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Background: Functional near infrared spectroscopy (fNIRS) is a versatile, noninvasive, and inexpensive tool that can be used to measure oxyhemoglobin (O 2 Hb) changes in the cortical brain caused by increasing bladder sensation during filling in upright posture. This study's purpose is to provide a rigorous methodologic template that can be implemented for comparative studies of fNIRS in the diagnosis and management of lower urinary tract symptoms including overactive bladder (OAB) and other forms of lower urinary tract dysfunction., Methods: Participants without any urologic conditions completed a validated oral hydration protocol facilitating and equilibrating natural bladder filling. First desire to void and real time bladder sensation (0-100%) were recorded using a Sensation Meter. A 24-channel fNIRS template simultaneously recorded prefrontal cortical O 2 Hb. Each channel was analyzed between "first desire" to void and 100% sensation, defined in this study as the period of "high sensation". Channels were sub-divided by cortical regions: right (nine channels), left (nine channels), middle (six channels)., Results: A total of eight participants (male: n=4, female: n=4) were enrolled with mean age 39±19.9 years and body mass index (BMI) of 25±3.93 kg/m 2 . There were no differences in age, BMI, race, or OAB survey scores based on biological sex. Signal acquisition improved with power bank use, postural head support for motion reduction, and head cap optimization. Acceleration-based concurrent motion measurement was effectively utilized to remove motion artifacts. O 2 Hb concentration patterns appeared irregular during low sensation and increased during high sensation after first desire across the frontal cortex., Conclusions: Employing a stepwise approach, this study defined a methodological guide for improved prefrontal fNIRS signal acquisition and analysis during bladder filling. The technique demonstrated that prefrontal fNIRS cortical O 2 Hb increases with elevated bladder sensation in normal subjects and sets the stage for comparative studies in individuals with OAB and other forms of lower urinary tract dysfunction., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-23-275/coif). LSB is funded by National Institute of Health Building Interdisciplinary Research Careers in Women’s Health (No. K12HD108269). JES, APK, and LS are funded by National Institute of Health (No. R21DK128649). JES and APK are funded by Virginia Commonwealth University Presidential Research Quest Fund (No. 295166). The other authors have no conflicts of interest to declare., (2023 Translational Andrology and Urology. All rights reserved.)

The loss of skeletal muscle strength mid-life in females is associated with the decline of estrogen. Here, we questioned how estrogen deficiency might impact the overall skeletal muscle phosphoproteome after contraction, as force production induces phosphorylation of several muscle proteins. Phosphoproteomic analyses of the tibialis anterior muscle after contraction in two mouse models of estrogen deficiency, ovariectomy (Ovariectomized (Ovx) vs. Sham) and natural aging-induced ovarian senescence (Older Adult (OA) vs. Young Adult (YA)), identified a total of 2,593 and 3,507 phosphopeptides in Ovx/Sham and OA/YA datasets, respectively. Further analysis of estrogen deficiency-associated proteins and phosphosites identified 66 proteins and 21 phosphosites from both datasets. Of these, 4 estrogen deficiency-associated proteins and 4 estrogen deficiency-associated phosphosites were significant and differentially phosphorylated or regulated, respectively. Comparative analyses between Ovx/Sham and OA/YA using Ingenuity Pathway Analysis (IPA) found parallel patterns of inhibition and activation across IPA-defined canonical signaling pathways and physiological functional analysis, which were similarly observed in downstream GO, KEGG, and Reactome pathway overrepresentation analysis pertaining to muscle structural integrity and contraction, including AMPK and calcium signaling. IPA Upstream regulator analysis identified MAPK1 and PRKACA as candidate kinases and calcineurin as a candidate phosphatase sensitive to estrogen. Our findings highlight key molecular signatures and pathways in contracted muscle suggesting that the similarities identified across both datasets could elucidate molecular mechanisms that may contribute to skeletal muscle strength loss due to estrogen deficiency.

