cost for propagation of pure male sterile seeds. These characteristics will significantly increase the value and transgenic safety of the third-generation hybrid rice technology.The online version contains supplementary material available at 10.1186/s12284-021-00510-y. Rf) genes that can specifically inhibit the function of the aberrant Mt gene  lines or photoperiod/thermo-sensitive genic male sterile (PTGMS) lines as female parent  and two  family members in rice     fusion with the amyloplastid targeting signal peptide ASP1 of α/β-hydrolase (LOC_Os01g39800) under the pollen-specific promoter of OsLSP3 (late-stage pollen-specific gene 3)   from Tity Fig. a,b. Planity Fig. a,c, and ity Fig. d-f. Afteity Fig. g. TransgThe moderate weight-reduction of the maintainer seeds made the weight-based sorting system feasible to separate the maintainer seeds from male sterile seeds for the third-generation hybrid rice technology. Although the maintainer plants were slightly weaker at the early stage, later growth and seed setting were not affected. Compared with the RFP-based seed sorting system, weight-based seed sorting system has the following advantages. First, the RFP system cannot sort out the maintainer seeds with weaker fluorescence signal, resulting in intermingling of the maintainer seeds with the NMS seeds. This not only leads to transgene escaping into environment, the presence of maintainer lines in the male sterile lines during hybrid seed production is also a problem. However, the weight-sorting machine only keeps the full seeds. The partially filled male sterile seeds are also sorted out together with the maintainer seeds, which ensures high quality of the male sterile seeds without any maintainer seeds. Even though a small fraction of the male sterile seeds are immingled with the maintainer seeds, it is not problematic to production, because the male sterile plants can only be pollinated by the maintainer line to produce the male sterile seeds. Second, the RFP-based seed sorting machines require complicate designs and are expensive for manufacturing and maintenance. Currently, the most efficient RFP-based seed sorting machine can process only ~ 35 kg seeds per hour (Song et al., amiRNA genes. This system shows obvious advantages over the RFP-based seed sorting system in accuracy, efficiency, and cost for propagation of male sterile seeds. The application of this system will increase the transgenic safety and makes the third-generation hybrid rice technology more profitable. While it is proved here in rice, the same strategy can be applied to other crops as well to expand the utilization of heterosis. In rice, manipulation of grain weight can be achieved by regulating starch synthesis. For other crops, manipulating seed weight is also possible by regulating seed size if such gene is available.In summary, we developed a weight-based seed sorting system for the third-generation hybrid rice technology by silencing the committed genes for starch synthesis via endosperm-specific expression of Additional file 1: Materials and Methods. Table S1. List of primers.


The aim of this study was to explore the influence of different intensities of acute aerobic exercise on brain activation in male methamphetamine (MA)-dependent patients during exercise. Twenty MA-dependent patients were divided randomly into two groups participating in 35 min of either moderate- or high-intensity aerobic exercise. Functional near-infrared spectral imaging (fNIRS) was used to detect hemodynamic changes in prefrontal cortex during the main 25-min exercise stage. The results revealed that high-intensity acute aerobic exercise aroused more cerebral oxygenation changes in the prefrontal cortex and left dorsolateral prefrontal cortex during exercise as compared with moderate-intensity exercise. Furthermore, there was a stronger positive connection observed between orbital frontal cortex and left dorsolateral prefrontal cortex in the high-intensity group than in the moderate-intensity group. Together these results suggest that for submaximal exercise intensities, high-intensity exercise may bring more benefits to male MA-dependent patients than moderate-intensity. Exercise program, especially aerobic exercise has been considered to be an effective treatment for drug abuse . PreviouAlthough a large number of relevant studies have shown positive effects of aerobic exercise on drug addicts, the neurological mechanisms remain unclear. Changes in the levels of oxygenated and deoxygenated hemoglobin in PFC that accompany exercise offer a possible neural basis . IncreasThus, this study aimed to explore the influence of different intensities of acute aerobic exercise on the activation of PFC in male methamphetamine (MA)-dependent patients. A fNIRS measure was used to detect hemodynamic changes in PFC during exercise. The effects of exercise of different intensities were systematically compared to explore whether there is still a positive relationship between intensity and exercise effect in MA-dependent patients.Twenty male participants aged 18–45 years were recruited from the Chinese Shiliping Drug Rehabilitation Bureau in Zhejiang. All subjects met the DSM-V criteria for MA-dependent according to the Structured