Exercises in virtual reality (VR) have recently become a popular form of rehabilitation and are reported to be more effective than a standard rehabilitation protocol alone. The aim of this study was to assess the efficacy of adjunct VR training in improving postural control in patients after total knee replacement surgery (TKR). Forty-two patients within 7–14 days of TKR were enrolled and divided into a VR group and a control group (C). The C group underwent standard postoperative rehabilitation. The VR group additionally attended twelve 30-min exercise sessions using the Virtual Balance Clinic prototype system. Balance was assessed on the AMTI plate in bipedal standing with and without visual feedback before and after the four-week rehabilitation. Linear measures and sample entropy of CoP data were analyzed. After four weeks of rehabilitation, a significant reduction in parameters in the sagittal plane and ellipse area was noted while the eyes remained open. Regression analysis showed that sample entropy depended on sex, body weight, visual feedback and age. Based on the sample entropy results, it was concluded that the complexity of the body reaction had not improved. The standing-with-eyes-closed test activates automatic balance mechanisms and offers better possibilities as a diagnostic tool. The complex interaction of somatosensory, visual and vestibular feedback networks, numerous brain regions, and the musculoskeletal system creates a system involved in postural control . PosturaOsteoarthritis is the most prevalent rheumatic joint disorder that results from breakdown of joint cartilage and underlying bone . OsteoarVirtual reality (VR) games use interactive computer environments that appear to be real to improve daily activities or functional movements . The patDiverse indicators have been explored in postural stability assessments, mostly based on the trajectory of the center of pressure (CoP). Classical approaches focus on CoP variability based on the notion that it displays extensive irregularities and nonstationary fluctuations during quiet standing. Therefore, linear tools, such as the CoP path length, sway velocity and area, quantify the amount of CoP movement during a specific task, independently of their order in the distribution. To better understand the physiology of postural control, nonlinear measures should be applied ,16. The The entropy family quantifies the regularity of a signal with predictable (for example periodic) signals resulting in low entropy and completely unpredictable signals resulting in high entropy. In addition to multiscale entropy (univariate or multivariate) and approximate entropy, sample entropy is one of the nonlinear parameters often used to calculate from CoP data. In contrast to approximate entropy, sample entropy does not depend on record length and is characterized by relative consistency . Sample The objective of the study was to assess the effect of adjunct VR-based training on changes in balance parameters, including classic measures and sample entropy, in the short period after knee arthroplasty.The study enrolled 42 patients within 7–14 days after total knee replacement surgery (TKR). All patients were operated on at the Orthopedic Department of Professor Adam Gruca Independent Public Teaching Hospital in Otwock. The inclusion criteria comprised (1) noncomplicated total knee replacement surgery because of primary knee osteoarthritis, (2) no other balance problems , (3) no current musculoskeletal complaints other than related to the operated joint, and (4) written consent to participate in the study.Patients meeting the inclusion criteria were divided using a block randomization technique  into twoAll patients qualified for the study attended a standard four-week protocol of stationary rehabilitation involving five rehabilitation sessions per week with each session lasting approximately 4 h. Rehabilitation procedures were performed by physiotherapists (A.K. and R.B.). Treatment included individual exercises , continuous passive motion exercises, gait and balance exercises, manual therapy and massage (with a focus on soft tissue and patellar and scar mobilization), cryotherapy in the operated area, laser therapy for scarring, magnetic field therapy and kinesiology taping applications.The study group additionally received 12 sessions (three sessions per week) of virtual reality games on the Virtual Balance Clinic (VBC) prototype system . The VR games were applied concurrently with other treatments. The VBC system consists of a balance plate and a “Kinect 2” camera. The balance plate allows the measurement of displacement of the center of pressure (CoP) in real time, and the “Kinect 2” camera is used to trace body movements. Accordingly, VBC software allows for qualifying each movement performed during exercises as “correct” or “false” or quantifying some movements as “partly correct”. The VBC system offers the choice of nine different games, including tasks, such as CoP movement in the sagittal or frontal plane or diagonally during standing with both legs parallel or toe to heel standing, maintaining balance during upper body movements (such as trunk rotations and/or arm movements), one leg standing, forward or side steps, or walking in place. Each session lasted 30 min and included three different games, and each patient played all games for the same length of time during the rehabilitation period. The level of difficulty was adjusted individually for each patient by a physiotherapist supervising the exercises. Each patient was assessed twice: before and after the four-week rehabilitation. All patients completed their rehabilitation protocol.The study protocol was approved by the Bioethics Committee of the Medical University of Warsaw (no. KB/28/2014). The study was conducted according to the ethical guidelines and principles of the Declaration of Helsinki.m + 1 points without allowing self-matches: B represents the total number of matches of length m, whereas A represents the subset of B that also matches for m + 1. SampEn was calculated using MATLAB  codes obtained from the Physionet tool [m = 2 and r = 0.2*(standard deviation of the data).The postural stability data for each subject were recorded using an AMTI AccuSway  plate with Balance Clinic software. Sample rate was set at 100 Hz. Each person completed three trials of bipedal standing with eyes open and three trials of bipedal standing with eyes closed. Each trial lasted 30 s with a one-minute rest between trials. We used the most popular parameters obtained with traditional linear and nonlinear methods, as proposed by various authors ,16,30. Tnet tool  with “dep-value was set at 0.05. The results of the patients’ second trials were analyzed. The second trial results were used because the patients would not always comply with the requirements on their first attempt and often reported fatigue on the third attempt. The Shapiro-Wilk test was used to assess the normality of all data distributions. Wilcoxon’s paired rank test was used to compare the effect of rehabilitation on the parameters of postural stability in each group. The study and control groups were subsequently compared using the Mann-Whitney U test to detect significant differences. In the last step, multi-variation analysis of regression using GRETL-GNU Regression, Econometric and Time-series Library version 2019a  was performed. The regression analysis was performed using the least squares method. The choice of the best model was based on the Akaike information criterion.Statistical analysis was performed using Statistica v. 13.1 , and the cut-off The results section is divided into three subsections. The first subchapter examines differences between the groups and the impact of rehabilitation protocols on postural control. The following section focuses on the visual feedback influence on the postural stability parameters. The last subchapter focuses on regression analysis.In the study group, which received additional VR-assisted rehabilitation with the VBC system, all linear parameters decreased in standing with eyes open and eyes closed . AdditioSampEn values in the study group decreased in the frontal plane and increased in the sagittal plane in both the closed- and open-eye tests. The same pattern was observed in the control group but only during standing with eyes closed. All these changes were not statistically significant.A comparison of the study and control groups failed to reveal statistically significant differences in the key anthropometric parameters of the subjects. Similarly, no significant differences were noted between the groups in the Mann-Whitney U test results with regard to the linear and nonlinear parameters and the influence of rehabilitation or visual feedback.p = 0.006) and path length (p = 0.001), whereas the control group demonstrated a significantly (p = 0.001) higher value of path length in the measurement with eyes closed.Analysis of the impact of visual feedback on the postural control parameters revealed much greater differences both before and after rehabilitation than those described in the previous section. In both groups before rehabilitation, closing eyes produced non-significantly higher values of all linear and nonlinear parameters. Additionally, the study group registered significantly higher values of SampEn for medio-lateral sway (p = 0.001), ellipse area (p = 0.017) and range of AP sway (p = 0.0301), whereas the control group additionally demonstrated higher SampEn for AP sway (p = 0.019) and ML sway (p = 0.014).Testing after the 4-week rehabilitation again revealed increased values of all parameters in standing with eyes closed. The study group registered significantly higher values of path length (p = 0.036), body mass  (p = 0.004) and age   a, visual= 0.004) b. No sigThis study aimed to assess the effect of adjunct VR-based training on balance parameters in patients undergoing rehabilitation shortly after total knee replacement surgery. Our results indicate that, unfortunately, no significant improvement in the postural stability parameters assessed was noted either following a standard rehabilitation protocol or an enhanced protocol with VR training. The underlying reason might have been that the four-week interval between surgery and assessment was possibly too short to demonstrate a significant clinical advantage of VR-enhanced rehabilitation. We have chosen 4-week interval between assessments because rehabilitation standards of the National Health Service after TKR include three- or four-weeks stationary rehabilitation protocol. Other studies have typically used rehabilitation protocols with at least an eight-week duration . AnotherTherefore, the present authors decided to use sample entropy as a more sophisticated research tool. The entropy family quantifies signal regularity . A more In summary, four weeks after TKR appears to be too early for an assessment of postural stability to produce coherent findings. This period was too short to obtain a significant improvement in balance, or such improvements can become visible over time. The sample entropy data indicate that the complexity of the body’s balance mechanisms did not improve.The presented study has some limitations, what include: small number of participants. Statistical power was in range 0.0561  up to 0.7901 . Thus, required number of participants should be much higher than 21 persons.