Introduction and Objectives: Urodynamics are the accepted gold standard for the evaluation of multiple forms of voiding dysfunction. However, the tests are expensive, invasive, poorly reproducible, and often prone to artifacts. Therefore, there is a pressing need to develop next-generation urodynamics. The purpose of this study was to develop a novel ex vivo porcine bladder urodynamics model with afferent pelvic nerve signaling that can be used as a preclinical surrogate for bladder sensation., Methods: Porcine bladders including the ureters and vascular supply were harvested from local abattoirs using an established protocol in both male and female animals. Ex vivo bladder perfusion was performed using physiologic MOPS (3-(N-morpholino) propanesulfonic acid) buffer solution. The pelvic nerve adjacent to the bladder was grasped with micro-hook electrodes and electroneurogram (ENG) signals recorded at 20 kHz. Bladders were filled with saline at a nonphysiologic rate (100 mL/min) to a volume of 1 L using standard urodynamics equipment to simultaneously record intravesical pressure. ENG amplitude was calculated as the area under the curve for each minute, and ENG firing rate was calculated as number of spikes (above baseline threshold) per minute. At the conclusion of the experiment, representative nerve samples were removed and processed for nerve histology by a pathologist (hematoxylin and eosin and S100 stains)., Results: A total of 10 pig bladders were used, and nerve histology confirmed the presence of nerve in all adequately processed samples. Vesical pressure, ENG firing rate, and ENG amplitude all increased as a function of filling. During filling tertiles (low fill: min 1-3, med fill: min 4-6, and high fill: min 7-10), normalized pressures were 0.22 ± 0.04, 0.38 ± 0.05, and 0.72 ± 0.07 (cmH2O). Similarly, normalized ENG firing rates were 0.08 ± 0.03, 0.31 ± 0.06, and 0.43 ± 0.04 spikes/minute, respectively, and normalized nerve amplitudes were 0.11 ± 0.06, 0.39 ± 0.06, and 0.56 ± 0.14) μV, respectively. Strong relationships between average normalized pressure values and averaged normalized ENG firing rate (r 2  = 0.66) and average normalized ENG amplitude (r 2  = 0.8) were identified., Conclusions: The ex vivo perfused porcine bladder can be used as a preclinical model for the development of next-generation urodynamics technologies. Importantly, the model includes a reproducible method to measure afferent nerve activity that directly correlates with intravesical pressure during filling and could potentially be used as a surrogate measure of bladder sensation., (© 2023 Wiley Periodicals LLC.)

Background: Prostate-specific antigen (PSA) testing remains controversial due to the debate about overdetection and overtreatment. Given the lack of published data regarding PSA testing rates in the population with spinal cord injury (SCI) within the US Department of Veterans Affairs (VA), there is concern for potential disparities and overtesting in this patient population. In this study, we sought to identify and evaluate national PSA testing rates in veterans with SCI., Methods: Using the VA Informatics and Computing Infrastructure Corporate Data Warehouse, we extracted PSA testing data for all individuals with a diagnosis of SCI. Testing rates were calculated, analyzed by race and age, and stratified according to published American Urological Association guideline groupings for PSA testing., Results: We identified 45,274 veterans at 129 VA medical centers with a diagnosis of SCI who had records of PSA testing in 2000 through 2017. Veterans who were only tested prior to SCI diagnosis were excluded. Final cohort data analysis included 37,243 veterans who cumulatively underwent 261,125 post-SCI PSA tests during the given time frame. Significant differences were found between African American veterans and other races veterans for all age groups (0.47 vs 0.46 tests per year, respectively, aged ≤ 39 years; 0.83 vs 0.77 tests per year, respectively, aged 40-54 years; 1.04 vs 1.00 tests per year, respectively, aged 55-69 years; and 1.08 vs 0.90 tests per year, respectively, aged ≥ 70 years; P < .001)., Conclusions: Significant differences exist in rates of PSA testing in persons with SCI based on age and race. High rates of testing were found in all age groups, especially for African American veterans aged ≥ 70 years., Competing Interests: Author disclosures The authors report no actual or potential conflicts of interest or outside sources of funding with regard to this article., (Copyright © 2023 Frontline Medical Communications Inc., Parsippany, NJ, USA.)