Diagnostic Interview, and has abstinent for at least 3 months. The exclusion criteria were a history of psychosis, physical or medical conditions that contraindicated exercise. Then, the subjects were divided randomly into two groups: moderate- and high-intensity exercise groups. The demographic characteristics of two groups are shown in Subjects in the two exercise groups were engaged in exercise programs that involved a 35-min exercise program by using cycle ergometer at moderate and high intensity, respectively, which was created in accordance with the recommendations of the American College of Sports Medicine . The intA multi-channel continuous-wave fNIRS instrument  was used to monitor hemodynamic activity during the 25-min main exercise stage. The fNIRS probe consisted of eight dual-wavelength sources (780 and 830 nm) and seven optical detectors. The sampling rate was 7.81 Hz. The probes were arranged in accordance with the 10–20 system, and each emitter was no more than 3 cm away from its corresponding detector. One source and one detector formed a channel, and 20 channels were formed in PFC . Informa1 was used to calculate Pearson’s correlation between the selected seed ROI and other ROIs to weigh the strength of functional connectivity of the pairwise relationships. Prior to any statistical analysis, the correlation values were converted to Fisher z-values using the Fisher z-transformation.Data were analyzed using Homer2 open source software , implemented in MATLAB using channel-based cubic spline interpolation was used to detect and remove motion artifacts . The comparison of mean [HbO] of each ROI during 25-min exercise vs. 0-value has been performed to test the existence of a group activation. Mean values for the behavioral variables were reported with standard errors. The statistical significance level was set to p < 0.05.Data of one participant in each of the two groups was excluded because of excessive artifacts; the remaining 18 participants were included in the final analyses. The Mann-Whitney U-test showed that the mean [HbO] of PFC in the high-intensity group (0.24 × 10–4 ± 0.17 × 10–4 mmol/L) was higher than that of the moderate-intensity group  was higher than that of the moderate-intensity group  was higher than that of the moderate-intensity group , the mean [HbO] of the high-intensity group in PFC , indicating that high-intensity acute aerobic exercise may enhance the connection between left DLPFC and OFC. No significant group differences were found in correlation between left DLPFC and other 4 ROIs.Group connectivity matrices are shown in The current study used fNIRS to explore the effect of moderate- and high-intensity acute aerobic exercise on changes in PFC activity in MA-dependent patients. We found that compared with moderate-intensity, high-intensity acute aerobic exercise induces more cerebral oxygenation changes in PFC during exercise, indicating that there may be a positive relationship between exercise intensity and exercise effect in MA-dependent patients. This is in line with previously published studies on healthy volunteers which found that high-intensity exercise promotes cerebral oxygenation changes in PFC when exercise intensity remains submaximal .Exercise is known to promote the growth of blood vessels in the brain and to increase blood flow in response to physiological and neuronal changes ; the excOur functional connectivity analysis showed that compared with moderate-intensity exercise, the positive connection between OFC and left DLPFC in high-intensity group had a higher tendency before correction. OFC is involved in processing motivation-related information and guiding behavior within the addiction loop, and is also responsible for monitoring information relating to reward or punishment and behavioral inhibition . People The current study has several limitations. Due to the particularity of the subjects there are restrictions on their recruitment, which leads to a slightly insufficient number of subjects participating in the experiment and some experimental results only showing marginal significance. Similarly, this study was limited by environmental conditions that only recruited male subjects and had a lack of female subjects. Previously published findings in healthy volunteers showed that overly high intensities may affect the efficiency of exercise interventions for cognitive function in female individuals, while male individuals can expect further improvements from highly intense programs . TherefoThe present study used the measurement of hemodynamic changes in PFC during acute aerobic exercise in MA-dependent subjects to demonstrate that high-intensity exercise induces more brain activation in PFC and more correlated activity between left DLPFC and OFC. Those results remind us that for submaximal exercise intensities, higher intensities may bring more benefits to male MA-dependent patients, such as the enhancement of brain activation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee, Shanghai University of Sport, Shanghai, China (102772019RT044). The patients/participants provided their written informed consent to participate in this study.YC designed the experiment and gave final approval. SG and CZ conducted the experiment. SG and YC analyzed the data and drafted the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