Caenorhabditis elegans mechanoreceptors located in ASG sensory neurons have been found to sense ambient temperature, which is a key trait for animal survival. Here, we show that experimental loss of xanthine dehydrogenase (XDH‐1) function in AIN and AVJ interneurons results in reduced cold tolerance and atypical neuronal response to changes in temperature. These interneurons connect with upstream neurons such as the mechanoreceptor‐expressing ASG. Ca2+ imaging revealed that ASG neurons respond to warm temperature via the mechanoreceptor DEG‐1, a degenerin/epithelial Na+ channel (DEG/ENaC), which in turn affects downstream AIN and AVJ circuits. Ectopic expression of DEG‐1 in the ASE gustatory neuron results in the acquisition of warm sensitivity, while electrophysiological analysis revealed that DEG‐1 and human MDEG1 were involved in warm sensation. Taken together, these results suggest that cold tolerance is regulated by mechanoreceptor‐mediated circuit calculation. Caenorhabditis elegans.A DEG/ENaC‐type mechanoreceptor‐mediated neural circuit regulates cold tolerance in  Caenorhabditis elegans is an ideal model for studying the neural circuitry underlying cold tolerance given its simple nervous system composed of only 302 neurons, whose connections are entirely known, as well as the range of well‐studied molecular and genetic approaches currently available Caenorhabditis elegans mutants have also been extensively used to identify key genes and determine the specific neurons at which they exert their effects C. elegans temperature response has been analyzed with respect to many phenomena, including dauer larva formation Temperature tolerance and acclimation are essential for all organisms, with such tolerance being controlled by multiple components such as the nervous system and muscles. C. elegans possesses an adaptive mechanism to tolerate cold external environments. For example, wild‐type worms grown at 15°C can survive at a temperature of 2°C, whereas those grown at 20°C or 25°C cannot  channels. TRPV1, for example, is known to detect regions of high temperature, while TRPA1 detects regions of low temperature + channel (DEG/ENaC) proteins comprise a diverse family of Na+ ion channels C. elegans homologue DEG‐1 is expressed in association with multiple mechanosensory neurons + potential across MDEG The degenerin/epithelial NaXanthine dehydrogenases (XDHs) are widely distributed in all eukaryotic organisms and bacteria, and are predicted to play important roles such as in purine catabolism C. elegans is thought to be a useful model for studying XDH.Xanthine dehydrogenase is expressed in the liver and small intestine in mammals xdh‐1 mutants XDH knockout worms exhibit abnormal cold tolerance and that their normal function can be recovered by expressing xdh‐1 cDNA in both AIN and AVJ interneurons. In vivo Ca2+ imaging also revealed that XDH‐1 acts as a positive temperature signal regulator in AIN and as a negative one in AVJ and that warm sensation by ASG sensory neurons via the mechanoreceptor DEG‐1 affects the neural activity of both AIN and AVJ interneurons. Moreover, the ectopic expression of DEG‐1 in the ASE, a non‐warm‐sensitive chemosensory neuron, resulted in the acquisition of the ability to sense warm sensation. In addition, two‐electrode voltage‐clamp recording of Xenopus oocytes expressing DEG‐1 demonstrated thermoreceptor‐like behavior. These results together suggest that DEG‐1, a DEG/ENaC‐type mechanoreceptor, is sufficient to confer warm responses and is required for the neural circuit calculation of the positive regulation of cold tolerance.We found that chr1 mutation associated with decreased cold tolerance following cultivation at 15°C   mutants possessed two point mutations in this domain: one in a conserved splicing acceptor site and the other in a nonconserved amino acid residue  Fig , and XDHity) Fig . FAD is ity) Fig . Iron–suity) Fig , and ouridue Fig . Yet anosite Fig ; we usedxdh‐1 mutants, we used the fluorescent ROS indicator molecule 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCF‐DA). Nonfluorescent H2DCF is converted to fluorescent 2′7′‐dichlorofluorescein (DCF) through interaction with intracellular ROS xdh‐1(ok3234), and daf‐2(e1370) ROS concentrations, using the latter daf‐2(e1370) mutants as a control. In wild‐type worms, ROS concentrations gradually increased during the assay, while xdh‐1(ok3234) concentrations remained normal (xdh‐1(ok3234) mutants do not experience fluctuation in ROS concentration . xdh‐1 cDNA expression in intestine and excretory cells, however, did not rescue cold tolerance  responsible for sues Fig  and founance Fig . This suxdh‐1 mutants was severer at the adult stage than that at the larval one (xdh‐1(ok3234) mutant cold tolerance, we conducted a time course assay for cold tolerance . XDH‐1 expression in approximately 70 neuron pairs driven by eight types of promoter also rescued abnormal cold tolerance of the xdh‐1 mutants . Likewise, expressing XDH‐1 in approximately 30 neuron pairs driven by eat‐4 and unc‐42 promoters achieved this rescue . Similar rescue phenomena of xdh‐1 mutants were observed by expressing XDH‐1 driven by eat‐4 or unc‐42 promoter . Both eat‐4 and unc‐42 promoters allowed XDH‐1 expression in ASH, AIN, and AVJ neurons . Furthermore, XDH‐1 expression in several pairs of neurons containing ASH, AIN, and AVJ restored the defect of xdh‐1 mutants , whereas XDH‐1 expression in three neurons containing ASH did not achieve this . We found that decreased cold tolerance of xdh‐1 mutants was rescued by the specific expression of XDH‐1 in both AIN and AVJ interneurons driven by inx‐17 and hlh‐34 promoters . However, the expression of XDH‐1 in either AIN or AVJ alone did not achieve this rescue . These results suggest that the function of XDH‐1 in both AIN and AVJ is required for normal cold tolerance.To determine the neuron type required for ters Fig . ExpressC. elegans behaviors. To explore possible involvement of AIN and AVJ functions in a behavior, chemotaxis toward AWA‐ or AWC‐sensed odorants was evaluated in xdh‐1(ok3234) mutants. Mutation of the xdh‐1 gene was found to have no influence on attraction toward AWA‐sensed diacetyl or AWC‐sensed benzaldehyde ‐type mechanoreceptor, resulted in particularly severe cold tolerance dysfunction   Fig A; howevetype Fig A and B.C. elegans. Ca2+ imaging revealed that wild‐type ASG responds to temperature changes by increasing intracellular Ca2+ concentration  and 31.0 ± 0.3°C for MDEG (n = 8)].Next, we conducted electrophysiological experiments to determine the thermosensitivity of DEG‐1 by applying the two‐electrode voltage‐clamp recording method to ytes Fig C–F. The ytes Fig . Conversytes Fig . These rxdh‐1 gene acts as a positive regulator of cold tolerance in C. elegans. Opposed functions of XDH‐1 in AIN and AVJ interneurons are a central in neural circuit calculation of cold tolerance. ASG sensory neuron is a functionally upstream interneuron of AIN and AVJ, and mechanoreceptor DEG‐1 in ASG is critical for temperature response of ASG. Ectopic expression of DEG‐1 in a gustatory neuron and Xenopus oocyte resulted in acquisition of warm sensitivity. Thus, the mechanoreceptor acts as an ambient temperature sensor in a neural circuit underlying the cold tolerance in C. elegans  specifically, which is located at right side of the head. Gong et alche‐1 gene encodes a C2H2‐type zinc finger transcription factor orthologous to Drosophila GLASS essential for photoreceptor cell differentiation, and CHE‐1 is required for determining the identity and function of the ASER and ASEL neurons che‐1 mutant defective in development of ASER and ASEL neurons showed normal cold tolerance glr‐3 mutant, we previously reported the analysis of GOA‐1 goa‐1 mutant showed abnormal cold tolerance, and its abnormality was rescued by expressing wild‐type goa‐1 cDNA in ASJ sensory neurons in goa‐1 mutant, in which GOA‐1 signaling downstream of GLR‐3 in ASER is remaining defective et alXenopus oocytes expressing DEG‐1 and its human homologue MDEG1 that both of them are capable to confer warm responsiveness. These results suggested that DEG‐1 confers warm‐sensing ability. Besides, other genetic and Ca2+ imaging studies in this study suggested that the function of DEG‐1 