Background: Seasonal influenza vaccination (SIV) of health care workers (HCWs) is critical in protecting patients' and HCWs' health. Our objective was to examine HCW SIV coverage and related determinants., Methods and Materials: A nationwide cross-sectional questionnaire survey was conducted among HCWs during the first half of 2021. The questionnaire (online or paper-based) included knowledge, attitude and practice questions regarding SIV, COVID-19 vaccines and vaccination., Results: Out of 6500 questionnaires administered, 2592 were completed (response rate: 39.9%). SIV coverage reached 69.4% (95% CI: 67.6-71.2%) based on self-reported vaccine uptake. Nurses and administrative staff were found to be more skeptical and have lower vaccine acceptance in comparison with physicians (aOR = 0.66 and aQR = 0.59, respectively). Other SIV hesitancy risk factors included working in secondary health care (aOR = 0.59) and working in northern Greece (aQR = 0.66). Determinants for SIV acceptance included being or living with high-risk people due to medical history (aOR = 1.84 and aOR = 1.46, respectively), positive attitudes towards routine vaccinations (aOR: 1.86), knowledge about COVID-19 vaccines (aOR = 1.53) and COVID-19 vaccine uptake (aOR = 3.45). The primary reason for SIV refusal was low risk perception (58.7%)., Conclusions: SIV coverage (2020/2021) was relatively high, but remained far from formal recommendations. Specific occupational groups were skeptical and low-risk perception was the main reason for vaccine refusal. Targeted policies should be developed and enforced.

Greece opened its points of entry on July 1, 2020, with specific guidelines for travellers arriving by sea, air or land. The aim of this article is to examine the effect of tourism on the long term course of the Coronavirus Disease 2019 (COVID-19) pandemic during the pre-vaccination era (June to December 2020) on the popular Greek island of Crete. To achieve this, a cross-sectional serosurvey, repeated at monthly intervals, was conducted to compare the seroprevalence in Crete with seroprevalence in the mainland of Greece. Crete welcomed nearly 2,000,000 travellers during the 2020 summer season. Left-over serum samples were collected and obtained from public and private laboratories located in Greece, including the island of Crete. These samples were tested for the presence of anti-SARS-CoV-2 IgG antibodies. A total of 55,938 samples were collected, 3,785 of which originated from Crete. In Crete, the seroprevalence ranged between 0% (June 2020) and 2.58% (December 2020), while the corresponding seroprevalence in Greece was 0.19% and 10.75%, respectively. We identified 4.16 times lower seropositivity in Crete (2.58%) in comparison with the mainland of Greece (10.75%) during December 2020. Moreover, the monthly infection fatality rate (IFR) in Crete was calculated at 0.09%, compared with 0.21% in mainland Greece for December 2020. The island of Crete presented more than four times lower seroprevalence than the mainland of Greece, despite being a highly attractive tourist destination. This evidence supports the idea that tourism may not have affected the long term course of the COVID-19 pandemic in Greece. However, due to contradicting results from previous studies, further investigation is needed., Competing Interests: 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. The author ML declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2023 Bogogiannidou, Koureas, Mouchtouri, Dadouli, Kyritsi, Vontas, Anagnostopoulos, Mina, Matziri, Vachtsioli, Papagiannakis, Archontakis, Leotsinidis, Theodoridou, Manios, Gikas, Speletas and Hadjichristodoulou.)

Repeated excessive alcohol consumption increases the risk of developing cognitive decline and dementia. Hazardous drinking among older adults further increases such vulnerabilities. To investigate whether alcohol induces cognitive deficits in older adults, we performed a chronic intermittent ethanol exposure paradigm (ethanol or water gavage every other day 10 times) in 8-week-old young adult and 70-week-old aged rats. While spatial memory retrieval ascertained by probe trials in the Morris water maze was not significantly different between ethanol-treated and water-treated rats in both age groups after the fifth and tenth gavages, behavioral flexibility was impaired in ethanol-treated rats compared to water-treated rats in the aged group but not in the young adult group. We then examined ethanol-treatment-associated hippocampal proteomic and phosphoproteomic differences distinct in the aged rats. We identified several ethanol-treatment-related proteins, including the upregulations of the Prkcd protein level, several of its phosphosites, and its kinase activity and downregulation in the Camk2a protein level. Our bioinformatic analysis revealed notable changes in pathways involved in neurotransmission regulation, synaptic plasticity, neuronal apoptosis, and insulin receptor signaling. In conclusion, our behavioral and proteomic results identified several candidate proteins and pathways potentially associated with alcohol-induced cognitive decline in aged adults., Competing Interests: The authors declare the following competing financial interest(s): Dr. Doo-Sup Choi is a scientific advisory board member to Peptron Inc. and Peptron had no role in the preparation, review, or approval of the manuscript nor the decision to submit the manuscript for publication. The remaining authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 The Authors. Published by American Chemical Society.)