An association between celiac disease and IgA nephropathy (IgAN) has been suggested. In celiac disease, in addition to circulating in serum, IgA-class tissue transglutaminase (tTG) autoantibodies are deposited in the small bowel mucosa and extraintestinal organs. In this case series of IgAN patients with or without celiac disease, we studied whether celiac disease-type IgA-tTG deposits occur in kidney biopsies. The study included nine IgAN patients, four of them with celiac disease. At the time of the diagnostic kidney biopsy serum tTG autoantibodies were measured and colocalization of IgA and tTG was investigated in the frozen kidney biopsies. Three IgAN patients with celiac disease had IgA-tTG deposits in the kidney even though in two of these the celiac disease diagnosis had been set years later. These deposits were not found in a patient with already diagnosed celiac disease following a gluten-free diet. Of the five non-celiac IgAN patients, three had IgA-tTG deposits in the kidney. We conclude that tTG-targeted IgA deposits can be found in the kidney biopsies of gluten-consuming IgAN patients but their specificity to celiac disease seems limited. Celiac disease, an immune-mediated enteropathy, is driven by the ingestion of cereals, wheat, rye, and barley containing gluten and characterized by a disease-specific autoantibody response targeting tissue transglutaminase (tTG) . During It has been proposed that celiac disease may be associated with IgA nephropathy (IgAN), globally the most common primary chronic glomerular disease ,10. The The association between celiac disease and IgAN is still being actively researched. In this case series we investigated whether celiac disease-type tTG-targeted IgA autoantibody deposits occur in the kidney biopsies of IgAN patients with or without concomitant or subsequent celiac disease.This retrospective study reports a case series of nine adult IgAN patients on whom diagnostic kidney biopsy was performed during the period 1981–1987 at Tampere University Hospital, Finland. IgAN was defined as glomerulonephritis with typical light microscopy features and IgA as the sole or main glomerular immunofluorescence finding . ®, Phadia, GmbH, Freiburg, Germany) according to manufacturers’ instructions. Values higher than 7.0 U/mL were regarded as positive.Stored frozen (−80 °C) serum samples taken at the time of kidney biopsy were used to identify IgA-class tTG autoantibodies using the ELIA Celikey assay  was approved by the Regional Ethics Committee of Pirkanmaa Hospital District. The study protocol follows the ethical guidelines of the Declaration of Helsinki. By definition, all nine IgAN patients in our case series had glomerular IgA deposits characteristic of IgAN a. Four oIn the kidney biopsies, celiac disease-type deposits characterized by colocalization of IgA and tTG were detected in all three patients with both IgAN and celiac disease on a normal gluten-containing diet . It is nOf the five IgAN patients without celiac disease, three had celiac disease-type IgA-tTG deposits around the proximal and distal tubuli in the kidney  without having elevated levels of serum tTG autoantibodies at the cross-sectional serologic evaluation at diagnosis . Of the The median follow-up for all patients was 28 (range 5–35) years . Four patients with IgAN and celiac disease were followed-up for a median 28 years (range 5–35) . Two of these had died and two suffered from severe loss of kidney function (eGFR 17 and 23 respectively). Five IgAN patients without celiac disease were followed-up for a median 20 years (range 10–35) . One had normal kidney function, two mild loss of kidney function, and two moderate renal impairment . None ofIn this case series, we found that, during ingestion of gluten, celiac disease-type tTG-targeted IgA deposits were present in the kidney biopsies of all patients with both IgAN and celiac disease and thus our results are in line with the case report by Costa et al. . Two of In our study the IgA-tTG deposits in the kidney were not specific to celiac disease patients since they were also found in three non-celiac IgAN patients without serum tTG autoantibodies. Although no data is available on the presence of extraintestinal IgA-tTG deposits in seronegative celiac disease, such patients have been reported to have such deposits in the small bowel mucosa . UnfortuIn our study the patients with IgAN and celiac disease seemed to have worse outcomes of IgAN than did the patients with IgAN only. This finding is interesting in the light of our earlier study and the Swedish study by Rehnberg and co-workers showing that prognosis in IgAN may be poorer with concomitant comorbid bowel disease ,30. HoweTo conclude, tTG-targeted IgA deposits were found in the kidney biopsies of gluten-consuming IgAN patients with or without known celiac disease. The significance of this interesting finding remains open and therefore in the future larger studies, preferably with more data on small bowel histochemistry and regular follow-up serology for tTG antibodies, are needed to investigate the association between celiac disease and IgAN in greater detail.