is involved in warm sensation of ASG, which affects downstream AIN and AVJ interneurons essential for cold tolerance.The results in this study showed that mechanoreceptor DEG‐1 is sufficient to confer temperature responsiveness when ectopically expressed in non‐warm‐sensitive ASE gustatory neuron and + current in Xenopus oocytes expressing DEG‐1 and its human homologue MDEG  in all experiments, KHR066/RB2575 flp‐17(ok3587) xdh‐1(chr1), KHR067/RB2379 xdh‐1/F55B11.1(ok3234), VC883 tag‐273(gk371), FX07280 tbc‐9(tm7280), KHR069 xdh‐1(chr1), CB1066 mec‐1(e1066), CB75 mec‐2(e75), CB1338 mec‐3(e1338), CB1339 mec‐4(e1339), CB1340 mec‐5(e1340), CB1472 mec‐6(e1342), CB2477 mec‐7(e1343), CB398 mec‐8(e398), CB1515 mec‐10(e1515), CB3284 mec‐12(e1605), TU55 mec‐14(u55), TU75 mec‐15(u75), TU265 mec‐17(u265), TU228 mec‐18(u228), TU38 deg‐1(u38), NC279 del‐1(ok150), DH246 let‐2(b246), VC1812 tab‐1(gk858), MT1098 unc‐105(n506), VC2633 degt‐1(ok3307), FX010725 pezo‐1(tm10725), PT8 pkd‐2(sy606); him‐5(e1490), TQ296 trp‐4(sy695), and CB49 unc‐8(e49). See We used the following t‐test (Welch). Multiple comparisons were performed using one‐way ANOVA tested with the Tukey–Kramer method and Dunnett's test. Dunnett's test was performed to compare the groups represented by the leftmost bar in the graphs with the other groups. Comparisons between other pairs of groups were performed using the unpaired t‐test (Welch) .Cold tolerance testing was conducted on six or more plates for 3 or more nonconsecutive days. All error bars in the figures indicate standard error of the mean (SEM). All statistical analyses assumed a normal distribution and were performed using parametric tests, the Tukey–Kramer method, Dunnett's test, or the unpaired Escherichia coli OP50 once the worms began laying eggs. Adults were removed after 16–24 h at 15°C, and progeny were left to mature for 120–130 h at 15°C. Before the next generation had hatched, plates containing fresh adult worms were counted after being placed on ice for 20 min, followed by transfer to a 2°C refrigerated cabinet  for 8–96 h. The temperature inside this cabinet was monitored using both digital and mercury thermometers. After cold stimulus, plates were either repeatedly transferred to a room‐temperature environment  or stored at 15°C overnight. We then counted living and dead worms to calculate survival rates.The cold tolerance assay was performed in accordance with previous reports Chemotaxis to volatile odorants was assayed in accordance with previous reports Lipids were extracted from synchronized cultures of adult worms and then transmethylated as described in previous studies 3. A few adult or larval worms were then placed on the gel. The gel was covered by glass, and fluorescent images were analyzed by confocal laser microscopy , using FV10‐ASW software .The following procedure was used to prepare samples for confocal microscopy: 2% (w/v) agarose gel on a glass micro‐slide was covered with 10 μl of 100 mM NaNrol‐6gf, pAK62 AIYp::gfp, or pKDK66 ges‐1p::nls::gfp as a transgenic marker at 30–50 ng/μl.Germline transformations were conducted as described previously 2DCF‐DA) . We used synchronized, counted adult worms cultivated at 15°C. Approximately 200–250 worms were harvested and washed in M9 buffer. Bacteria (OP50) were removed by three repeated washes, after which the resulting fluid was centrifuged at low speed. Animals were homogenized in 400 μl of PBS with 0.1% Tween 20 on ice using a glass homogenizer. To measure fluorescence, we used a fluorescence microplate reader  every 10 min for 1 h with an excitation wavelength of 485 nm and an emission wavelength of 535 nm at 15°C.Reactive oxygen species levels were quantified using the cell‐permeable, nonfluorescent probe 2′,7′‐dichlorofluorescein diacetate , pgp‐12p (3.5 kb), ges‐1p (3.3 kb), xdh‐1p (3.4 kb), dat‐1p (0.7 kb), osm‐6p (2 kb), ncs‐1p (3.1 kb), glr‐1p (5.4 kb), unc‐8p (4.2 kb), unc‐47p (0.3 kb), acr‐2p (3.4 kb), eat‐4p (6.4 kb), unc‐42p (3 kb), unc‐86p (3.6 kb), ocr‐4p (4.8 kb), ceh‐10p (3.5 kb), sra‐6p (3.8 kb), lim‐4p (3.6 kb), ser‐2p (4.1 kb), inx‐17p (1.2 kb), and hlh‐34p (2.5 kb), were inserted upstream of pNTN027 xdh‐1 cDNA, to create pNTN034, 035, 036, 046, 047, 048, 049, 050, 051, 052, 053, 054, 055, 057, 059, 060, 061, 063, 064, 067, and 068 plasmids, respectively, for cellular experimentation. pNTN075 hlh‐34p::yc3.60 contains the 2.5 kb hlh‐34p gene and the yc3.60 gene. pNTN106 gcy‐5p::deg‐1 cDNA contains the gcy‐5 promoter received from Dr. Yuichi Iino, the University of Tokyo. pNTN116 contains the inx‐17 promoter, the yc3.60 gene, and the 3′‐UTR of the let‐858 gene. pNTN123 gcy‐21p::yc3.60 contains the 1,403 bp upstream promoter sequence of the gcy‐21 gene amplified by PCR from the wild‐type genome, which was created by replacing the hlh‐34p gene of pNTN075 with gcy‐21p. Previous reports described the gcy‐21p::gfp construct containing the first and second exons and the first intron as inducing the expression of GFP strongly in ASG and weakly in ADL. However, the gcy‐21p::gfp construct excluding all exons and introns induces GFP expression in ASG only. We therefore used gcy‐21p as an ASG‐specific promoter. pNTN126 gcy‐21p::deg‐1 cDNA contains the 1,403 bp upstream promoter sequence of gcy‐21 and deg‐1 cDNA. pMIU34 flp‐6p::CeG‐CaMP8 contains a 2,680 bp upstream promoter sequence for the flp‐6 gene and G‐CaMP8 that is codon‐optimized for C. elegans (CeG‐CaMP8). deg‐1 cDNA was inserted into a pGEMHE vector containing Xenopus beta‐globin 5′‐ and 3′‐UTR for electrophysiological recording (pNTN119). MDEG cDNA was inserted into a pGEMHE vector containing Xenopus beta‐globin 5′ and 3′ UTR for electrophysiological recordings (pNTN125). pNTN143 ceh‐10p::nCre contains the ceh‐10 promoter sequence and the nCre sequence. pNTN144 hlh‐34p::LoxP::xdh‐1 cDNA::LoxP contains the hlh‐34 promoter sequence, xdh‐1 cDNA, sandwiched between two LoxP‐flanked stop sequences. pNTN145 inx‐17p::LoxP::xdh‐1 cDNA::LoxP contains the inx‐17 promoter sequence, xdh‐1 cDNA, also sandwiched between two LoxP‐flanked stop sequences. pNTN159 nmr‐1p::yc3.60 contains 5 kb of upstream nmr‐1 gene sequence including the first five codons and yc3.60.pNTN020 deg‐1 cRNA and MDEG cRNA were separately injected into oocytes and incubated at 18°C for 3–6 days before electrophysiological recordings were performed. We held the membrane potential at −80 mV and recorded the macroscopic current using the two‐electrode voltage‐clamp technique with a bath clamp amplifier  and pClamp software  in bath solution containing 100 mM NaCl, 2 mM MgCl2, and 10 mM HEPES (pH 7.3). Temperature stimulation was regulated using a lab‐made temperature controller with a range of 10–35°C and was monitored by both a thermistor probe adjacent to the oocytes and a thermometer . An Arrhenius plot was created, indicating the current amplitude induced by temperature changes on the y‐axis  versus the inverse of temperature on the x‐axis . Temperature thresholds were determined by the intersection of the two linear regions (magenta lines), and all thresholds were then averaged. As a negative control experiment, we used amiloride, an inhibitor of DEG/ENaC ion channel, and oocytes incubated at 18°C in bath solution with 500 μM amiloride (Sigma‐Aldrich) for 48 h.In vivo Ca2+ imaging was performed in accordance with previous reports 2+ indicators. When using GCaMP8, we co‐expressed tag‐RFP (pKOB006 gcy‐5p::tag‐RFP) to measure the fluorescence ratio between GCaMP and tag‐RFP 2+ indicator in one or more neurons were glued to a 2% (w/v) agar pad on glass, immersed in M9 buffer, and covered by a cover glass. Cyan (CFP) and yellow (YFP) fluorescence by YC3.60, and green (GCaMP8) and red (tag‐RFP) fluorescence were simultaneously captured using an EM‐CCD camera, EVOLVE512 . Changes in intracellular Ca2+ concentration were measured as the yellow/cyan fluorescence ratio for YC3.60 or green/red fluorescence ratio for GCaMP8. See NT, AO, KO, AKa, YM, AT, YF, and AKu performed the experiments; NT, AO, AT, YF, and AKu designed the experiments, interpreted the results, and wrote the final report.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Source Data for AppendixClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 4Click here for additional data file.