Protein phosphorylation is important in skeletal muscle development, growth, regeneration, and contractile function. Alterations in the skeletal muscle phosphoproteome due to aging have been reported in males; however, studies in females are lacking. We have demonstrated that estrogen deficiency decreases muscle force, which correlates with decreased myosin regulatory light chain phosphorylation. Thus, we questioned whether the decline of estrogen in females that occurs with aging might alter the skeletal muscle phosphoproteome. C57BL/6J female mice (6 mo) were randomly assigned to a sham-operated (Sham) or ovariectomy (Ovx) group to investigate the effects of estrogen deficiency on skeletal muscle protein phosphorylation in a resting, noncontracting condition. After 16 wk of estrogen deficiency, the tibialis anterior muscle was dissected and prepped for label-free nano-liquid chromatography-tandem mass spectrometry phosphoproteomic analysis. We identified 4,780 phosphopeptides in tibialis anterior muscles of ovariectomized (Ovx) and Sham-operated (Sham) control mice. Further analysis revealed 647 differentially regulated phosphopeptides (Benjamini-Hochberg adjusted P value < 0.05 and 1.5-fold change ratio) that corresponded to 130 proteins with 22 proteins differentially phosphorylated (3 unique to Ovx, 2 unique to Sham, 6 upregulated, and 11 downregulated). Differentially phosphorylated proteins associated with the sarcomere, cytoplasm, and metabolic and calcium signaling pathways were identified. Our work provides the first global phosphoproteomic analysis in females and how estrogen deficiency impacts the skeletal muscle phosphoproteome.

Background: Mental health problems are one of the leading causes of disease burden worldwide, and are mainly diagnosed and treated in general practice. It is unclear however, how general practitioners (GPs) identify mental health problems in their patients. The aim of this study was to explore how patients' self-reported levels of mental distress correspond with psychological diagnoses made by their GPs, and associations with sex, age, number of consultations, and somatic symptom diagnoses., Methods: A questionnaire study coupled with retrospective and prospective cohort data from 553 patients aged 16-65 years in six GP offices in Oslo, Norway during 21 months in 2014-2016., Results: We found that 73.3% of patients with self-reported high levels of mental distress versus only 13.3% of the patients with low levels of mental distress had received a psychological diagnosis (p < 0.01). We found an increase in number of consultations for the group with high levels of mental distress regardless of having received a psychological diagnosis (p < 0.01). There was also an increase in number of somatic symptoms (p = 0.04) and higher number of females (0.04) in this group. 35% of patients had received one or more psychological diagnosis by their GP. Mean CORE-10 score, being female and a high number of consultations was associated with having received a psychological diagnosis. In the adjusted analyses high CORE-10 score and a high number of consultations still predicted a psychological diagnosis., Conclusions: We found a clear association between self-reported mental distress and having received a psychological diagnosis amongst the participants, and the probability for being identified increased with increasing levels of mental distress, and increasing number of visits to their doctor. This suggests that GPs can identify patients with high levels of mental distress in general practice in an adequate way, even though this can sometimes be a complex issue., Trial Registration: Trial registration The main study was retrospectively registered in ClinicalTrials.gov on August 10 2019 with identification number NCT03624829., (© 2022. The Author(s).)

Bone density decreases rapidly after spinal cord injury (SCI), increasing fracture risk. The most common fracture sites are at the knee (i.e., distal femur or proximal tibia). Despite this high fracture incidence, knee-specific scans for bone density using dual x-ray absorptiometry (DXA) were not available until 2014 and are still not routinely used in clinical practice today. This has made it difficult to determine the rehabilitation efficacy and hindered understanding of the long-term changes in knee areal bone density. The purpose of this investigation was to compare areal bone mineral density values for the knee from both total-body and knee-specific DXA scans in persons with SCI. A total of 20 participants (16 males) >1 yr-post spinal cord injury received two DXA scans; a total-body scan and a knee-specific scan. Standardized methods were used to create regions of interest to determine bone density of four regions - the epiphysis and metaphysis of the distal femur and proximal tibia - from the total-body scan. Linear regressions and Bland-Altman analyses were conducted to determine the correlation (r 2 ) and agreement (mean bias ± 95% level of agreement) respectively between the two scan types for each region. Linear regression analyses showed strong significant (p < 0.001) relationships between the two scan types for the distal femur epiphysis (r 2  = 0.88) and metaphysis (r 2  = 0.98) and the proximal tibia epiphysis (r 2  = 0.88) and metaphysis (r 2  = 0.99). The mean bias ± 95% level of agreement were distal femur epiphysis (0.05 ± 0.1 g/cm 2 ) and metaphysis (0.02 ± 0.04 g/cm 2 ); proximal tibia epiphysis (-0.02 ± 0.1 g/cm 2 ) and metaphysis (0.02 ± 0.03 g/cm 2 ). Results suggest knee-specific bone density can be assessed using a total-body DXA scan. This may allow for more comprehensive use of DXA scans which would reduce the burden of multiple site-specific scans for persons with SCI and enable more widespread adoption of knee bone density assessment in this population., (Copyright © 2021 The International Society for Clinical Densitometry. Published by Elsevier Inc. All rights reserved.)

Background: Nation-wide SARS-CoV-2 seroprevalence surveys provide valuable insights into the course of the pandemic, including information often not captured by routine surveillance of reported cases., Methods: A serosurvey of IgG antibodies against SARS-CoV-2 was conducted in Greece between March and December 2020. It was designed as a cross-sectional survey repeated at monthly intervals. The leftover sampling methodology was used and a geographically stratified sampling plan was applied., Results: Of 55,947 serum samples collected, 705 (1.26%) were found positive for anti-SARS-CoV-2 antibodies, with higher seroprevalence (9.09%) observed in December 2020. Highest seropositivity levels were observed in the "0-29" and "30-49" year age groups. Seroprevalence increased with age in the "0-29" age group. Highly populated metropolitan areas were characterized with elevated seroprevalence levels (11.92% in Attica, 12.76% in Thessaloniki) compared to the rest of the country (5.90%). The infection fatality rate (IFR) was estimated at 0.451% (95% CI: 0.382-0.549%) using aggregate data until December 2020, and the ratio of actual to reported cases was 9.59 (7.88-11.33)., Conclusions: The evolution of seroprevalence estimates aligned with the course of the pandemic and varied widely by region and age group. Young and middle-aged adults appeared to be drivers of the pandemic during a severe epidemic wave under strict policy measures.

After spinal cord injury (SCI) physical activity levels decrease drastically, leading to numerous secondary health complications. Exoskeleton-assisted walking (EAW) may be one way to improve physical activity for adults with SCI and potentially alleviate secondary health complications. The effects of EAW may be limited, however, since exoskeletons induce passive movement for users who cannot volitionally contribute to walking. Trans-spinal stimulation (TSS) has shown the potential to enable those with even the most severe SCI to actively contribute to movements during EAW. To explore the effects of EAW training on improving secondary health complications in persons with SCI, participants with chronic ( n = 8) were enrolled in an EAW program 2-3 times per week for 12 weeks. Anthropometrics (seated and supine waist and abdominal circumferences (WC and AC), body composition assessment (dual exposure x-ray absorptiometry-derived body fat percent, lean mass and total mass for the total body, legs, and trunk), and peak oxygen consumption (VO 2 during a 6-minute walk test [6MWT]) were assessed before and after 12 weeks of EAW training. A subset of participants ( n = 3) completed EAW training with concurrent TSS, and neuromuscular activity of locomotor muscles was assessed during a 10-m walk test (10MWT) with and without TSS following 12 weeks of EAW training. Upon completion of 12 weeks of training, reductions from baseline (BL) were found in seated WC (-2.2%, P = 0.036), seated AC (-2.9%, P = 0.05), and supine AC (-3.9%, P = 0.017). Percent fat was also reduced from BL for the total body (-1.4%, P = 0.018), leg (-1.3%, P = 0.018), and trunk (-2%, P = 0.036) regions. No effects were found for peak VO 2 . The addition of TSS for three individuals yielded individualized responses but generally increased knee extensor activity during EAW. Two of three participants who received TSS were also able to initiate more steps without additional assistance from the exoskeleton during a 10MWT. In summary, 12 weeks of EAW training significantly attenuated markers of obesity relevant to cardiometabolic health in eight men with chronic SCI. Changes in VO 2 and neuromuscular activity with vs. without TSS were highly individualized and yielded no overall group effects., Competing Interests: Conflict of Interest: 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.