Long noncoding RNAs (lncRNAs) play very important roles in cell differentiation. Our recent study has demonstrated that a novel lncRNA named lnc-OAD modulated 3T3-L1 adipocyte differentiation. In the present study, we examined the roles of lnc-OAD in bone morphogenetic protein 2- (BMP-2-) induced osteoblast differentiation. Lnc-OAD expression was increased during BMP-2-induced osteoblast differentiation in C3H10T1/2 mesenchymal stem cells and MC3T3-E1 preosteoblast cells. Knockdown of lnc-OAD expression by specific siRNA remarkably decreased early osteoblast differentiation. In addition, stable knockdown of lnc-OAD by lentivirus vector also significantly inhibited late osteoblast differentiation and matrix mineralization in vitro. Conversely, stably overexpressed lnc-OAD with lentiviral vector accelerated osteoblast differentiation. Mechanistically, knockdown of lnc-OAD reduced significantly the phosphorylation of AKT and the expression of Osterix induced by BMP-2, while overexpression of lnc-OAD enhanced the phosphorylation of AKT and the expression of Osterix. Taken together, our study suggests that lnc-OAD plays an important role in modulating BMP-2-induced osteoblast differentiation via, at least in part, regulating the AKT-Osterix signaling axis. Osteoblast commitment and differentiation from mesenchymal stem cells (MSCs) is a complex multistep process controlled by intricate signaling cascades and cellular molecules , 2. In tβ-catenin/TCF1/Runx2 signaling [β-catenin signaling pathway [Long noncoding RNAs (lncRNAs), a novel class of RNA with no significant protein-coding potential, have been recently identified as key mediators of many biological processes though diverse mechanisms such as transcription regulation and epigenetic chromatin modification . In receignaling . LncRNA ignaling . HOTAIR  pathway , 15. Lnc pathway . AlthougRecently, we have demonstrated that the lnc-OAD, one newly identified lncRNA transcribed from mouse 1700018A04Rik gene, modulated adipogenesis [β-glycerophosphate were purchased from Sigma-Aldrich . Antibody sources were as follows: p-ERK1/2 , ERK1/2 (#4695), p38 (#9212), p-p38 , and Runx2 (#12556) were obtained from Cell Signaling Technology ; AKT (ab32505), p-AKT , and ALPL (ab108337) were purchased from Abcam ; Osteocalcin (sc-390877), and Osterix (sc-393325) were provided by Santa Cruz Biotechnologies ; β-actin (AF5001), HRP-labeled goat anti-mouse (A0216), and anti-rabbit (A0208) IgG were obtained from Beyotime Biotechnology .Mouse C3H10T1/2 mesenchymal stem cells and mouse MC3T3-E1 preosteoblast cells were obtained from Chinese Academy of Science Cell Bank . Recombinant human BMP-2  was provided by Proteintech Group, Inc. . TRIzol reagent and Lipofectamine RNAiMAX were obtained from Thermo Fisher Scientific . p-nitrophenyl phosphate (p-NPP), ascorbic acid, and μg/mL ascorbic acid, and 10 mM β-glycerophosphate.C3H10T1/2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum . MC3T3-E1 cells were cultured in a-MEM medium without ascorbic acid  containing 10% FBS. Both cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. To induce early osteoblast differentiation, cells grown to 90% confluence were replaced with differentiation medium containing DMEM (for C3H10T1/2) or a-MEM (for MC3T3-E1), 2.5% FBS, and 100 or 200 ng/mL rhBMP-2, and the differentiation medium was changed every 2-3 days. To induce terminal osteoblast differentiation and mineralization, cells were cultured in osteoblast mineralization medium containing 2.5% FBS, 100 ng/mL rhBMP-2, 50 μL cell extracts were added and incubated with 100 μL of p-nitrophenyl phosphate (p-NPP) for 30 min and stopped by adding 100 μL of 1 N NaOH, and then, absorbance was read at 405 nm. The protein content of cell extracts was measured by the BCA method. The OD405 values was standardized by protein content, and then, the relative ALP activity was presented as fold change to the control group. For ALP staining, cells were washed with PBS twice, fixed with 70% ethanol for 20 min. After washing three times with PBS, cells were incubated with a mixture of 0.1 mg/mL napthol AS-MX phosphate and 0.6 mg/mL Fast Blue BB salt for 30 min at room temperature.Levels of early osteoblast differentiation were determined by alkaline phosphatase (ALP) activity assay and ALP staining analysis. For ALP activity assay, cells were cultured in 24-well plates and induced for early osteoblast differentiation. Cell extracts were prepared with CytoBuster™ protein extraction reagent and centrifuged at 15,000g for 5 minutes. Then, 10 MC3T3-E1 cells were seeded into 24-well plates, and when they reached about 90% confluence, the osteoblast mineralization medium was added to induce mineralization. On day 10, cells were fixed with 70% ethanol for 20 min and stained with 0.5% Alizarin red S solution  for 10 min at room temperature. For quantification of staining, the AR-S stain was extracted using 10% cetylpyridinium chloride for 15 min and quantified by measuring its absorbance at 540 nm. Relative AR-S stain was calculated as fold change of the control group.Small interfering RNAs (siRNAs) specific for mouse lnc-OAD and negative control siRNAs (si-NC) were synthesized by Shanghai GenePharma Co. Ltd. . In order to prolong the time of siRNA stabilization, siRNAs were chemically modified with 2′-O-methylation (2′-OMe). The nucleotide sequences for mouse lnc-OAD siRNA were si-lnc-OAD #1: sense, 5′-CCAGGUGUGUCCUGUGAUUTT-3′; antisense, 5′-AAUCACAGGACACACCUGGTT-3′; si-lnc-OAD #2: sense, 5′-CCUACUGAAGCAUGGCUAUTT-3′; antisense, 5′-AUAGCCAUGCUUCAGUAGGTT-3′; si-NC: sense, 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. After cells were grown to 40–60% confluence, siRNA duplexes with either lnc-OAD siRNA or si-NC were transfected into the cells using Lipofectamine RNAiMAX according to the manufacturer's protocol.μg/mL for MC3T3-E1 and 3 μg/mL for C3H10T1/2). The knockdown or overexpression efficiency was evaluated by qRT-PCR analysis.Lentivirus particles for shRNA-mediated knockdown of lnc-OAD (sh-Lnc-OAD) and negative control sh-NC were provided by Shanghai Genechem Co. Ltd. . The shRNA sequences were designed, and stable lnc-OAD knockdown cells were generated as our previously described . For genβ-Actin. All real-time PCR primer sequences were as follows: lnc-OAD ; Runx2 ; Osterix ; β-actin .The cellular total RNA was extracted with Trizol Reagent according to the manufacturer's instructions. Then, the total RNA was synthesized cDNA by using PrimeScript RT reagent Kit with gDNA Eraser , and real-time PCR was conducted using one step SYBR® PrimeScript™ RT-PCR Kit II . The gene expression levels were normalized to endogenous control https://imagej.nih.gov/ij/).Western blot analysis was carried out as our previously described . Brieflyt-test or one-way analysis of variance followed by Dunnett's comparison test  or Dunnett's T3 comparison test , and P < 0.05 was considered statistically significant.All data were expressed as mean ± SEM for each group with three biological replicates. Differences between groups were analyzed using Student's To better understand the function of lnc-OAD, we carried out genome location analysis and detected its expression in mouse cell lines from different tissue sources. In the UCSC Genome Browser on NCBI37/mm9 Assembly, lnc-OAD, which transcribed from 1700018A04Rik gene, was mainly labeled as ENSMUST00000150418/NR_029439.1 or ENSMUST00000126138, and we named them as lnc-OAD.1 and lnc-OAD.2, respectively. Lnc-OAD was flanked by the Forkhead box Q1 (FOXQ1) and Forkhead box F2 (FOXF2) loci . qRT-PCRWe previously performed lncRNA microarray analysis and identified a series of differentially expressed lncRNAs during BMP-2-induced osteoblast differentiation in mouse C3H10T1/2 mesenchymal stem cells. Among them, lnc-OAD was one of the upregulated expression lncRNAs. To determine the role of lnc-OAD in BMP-2-induced osteoblast differentiation, we detected its expression level using quantitative RT-PCR. As shown in Figures To clarify the potential biological role of lnc-OAD in osteoblast differentiation, we knocked down lnc-OAD using two different sets of siRNAs targeting lnc-OAD (si#1 and si#2). As shown in Figures To further determine the effect of lnc-OAD in osteoblast differentiation, we generated stably overexpressed lnc-OAD C3H10T1/2 and MC3T3-E1 cell lines using lentiviral vectors. qRT-PCR confirmed dramatically elevated expression of lnc-OAD mRNA in C3H10T1/2  and MC3TNext, we examined whether lnc-OAD was required for late osteoblast differentiation. According to the Alizarin Red S staining, the lnc-OAD gene stably knockdown cells showed a drastic decrease in mineralized nodule formation Figures , while tOsteoblast differentiation was coregulated by a series of important transcription factors such as Runt-related transcription factor 2 (Runx2) and zinc finger transcription factor Osterix. To further investigate the mechanism by which lnc-OAD might regulate osteoblast differentiation, we detected the expression of above important transcription factors in osteogenic differentiation. As shown in μM AKT inhibitor MK-2206 clearly decreased the ALP staining and the protein expression of Osterix induced by BMP-2, which further confirmed that AKT signaling pathway activation was necessary for BMP-2-induced osteoblast differentiation. Next, we examined whether the AKT phosphorylation induced by BMP-2 was modulated by lnc-OAD, as shown in Figures Given that previous studies have demonstrated that AKT signaling pathway was required for BMP-2-induced osteoblast differentiation and AKT pathway could regulate the expression of Osterix , 22, in Thus, based on all current data, we proposed a model to explain the possible mechanism of lnc-OAD regulating BMP-2-induced osteoblast differentiation as follows : BMP-2 wBMP-2, a member of the BMPs subfamily and secreted by osteoblasts, has a promoting effect on osteoblast differentiation . In the Many lines of evidence suggested that osteoblast differentiation was precisely controlled by a series of transcription factors such as Runx2, Osterix, Dlx2, Dlx5, and Atf4 , 25. AmoPrevious studies have demonstrated that BMP-2 could regulate multiple signaling pathways inducing osteoblast differentiation such as AKT, MAPK, and Smad , 9. PI3KIn summary, the present study showed that the newly identified long noncoding RNA lnc-OAD was required for BMP-2-induced osteoblast differentiation. Furthermore, the AKT-Osterix signal axis was identified as an important mechanism of lnc-OAD regulating BMP-2-induced osteoblast differentiation. These results might be helpful to further understand the regulation of osteogenic differentiation network by long noncoding RNA. However, further in vivo study was still required to fully elucidate the role of lnc-OAD in osteoblast differentiation and bone development.