Skeletal muscle stiffness is altered after spinal cord injury (SCI). Assessing muscle stiffness is essential for rehabilitation and pharmaceutical interventions design after SCI. The study used magnetic resonance elastography to assess the changes in stiffness after chronic SCI compared to matched able-bodied controls and determine its association with muscle size, spasticity, and peak torque in persons with SCI. Previous studies examined the association between muscle stiffness and spasticity, however, we are unaware of other studies that examined the effects of muscle composition on stiffness after SCI. Ten participants (one female) with chronic SCI and eight (one female) matched able-bodied controls participated in this cross-sectional study. Magnetic resonance elastography was utilized to monitor stiffness derived from shear waves propagation. Modified Ashworth scale was used to evaluate spasticity scores in a blinded fashion. Peak isometric and isokinetic torques were measured using a biodex dynamometer. Stiffness values were non-significantly lower (12.5%; P = 0.3) in the SCI group compared to able-bodied controls. Moreover, stiffness was positively related to vastus lateralis whole muscle cross-sectional area (CSA) (r 2 = 0.64, P < 0.005) and vastus lateralis absolute muscle CSA after accounting for intramuscular fat (r 2 = 0.78, P < 0.0007). Stiffness was also positively correlated to both isometric (r 2 = 0.55-0.57, P < 0.05) and isokinetic peak (r 2 = 0.46-0.48, P < 0.05) torques. Our results suggest that larger clinical trial is warranted to confirm the preliminary findings that muscle stiffness is altered after SCI compared to healthy controls. Stiffness appeared to be influenced by infiltration of intramuscular fat and modestly by the spasticity of the paralyzed muscles. The preliminary data indicated that the relationship between muscle stiffness and peak torque is not altered with changing the frequency of pulses or angular velocities. All study procedures were approved by the Institutional Review Board at the Hunter Holmes McGuire VA Medical Center, USA (IRB #: 02314) on May 3, 2017., Competing Interests: None

Background: Patients with mental health problems often present with somatic symptoms when visiting their general practitioner (GP). Somatic presentations may challenge correct diagnosing of mental health disorders in general practice, where most of these disorders are treated., Objective: Explore the associations between common psychological diagnoses and somatic symptom diagnoses in Norwegian urban general practice., Methods: A retrospective cohort study including electronic medical data from 15 750 patients aged 16-65 years from 35 GPs in six GP offices in Oslo, Norway, during 12 months in 2014-2015. We explored prevalences and associations between anxiety-, depression-, and stress-related diagnoses, and somatic symptom diagnoses., Results: Patients with anxiety-, depression- and stress-related diagnoses had a mean number of 2.9±3.6 somatic symptom diagnoses during the 12 months, compared to 1.9±2.5 for patients without any psychological diagnoses (P < 0.001). The mean number of somatic symptoms was significantly higher for the different psychological diagnoses viewed separately, for both sexes and different age groups. There was an increase in probability for anxiety, depression, or stress-related diagnoses with an increasing number of somatic symptom diagnoses during the 12 months. We found a significant increase in somatic symptom diagnoses from ICPC-2 chapters: General and unspecified, digestive, cardiovascular, musculoskeletal, neurological, urological, female genital disorders and social problems. Associated symptom patterns were different for each of the included psychological diagnoses., Conclusions: This study shows that patients with anxiety, depression- and stress-related diagnoses present with increased and characteristic somatic symptoms compared to patients without these diagnoses in general practice., (© The Author(s) 2021. Published by Oxford University Press.)