GCK result in a persistent, mildly raised glucose from birth, but it is usually diagnosed in adulthood as maturity-onset diabetes of the young (MODY), where hyperglycemia is often an incidental finding. The hyperglycemia of GCK-MODY is benign and does not require treatment, but is important to be aware of, particularly in females where it has implications for managing pregnancy. We present three cases of neonatal hyperglycemia resulting from a heterozygous mutation in GCK, illustrating its clinical presentation and evolution in early life. In summary, as with adults, neonatal hyperglycemia is an incidental finding, does not require treatment and has no adverse consequences for health. Neonates and their parents should be referred for genetic testing to confirm the diagnosis, avoid a label of diabetes and enable pregnancy counselling for females found to be affected.Heterozygous mutations in  GCK, are common  and result in partial glucokinase deficiency2. This causes mild hyperglycemia (tightly regulated between 5.5-8 mmol/L3), often diagnosed incidentally in adulthood as maturity-onset diabetes of the young (MODY)4. The degree of hyperglycemia is uniform irrespective of mutation type5, and treatment of hyperglycemia is not required6, as it does not result in the micro- or macrovascular complications seen with type 1 and type 2 diabetes7. However, it does have implications for women of reproductive age, since fetuses who have not inherited the mutation from their mother secrete higher levels of insulin in response to maternal hyperglycemia, so are at risk of macrosomia and its associated pregnancy complications9. GCK-MODY is frequently diagnosed in pregnancy, either because of routine screening for gestational diabetes or a history of fetal macrosomia10.Heterozygous inactivating mutations in the gene encoding the glucose sensing enzyme glucokinase, 11. It can be permanent (PNDM), requiring lifelong treatment, or transient (TNDM), where diabetes remits but typically returns in childhood or early adulthood12. The clinical presentation of NDM is usually severe, with marked hyperglycemia (frequently >30 mmol/L) and ketoacidosis13, and treatment is required to maintain euglycemia.Neonatal diabetes (NDM) is diagnosed in the first six months of life and affects approximately 1 in 100,000 live births21. Obtaining a genetic diagnosis is important, since prognosis and approach to monitoring and treatment are dependent on the underlying genetic cause25.NDM may be isolated or diagnosed in association with other conditions or congenital anomalies. NDM has a known genetic etiology in almost 90% of cases, with a mutation in one of 28 known disease-causing genes affecting glucose metabolism, insulin secretion or pancreatic developmentGCK result in complete glucokinase deficiency and are an infrequent cause of PNDM, accounting for ~3% of cases with a confirmed genetic diagnosis14, but an important differential diagnosis of hyperglycemia in infants with very low birth weight 26. This contrasts with heterozygous inactivating mutations in GCK which cause GCK-MODY, where the degree of fetal insulin secretion and its effect on birth weight is determined by the parent-of-origin of the mutation8. Birth weight is reduced by approximately 500 g when the mother is unaffected and the fetus has inherited the mutation from the father, whereas birth weight is typically in the normal range when inherited from a mother with GCK-MODY9.Bi-allelic inactivating mutations in 27 and genetic screening for suspected GCK-MODY28 have shown that hyperglycemia is present from birth in affected individuals. However, it is rare for it to be recognised in neonates and considered as a cause of neonatal hyperglycemia in the absence of a known family history. Here, we present three cases of neonatal hyperglycemia who did not have a family history of GCK-MODY and were referred for genetic testing for NDM. We describe how GCK-MODY can present in newborns and emphasise the importance of considering it in cases of asymptomatic, mild neonatal hyperglycemia not requiring treatment.Family studies in GCK-MODY pedigreesGCK (NM_000162.3). The GCK mutations were identified either by PCR amplification followed by Sanger sequencing (primers available on request) or by analysis of all the known NDM genes using an in-house targeted next generation sequencing (tNGS) assay as previously described29. Variant classification was performed according to the ACMG international guidelines for variant interpretation30. There was no known history of a genetic cause for diabetes in their families. Their parents were genotyped once a mutation in GCK was identified in the child.Out of 2,203 individuals identified as having hyperglycemia in the first six months of life and referred to Exeter Genomics Laboratory for genetic testing of NDM, we identified three individuals with heterozygous inactivating mutations in st standards31 and most recent available weight and height SDS were calculated using the WHO Child Growth Standards or Reference33. Informed consent was received from their parents and ethics approval for research was provided by the North Wales Research Ethics Committee.Clinical data  was collABCC8, KCNJ11 and INS genes and was screened for a methylation defect at the 6q24 locus. The results of these tests were negative, so targeted next generation sequencing (tNGS) of a panel of 28 known NDM genes29 was performed and a heterozygous missense variant in GCK (c.1225G>T (p.D409Y)) was identified . Pregnancy and delivery were uneventful, but he was admitted to hospital with signs suggestive of bowel obstruction at four months of age. He was diagnosed with Hirschsprung’s disease and underwent surgery. His blood glucose prior to surgery was 8 mmol/L and varied between 11 and 16 mmol/L during surgery. He did not require insulin treatment. At six months of age his case was reviewed by a Pediatric Endocrinologist who established that his paternal grandmother had a history of type 2 diabetes but there was no other significant family history. His HbA1c was 47.5 mmol/mol, C-peptide was 190 pmol/L and an autoantibody screen was negative. He was referred for genetic testing for NDM. He first underwent Sanger sequencing of the entified . This vaGCK was performed, which revealed a heterozygous GCK nonsense mutation (c.1183G>T (p.E395*)), which was also present in her mother . Pregnancy and delivery were uneventful. The girl’s blood glucose was measured during a viral illness at four months of age and found to be raised at 6 mmol/L. No treatment was given and her blood glucose was tested intermittently in childhood  before a specialist review at six years and seven months of age. She was of a normal height and weight , but was noted to have some signs of early pubarche (Tanner Stage 2). At the time her HbA1c was 49.7 mmol/mol, serum C-peptide was 255 pmol/L and an autoantibody screen, including GAD, IA2 and ICA, was negative. Her mother had been diagnosed with gestational diabetes (GDM) in a previous pregnancy at 19 years old and was subsequently diagnosed with type 2 diabetes aged 23 years. The mother was treated with a low carbohydrate diet (including in her pregnancy with the proband) until the age of 26 years, when she was started on metformin and saxagliptin. In addition to her mother’s history of diabetes, the girl’s maternal grandfather had been diagnosed with type 2 diabetes aged 46 years. In view of the history, Sanger sequencing of r mother . A samplGCK (c.121A>G (p.M41V)) and 6q24 MS-MLPA was negative. The GCK variant was classified as likely pathogenic. The proband had inherited this mutation from her mother, confirming a diagnosis of GCK-MODY in both. At her most recent assessment at the age of 15 months, her hyperglycemia remained untreated and she was well . Her mother was treated for GDM with insulin in pregnancy. The infant’s blood glucose was measured as a routine screen in the first 24 hours of life and was found to be high; pre-feed blood glucose levels ranged between 5.5 and 10 mmol/L and the highest measured was 12.6 mmol/L. There was no ketonuria and her serum C-peptide was 96 pmol/L pre-feed and 536 pmol/L post-feed. She was noted to have macroglossia, but was otherwise well and her hyperglycemia did not require treatment. She was referred for genetic testing for NDM and underwent screening of the known genes by tNGS and methylation-specific MS-MLPA of the 6q24 locus. A heterozygous novel missense variant was identified in was well .28. Previous series where genetic screening was performed in individuals with incidental hyperglycemia have identified children who presented as young as one year of age36. However, referral for genetic testing with suspected NDM is rare, and these cases confirm the importance of considering GCK-MODY as a cause of neonatal hyperglycaemia.In this series of GCK-MODY diagnosed in cases referred for genetic testing for suspected NDM, we have shown that neonatal hyperglycemia is an incidental finding, does not require treatment and follows a benign course in childhood. Retrospective studies in individuals with a known family history or clinically suspected GCK-MODY have previously confirmed that hyperglycemia can occur from birth37. The birth weight for the infant in Case 2 was normal, despite her mother’s history of diabetes diagnosed after screening in a previous pregnancy. Therefore, for a hyperglycemic infant of normal or low birth weight born to a mother with GDM, particularly where maternal hyperglycemia has failed to respond to insulin treatment in pregnancy38 and persists postpartum, GCK-MODY is an important consideration. Neonatal hypoglycemia and macrosomia may be evident in the infant who has not inherited the same GCK mutation as their mother9, so a high birth weight and history of neonatal hypoglycemia in siblings may also provide a useful clue to the diagnosis. Where the mutation is inherited from the father, birth weight is reduced by approximately 500 g on average8, but this was not seen in Case 1, and his father (who he had inherited the GCK mutation from) was not known to have diabetes. It is very common for adult males to be unaware of their diagnosis, since the hyperglycemia of GCK-MODY does not cause symptoms or complications, so it may be less clear-cut when the mutation is inherited from the father. However, where there is a strong family history of “type 2 diabetes” without other clinical features or complications and diagnosed at a young age (as was seen in Case 2), this should also raise suspicion for GCK-MODY.In pregnancies affected by GCK-MODY, as fetal growth depends on the parent-of-origin of the mutation, birth weight combined with parental history can provide useful clues to the underlying diagnosis in an otherwise well infant with hyperglycemia. However, birth weight is not always a reliable indicator, as fetal insulin- and non-insulin-mediated growth will have different effects between different individuals. In Case 3, the infant had a birth weight lower than might be expected for a mother who had GDM requiring insulin. It is possible that maternal insulin treatment contributed to the lower birth weight in this case40, but this is a variable and imprecise sign which has no known relation to GCK-MODY. Overall, the presence of other features emphasises that these children are at risk of other pediatric conditions and these should not be attributed to GCK-MODY.We found that GCK-MODY was a rare diagnosis for infants with neonatal hyperglycemia and without a known family history referred for genetic testing (~0.1%). These three cases also had other clinical conditions or signs which might have prompted the clinicians to consider an underlying genetic aetiology. Hirschsprung’s disease and possibly precocious puberty were present in Case 1 and 2, respectively, but these are not related to hyperglycemia and have not been reported to be typical clinical features for individuals with NDM. The infant in Case 3 had macroglossia, which has been identified as a feature of some cases of 6q24 TNDM3. Recent measures of height and weight in the cases reported here were also within the normal range, consistent with that seen in adults with GCK-MODY41.Hyperglycemia is stable in GCK-MODY, as shown by Case 2 where her glucose was maintained within a tight range when tested at different points in early childhood, and no progression in HbA1c levels in the absence of treatment in Cases 1 and 2. This is consistent with the adult literature, where HbA1c is relatively stable, showing a slight rise in later life which is also seen in individuals without diabetes1, it can be identified at birth. As blood glucose monitoring becomes more frequent, and if neonatal bloodspot glucose becomes routine42, it may be diagnosed more often in the future. The distinguishing features are a mild hyperglycemia that does not require treatment or cause illness, unlike that seen in other cases of NDM, including complete glucokinase deficiency caused by bi-allelic inactivating mutations. Furthermore, the combination of hyperglycemia with a birth weight within the normal range, particularly where their mother also had GDM, and a strong family history of early-onset, non-progressive “type 2 diabetes” should raise suspicion for GCK-MODY. Genetic diagnosis is useful, since unnecessary treatment and monitoring are avoided. Additionally, for female infants and mothers of probands found to have the mutation, it allows preparation for future pregnancies, where antenatal management differs to typical cases of diabetes in pregnancy9.In summary, although GCK-MODY is more commonly identified in adults as an incidental finding of hyperglycemia


AR) gene expression on the clinical features and progression of PTC. The levels of AR mRNA and protein in frozen, formalin-fixed, paraffin-embedded tissue samples from PTC and adjacent normal thyroid tissue were assessed by quantitative real-time polymerase chain reaction and immunohistochemical staining, respectively, and the relationships between AR expression and clinical features were analyzed. The thyroid cancer cell lines, BCPAP and TPC-1, were used to evaluate the effects of AR on the regulation of cell migration, and key epithelial–mesenchymal transition (EMT) markers. AR mRNA expression was significantly higher in normal thyroid tissue from men than women. The sex difference in AR mRNA expression diminished during PTC tumorigenesis, as AR mRNA expression levels were lower in PTC than normal thyroid tissues from both men and women. AR mRNA expression was significantly decreased in PTC patients with higher risk and in those with extrathyroidal extension. Overexpression of AR in BCPAP cells decreased cell migration and repressed the EMT process by down-regulating mRNA expression of N-cadherin, Snail1, Snail2, Vimentin, and TWIST1 and up-regulating E-cadherin gene expression. These results suggest that suppression of the androgen–AR axis may lead to aggressive tumor behavior in patients with PTC.Male gender is a risk factor for mortality in patients with papillary thyroid carcinoma (PTC). This study investigated the impact of androgen receptor ( Thyroid cancer is the most common endocrine malignancy, with increasing prevalence over time ,2,3. PapAR) has been first evidenced for the expression of both normal thyroid tissue and malignant thyroid tumors [The sex hormone receptors are a group of steroid hormone receptors that interact with sex hormones, including androgens, estrogen, and progesterone . Sex sted tumors ,15,16, lAR is associated with the development and progression of PTC and AR has tumor suppressive function in the metastasis of PTC.These intriguing observations suggest that sex steroids and/or their receptors may play central roles in regulating the development of thyroid cancers. Better understanding of the molecular mechanisms of sex hormone receptors involved in PTC initiation and progression may aid in the identification of novel therapeutic targets and molecular markers. The current study presents the evidence to show that low expression of AR, estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), and progesterone receptor (PR), were analyzed in these samples by qRT-PCR. While there is marginal or no significant difference in the ERα, ERβ, and PR mRNA expression between PTC and the surrounding normal parenchyma, the level of AR mRNA expression was significantly lower in PTC than in the surrounding normal parenchyma  had extrathyroidal extensions, 59 (43.0%) had lymph node metastases, and 30 (21.9%) were classified as a high risk group. Understanding the molecular mechanisms that regulate the biology of PTC in men and women may help identify novel targets for therapeutic intervention. Although  cancers ,17, its d 29 men , and theAR mRNA expression was higher in normal thyroid glands of men than women  showed that there were 3 independent risk factors for extrathyroidal extension, including age (>55 y/o) ; lymph node metastasis ; and expression of AR , suggesting that AR expression is an independent factor for extrathyroidal extension in PTC  or a control vector (pSG5) into BCPAP and TPC-1 cells. Transfection of pSG5-AR, but not pSG5, markedly enhanced the expression of AR mRNA and protein .Because pression , providiAR has crucial roles in many types of cancer, as shown by sex difference [AR was less understood in PTC. The present study found that AR mRNA expression in paired normal thyroid tissue was higher in men than women but it was significantly lower in PTC specimens in both men and women. We further found that reduced AR mRNA expression was associated with high cancer risk and extrathyroidal extension in patients in PTC  which lead to regulation of EMT process [E-cadherin expression was shown to correlate with hypermethylation of E-cadherin 5’ CpG island and a similar study reported that activation of EMT by androgen deprivation therapy in metastatic prostate cancer [AR  and short duration of follow up period (average less than 5 yrs), therefore, the role of AR in sex differences for PTC’s survival and recurrence is difficult to determine. The evaluation of the relationship between long-term clinical outcome and androgen–AR axis is on the way to further investigate.There have been few studies reported evaluating gender discrepancies between This prospective study analyzed 137 patients with PTC who were treated at the Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan, between August 2013 and October 2018. Samples from these patients were selected based on the clinical features of the patients and access to prospectively collected tissue collections by one of the authors (Dr. Shun-Yu Chi). The 137 patients included 108 women and 29 men, ranging in age from 18–78 years. Patients aged <18 years or >80 years were excluded, as were patients with follicular carcinoma, medullary cancer and anaplastic cancer. Details of the clinical features of the PTC patients in this study are presented in Tissue samples were snap-frozen in liquid nitrogen at the time of total thyroidectomy, and subsequently stored in liquid nitrogen. The American Joint Commission on Cancer-International Union Against Cancer criteria were utilized for risk classification based on each patient’s clinicopathologic risk factors . PatientTM  and thyroid cancer cell lines using Quick-RNATM Mini Prep kit . The samples were processed by the manufacturer’s instructions. The primers for real-time PCR were: h18S, forward (5′-GTAACCCGTTGAACCCCATT-3′) and reverse (5′-CCATCCAATCGGTAGTAGTG-3′); AR, forward (5′-CCTGGCTTCCGCAACTTACAC-3′) and reverse (5′-GGACTTGTGCATGCGGTACTCA-3′); ERα, forward (5′-AGTTGGCCGACAAGGAGTTG-3′) and reverse (5′-CGCACTTGGTCGAACAGG-3′); ERβ, forward (5′-TACTGACCAACCTGGCAGACAG-3′) and reverse (5′-TGGACCTGATCATGGAGGGT-3′); and PR, forward (5′-AGCCGGTCCGGGTGCAAG-3′) and reverse (5′-CCACCCAGAGCCCGAGGG-3′). The protocol for real-time PCR has been described [Total RNA was extracted from surgical specimens which stored in DNA/RNA Shiledescribed .Surgical specimens of PTC were routinely fixed in 10% formalin, embedded in paraffin, and sectioned at a thickness of 2.5 μm. The tissue sections were deparaffinized in xylene, dehydrated in a graded ethanol series, and heated in boiling 0.01 M citrate buffer (pH 6.0) for 15 min in a microwave oven for antigen retrieval. After cooling to room temperature, endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 30 min. The sections were incubated with primary rabbit monoclonal anti-AR antibody  diluted in Antibody Diluent Reagent Solution  at 4 °C overnight, rinsed in 0.1% TBS-T, stained with Dako REAL™ EnVision™ Detection System, Peroxidase/DAB, Rabbit/Mouse as a chromogen, and counterstained with hematoxylin. The sections were photographed on a Nikon Eclipse 50i microscope  connected to a Nikon DS-Fi1 color digital camera . Staining intensity was measured by a pathologist (Dr. Shun-Chen Huang) at Chang Gung Memorial Hospital using light microscopy. The section containing both normal thyroid and tumor part was chosen for reading. Ten low power (10X objective lens) fields of each part were selected to evaluate its intensity and percentage. The staining intensity was graded as 0, negative; 1+, weakly positive; 2+, moderately positive; 3+, strongly positive. The percentage of positively stained cells were classified as grade 1, <10%; grade 2, ≥10% and ≤25%; grade 3, >25% and ≤50%; grade 4, >50% and ≤75%; and grade 5, >75% . The fin2. The human thyroid cancer cell lines BCPAP  and TPC-1 and the normal thyroid cell line Nthy-ori-3-1  were routinely cultured in RPMI1640 containing 10% fetal bovine serum (FBS), 1x antibiotic-Antimycotic (Gibico cat# 15240112), and 2 mM L-glutamine  at 37 °C in a humidified chamber containing 5% COAR over-expression plasmid (pSG5-AR) and its control vector (pSG5) were used to transfect the BCPAP and TPC-1 cells as previously described [5 cells per well in 6-well plates in antibiotic-free medium for 24 h prior to transfection. The cells were transfected with pSG5-AR or pSG5 using DharmaFECTTM transfection reagent . The cells were collected 24 h later, and the protein and mRNA expression of AR was quantified.The escribed ,43. BrieCells transfected with plasmids for 24 h were lysed in RIPA buffer containing protease, phosphate inhibitor cocktail  and 1 mM phenylmethylsulphonyl fluoride (PMSF). Samples of 100 μg total protein were separated on 8% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes , which were incubated with primary mouse monoclonal anti-AR antibody  and anti-β-actin antibody  at 4 °C overnight, followed by incubation with goat-anti-mouse HRP-conjugated secondary antibody at room temperature for 1 h . Bands were visualized using an Enhanced ChemoLuminescence kit  according to the manufacturer’s instructions. Relative levels of AR expression were determined by normalization to the expression of β-actin. Densitometric analysis of the protein bands was performed using Bio-Rad Quantity One 1-D Analysis software.AR (pSG5-AR) or a control vector (pSG5) for 24 h were seeded at 5 × 104 cells/well in the upper chambers, with the lower chambers filled with culture medium containing 10% FBS, followed by incubation at 37 °C for 24 h in a humidified incubator containing 5% CO2. Cells that did not pass through the filter were wiped off. Cells that had migrated to the reverse side of the filter were fixed with methanol for 10 min, washed with PBS, stained with Giemsa solution (Millipore) for 30 min. Cells were photographed and counted under a light microscope with a 10x objective lens with the selective use of a 10× objective lens for confirmation. The number of migrated cells was counted by analyzing 3 random fields of the membranes per condition.Cell migration assay were performed in transwells in 24 well plates. Transwells  with 8uM Polyethylene Terephthalate membrane used for cell migration. Thyroid cancer cells transfected with a vector overexpressing AR expression in paired cancer and normal tissue from patients with PTC, were compared using Wilcoxon test. Correlations between AR expression and tumor characteristics were assessed by Mann–Whiney U-test analysis. Variables were enrolled in multiple logistic regressions with forward stepwise procedure to identify independent risk factors for extrathyroidal extension. The Spearman correlation coefficient was used to determine the correlation of AR and E-cadherin expression level. All statistical analyses were performed using SPSS software . All p-values were two sided, with p-values < 0.05 considered statistically significant.Continuous variables, including AR may play a pivotal role in thyroid cancer tumorigenesis, as AR mRNA expression levels were lower in PTC than normal thyroid tissues. In addition, the pattern of low AR expression was associated with high cancer risk and extrathyroidal extension during the tumor progression in papillary thyroid carcinoma. As growing number of clinical studies continue and further study is needed to further elucidate the relationship between AR and PTC, our data in this study suggest that the potential importance of activation of androgen–AR axis and its downstream signaling pathways may serve as a novel therapeutic target in advanced and metastatic thyroid carcinomas.To sum up, our data indicate that


Rationale: Lu-177-PSMA-617 radioligand therapy (RLT) is currently under approval for treatment of metastatic castration resistant prostate cancer (mCRPC) patients with late stage disease. However, previous studies demonstrated both heterogeneity of prostate specific membrane antigen (PSMA) expression, as well as response to PSMA treatment among mCRPC patients. Thus, there is an unmet need for identifying predictive parametres prior or under PSMA-RLT treatment. We therefore aimed to correlate several clinical and molecular parameters with response to PSMA treatment in a cohort of mCRPC patients undergoing PSMA RLT followed by a detailed analysis of promising candidates.Methods: Nineteen patients, median age 68.8 years (range: 56.9 - 83.3) with mCRPC were included in this study. We performed baseline analysis of clinical parameters based on PSMA PET/CT, (metabolic tumor volume (MTV), total tumor volume (TTV)), serum PSA, ALP, LDH and gene expression analysis of circulating tumor cells (expression of AR full length (AR-FL), AR splice variant 7 (AR-V7), PSA and PSMA) as well as common markers for neuroendocrine differentiation (NED).Results: Patients presented with bone, lymph node, and visceral metastases . All patients were pretreated with docetaxel, either abiraterone or enzalutamide, or both. Biochemical response in terms of PSA decline ≥50 or ≥30% was observed in 42% and 63%, respectively. There were significant correlations between PSA and PSMA mRNA expression, as well as tumor volumes (both MTV and TTV), AR-FL and AR-V7 mRNA expression. However, there was no correlation with response to PSMA treatment. Furthermore, none of these parameters was significantly correlated with baseline serum PSA values. Common NED markers were shown to be specifically high expressed and revealed impact on OS independent from AR-V7 gene expression.Conclusion: We demonstrate that AR-FL and its splice variant AR-V7 might serve as prognostic biomarkers displaying high tumor burden in mCRPC patient prior to PSMA-RLT. Contrary, PSMA, which has been discussed as a biomarker for PSMA targeted treatment, does not display strong prognostic ability - at least on the mRNA level. Surprisingly, none of these parameters correlates to response to PSMA treatment. In contrast, commom NED markers such as SYP and ENO2 as well as FOXA1 expression level seem to predict OS, but not PFS, more reliably. We admit that a limitation of our study is the focus on mRNA expression of potential biomarkers only. Further investigations analyzing the potential role of protein expression of these markers are therefore warranted. However, in order to understand the efficacy and potential of 177Lu-PSMA-RLT, further prospective investigations have to be initiated leading to more insights of PSMA-RLT mechanisms. Additionally, the usage of 177Lu-PSMA-RLT is still lacking reliable prognostic biomarkers.Prostate cancer (PC) is the most frequent cancer in men worldwide 177Lu-PSMA-RLT. Inhibition of AR by novel hormonal treatment has been described to induce expression of PSMA There is evidence that PSMA expression is induced by AR signaling ® system  prior to therapy is recommended by the current Prostate Cancer Working Group (PCWG3) guidelines. One approach for establishment of novel biomarkers are analyses of circulating tumor cells (CTCs) obtained by liquid biopsy approaches pos patients pos patients from AR-V7neg individuals, however, is poorly reported. An intriguing phenomenon associated with loss of AR signaling is the development of a neuroendocrine signature after tumor progression within the castration-resistant stage. This NED might be connected with loss of AR-V7 expression 177Lu-PSMA radioligand therapy.Both AR as well as PSMA are expressed in CTCs of prostate cancer, though expression tends to be heterogeneous between patients and within one single patient 177Lu-PSMA-RLT were prospectively enrolled in this study. Inclusion criteria were at least one line of chemotherapy and treatment with either abiraterone or enzalutamide, or both. Blood samples were collected prior to the first cycle of 177Lu-PSMA application.Nineteen patients with mCRPC, eligible for 177Lu-PSMA-617 RLT was individually made in each case by an interdisciplinary tumor board. 177Lu-PSMA-617 RLT was offered in case of exhausted recommended therapeutic options and disease progression according to guidelines for mCRPC management 177Lu-PSMA-617 therapy. All patients gave their written informed consent prior to treatment and blood sampling. This study was approved by the local ethics committee . Overall survival (OS) was determined as the lifespan in months from the start of PSMA-RLT to death or last contact. PSA decline was calculated as the change of PSA serum levels from baseline compared to the respective time-point.The decision on Enrichment of circulating tumor cells as well as analysis of expression of AR, AR-V7 and PSA has been performed as described elsewhere A known disadvantage during the process of CTC enrichment from blood samples is the non-specific capture of leukocytes AR-FL, KLK3-PSA, FOXA1, ENO2, SYP and NCAM expression were analyzed using TaqMan assays  AR-V7 status was analyzed by using a previously described custom-made TaqMan PCR assay (2). All qPCR runs were performed along with TaqMan PCR assays for housekeeping gene RPL37A (Hs01102345_m1) .Expression levels of biomarker genes for NED  were analyzed in healthy blood donor samples. In case of expression in healthy individuals, the delta Ct value between marker gene and RPL37A was assesed. The median delta Ct of all healthy controls along with a twofold standard deviation was recalculated to determine a threshold for CTC specific expression levels of NED genes.Relative copy number levels of AR, AR-V7, PSA, PSMA, FOXA1, ENO2, SYP and NCAM were calculated using the following equation: 2^. Expression scores were determined using the whole range of relative copy numbers, which were subdivided by logarithmic separation . The PSA expression level was used to determine the CTC score, demonstrating a relative level of CTC load. Relative gene expression was determined with PSA gene expression as baseline after normalization of PSA mRNA expression to the housekeeper gene RPL37A.18F-PSMA-1007 was produced in a GE TracerLab MX synthesizer according to the one-step procedure described by Cardinale et al. and standard operation procedures (SOP), which have been described previously 177Lutetium  in-house according to radiosynthesis procedures described previously 177Lu-PSMA-617 uptake and retention in tumor lesions at least one whole-body planar scintigraphy scan and a SPECT/CT scan of the thorax and abdomen was performed 48h after injection. 177Lu-PSMA-617 RLT was repeated every 6-8 weeks until progression or death.The commercially available GMP precursor PSMA-617  was labelled with Statistical analysis was performed using GraphPad Prism7. The cohort size (n=19) and its subgroups were too small for normality testing. Therefore, all group comparisons were performed by non-parametric t-test (Mann-Whitney). Pearson correlation analysis was performed using the RStudio corrplot package. Kaplan-Meier survival analysis and plotting were performed using SPSS . P-values <0.05 were considered statistically noticeable.A total of 19 patients with a median age of 68.8 years (range 56.9-83.3 years) were selected. All patients had confirmed mCRPC and had underwent previous novel hormonal treatment using abiraterone (94%), enzalutamide (68%) or both (63%). Chemotherapy with docetaxel and/or cabazitaxel was obligatory. Twelve patients (63%) presented with a health status of ECOG 0-1, five patients (26%) with ECOG 2, and one patient (5%) with ECOG 3. The median overall survival of this cohort was 9.9 months  was 4.5 months (95% CI: 4.4-4.5) A+B. For All patients displayed the presence of CTCs by KLK3-PSA signals, which demonstrated both a late stage of disease, as well as a high tumor load or metastatic burden.177-PSMA-therapy. Fifteen patients demonstrated a decline of PSA serum levels (84%), 9 patients (63%) presented with a best PSA decline of >30% and 8 patients (42%) with a PSA decline of ≥50%  at the onset of LuRelative copy number data for CTCs (by PSA score), PSMA, AR and AR-V7 mRNA, baseline serum PSA levels, OS as well as parameters from PSMA PET-CT imaging were subjected to a global correlation analysis Figure B. OS shopos (n=11) and AR-V7neg (n=8) patients.In a global analysis relative copy numbers of AR and PSMA correlate with tumor load, reflected by MTV and total lesions, which was not detected in case of AR-V7, which correlates to total lesions but not to MTV. To further elucidate the role of AR-V7, the cohort was subdivided into AR-V7neg patients  and negative lesions in 12.5% (1 patient) of this group  was detected in patient samples only, whereas SYP and ENO2 expression were also detected in healthy blood donor samples. However, threshold determination revealed a significantly higher expression level in a subgroup of patients (NEDhi) compared to healthy controls (SYP (p<0.0005) and ENO2 (p<0.00005)). Relative copy numbers of FOXA1 were significantly higher within the subgroup of AR-V7 positive patients, whereas no significant differences in relative copy numbers were detected for ENO2 and SYP between AR-V7 subgroups. Nonetheless, patients expressing SYP and ENO2 above threshold had a significantly shorter OS regardless of AR-V7 expression (p<0.05), while this was not observed for PFS. The highest rate of PSMA positive lesions was detected within the AR-V7pos subgroup  by blinded image rating. Contrary, the AR-V7neg subgroup displayed variable or even PSMA negative lesions  for AR-V7 positive patients, while these correlations were absent in AR-V7s Figure C and D. S Figure  E+F. In is group . AR-V7ne177-PSMA-therapy.This study, to the best of our knowledge, provides a first clinical and molecular CTC analysis of mCRPC patients prior to LuThe membrane spanning PSMA protein has been extensively investigated as a potential biomarker in mCRPC The AR splice variant AR-V7 is a novel prognostic biomarker for mCRPC. AR-V7 has been reported to predict shorter OS and PFS in patients treated with abiraterone and enzalutamide All studies on AR-V7 display a significant number of patients not responding to antihormonal treatment despite a lack of AR-V7 The neuroendocrine state has been characterized by lack of PSMA expression and therefore less effective PSMA-targeted therapies as well as PSMA-supported imaging. However, the onset of NED is difficult to predict and the mechanisms enabling NED are still unclear. NED can be identified by markers, e.g. enolase 2 (ENO2), chromogranin (CgA), synaptophysin (SYP), and CD56 (NCAM) in tissue biopsy staining. In these settings, it is usually accompanied with loss of PSMA and AR expression concomitant with decrease of expression of AR target genes, e.g. FOXA1. Thus, tumor cells expressing high levels of AR and AR-V7 represent the adenocarcinoma state of prostate cancer, still relying on functional AR axis signaling. In case of mCRPC this might even be accompanied by AR-V7 expression, ultimately regulating AR target gene expression. Yet, absent or low AR levels as well as lack of AR-V7 expression might lead to decrease of FOXA1 expression, which in turn might be indicative for NED neg compared to AR-V7pos patients, but no difference regarding ENO2 and SYP. Further, high expression of SYP and ENO2 seems to significantly affect OS, which might be indicative for a more aggressive disease with attributes of NED. To support this hypothesis we included common serum markers such as LDH, ALP and, of course, PSA in our analysis. Significantly lower PSA serum levels in AR-V7neg compared to AR-V7pos patients support the hypothesis of an AR-independent, yet emerging neuroendocrine tumor phenotype pos patients with low NED gene expression. Higher LDH serum levels have been reported for active tumor metastatic activity and association with shorter OS neg mCRPC and those patients have to be subjected to a NED-targeted diagnostic panel. Somatostatin-targeting imaging such as 68Ga-DOTATATE-PET and screening for NED biomarkers in CTCs should be considered as clinical options. Using liquid biopsy, changes in gene and protein expression in CTCs can easily be followed. Any changes could subsequently be considered for making reasonable decisions regarding therapy options and changes, thus, rendering PC therapy highly personalized and flexible.In this study, a significant correlation between AR-V7 and FOXA1 expression was detected. Additionally, AR-V7 negative patients displayed significantly increased NED genes, e.g. ENO2, SYP. However, 17/19 (89.5%) and 19/19 (100%) of the patients enrolled in this study displayed AR or PSMA expression, respectively. Given the technical limitations of our approach - not to be able to determine gene expression on a single cell level - some cells might have undergone NED, whereas other cells may still phenotypically reveal an adenocarcinoma stage. Nevertheless, lack of AR-V7 was also accompanied with variable PSMA expression in tumor lesions. Thus, we hypothesize that the loss of AR-V7 might be an indicator for an onset of NED. Expression of SYP and ENO2 in patients as well as in healthy donors does not relate to any the presence nor malignity of the tumor, while FOXA1 seems to be specific for adenocarconima CTCs. We observed a significant drop in FOXA1 levels in AR-V7177Lu-PSMA-RLT on NED marker expression should be monitored as well. Previous work has identified CTC count during abiraterone treatment as a useful marker for disease progression and survival Relative expression levels of cAR, cPSMA and cPSA in CTCs seem to be linked to the actual tumor and metastasis load, represented by their correlation with imaging parameters. In this context, changes of marker gene expression by serial analyses during course of treatment might become a clinical tool of real-time monitoring. Moreover, the effect of On top, analytical and clinical validation has to be performed rigorously before drawing clinical conclusion. We hypothesized PSMA as a potential biomarker for patients which undergo PSMA directed therapy. However, we have to admit, that PSMA expression at baseline does not correlate to response to PSMA targeted therapy. This might rely on detection of PSMA mRNA only without evaluation of functional PSMA protein expression. Thus, future approaches have to focus on detection of PSMA protein rather than mRNA transcripts in CTCs.Further, according to this study, high transcriptional PSA levels may point at high cAR levels in CTCs. However, serums PSA, as well as its decline, do not display any relation to parameters identified by the molecular analysis of CTCs nor to PSMA-PET-CT imaging data. Therefore, sole PSA response as a predictor might be misleading, as it is not necessarily linked to clinical benefit. Besides, low levels of PSA are also associated with NED Additionally, PSMA- independent imaging methods, such as somatostatin-targeted DOTATATE PET, might give more insights into the phenotypic nature of distinct metastatic lesion and should therefore be included into the diagnostic panel 177Lu-PSMA-therapy, this study points at a relation of the molecular diagnostic CTC footprint to tumor load measured by PSMA-PET-CT imaging. However, this approach turned out less useful in terms of prediction of therapy response, survival and time to progression. Therefore, liquid biopsy collection is recommended not only prior to 177Lu-PSMA-therapy, but throughout the therapy course in order to detect changes in certain parameter transcription levels. To complete the diagnostic panel CTC proteomics should definitely be included into the analysis. Further, application of PSMA-RLT is questionable in late stage mCRPC, accompanied by loss of AR axis signaling and drop of FOXA1 levels. Also, decreasing serum PSA levels should be considered carefully and viewed in a context with NED markers in order enable distinction between true response and progression to neuroendocrine PC.Using relative transcriptional levels of novel biomarkers under investigation such as PSMA and AR in liquid biopsies from patients with mCRPC prior to Supplementary figures and tables.Click here for additional data file.