A serosurvey of IgG antibodies against SARS-CoV-2 was conducted in Greece between May and August 2020. It was designed as a cross-sectional survey and was repeated at monthly intervals. The leftover sampling methodology was used and a geographically stratified sampling plan was applied. Of 20,110 serum samples collected, 89 (0.44%) were found to be positive for anti-SARS-CoV-2 antibodies, with higher seroprevalence (0.35%) observed in May 2020. The highest seroprevalence was primarily observed in the "30-49" year age group. Females presented higher seroprevalence compared to males in May 2020 (females: 0.58% VS males: 0.10%). This difference reversed during the study period and males presented a higher proportion in August 2020 (females: 0.12% VS males: 0.58%). Differences in the rate of seropositivity between urban areas and the rest of the country were also observed during the study period. The four-month infection fatality rate (IFR) was estimated to be 0.47%, while the respective case fatality rate (CFR) was at 1.89%. Our findings confirm low seroprevalence of COVID-19 in Greece during the study period. The young adults are presented as the most affected age group. The loss of the cumulative effect of seropositivity in a proportion of previous SARS-CoV-2 infections was indicated.

A COVID-19 outbreak occurred among residents of a Roma settlement in Greece (8 April-4 June 2020). The aim of this study was to identify factors associated with an increased risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and to evaluate the effectiveness of control measures implemented. Data were analyzed from individuals that were tested for SARS-CoV-2 during contact tracing, population screening or hospital visits. RT-PCR was used for the detection of SARS-CoV-2 in oropharyngeal samples. Risk factors for household secondary attack rates (SAR) and hospitalization with COVID-19 were examined using chi-square tests, Fisher's exact tests and logistic regression analyses. During the outbreak, 142 cases, 20 hospitalizations and 1 death were recorded, with a total of 2273 individuals tested. The risk of hospitalization was associated with age (OR: 1.04, 95% CI: 1.02-1.07) and Cycle threshold (Ct) values (OR for a decrease in Ct values by 1: 1.18, 95% CI: 1.07-1.31). Household SAR was estimated at 38.62% (95% CI: 32.50-45.01%). After the designation of an isolation facility for cases, household SAR declined from 74.42% to 31.03%. Household size was associated with the risk of infection (OR: 2.65, 95% CI: 1.00-7.07). The presence of COVID-19 symptoms among index cases was correlated with higher transmission (OR: 23.68, 95% CI 2.21-253.74) in multivariate analysis, while age was found to be associated with SAR only in univariate analysis. Roma communities can be particularly vulnerable to the spread of SARS-CoV-2. In similar settings, symptomatic cases are more important transmitters of SARS-CoV-2. Within these communities, immediate measures should be implemented to mitigate disease spread.

Significance: After a dilated eye examination, many patients experience symptoms of prolonged light sensitivity, blurred vision, and cycloplegia associated with pharmacological mydriasis. Phentolamine mesylate ophthalmic solution (PMOS) may expedite the reversal of mydriasis in patients, potentially facilitating return to functional vision and reducing barriers to obtaining dilated eye examinations., Purpose: The protracted reversal time after pharmacologically induced pupil dilation impairs vision. We tested the hypothesis that PMOS rapidly reduces pupil diameter in this acute indication., Methods: In this double-masked placebo-controlled, randomized, two-arm crossover phase 2b trial, we evaluated the effects of one drop of 1% PMOS applied bilaterally in subjects who had their pupils dilated by one of two common mydriatic agents: 2.5% phenylephrine or 1% tropicamide. End points included change in pupil diameter, percent of subjects returning to baseline pupil diameter, and accommodative function at multiple time points., Results: Thirty-one subjects completed the study (15 dilated with phenylephrine and 16 with tropicamide). Change in pupil diameter from baseline at 2 hours after maximal dilation with 1% PMOS was -1.69 mm and was significantly greater in magnitude compared with placebo for every time point beyond 30 minutes (P < .05). At 2 hours, a greater percentage of study eyes given 1% PMOS returned to baseline pupil diameter compared with placebo (29 vs. 13%, P = .03), which was this also seen at 4 hours (P < .001). More subjects treated with PMOS in the tropicamide subgroup had at least one eye returning to baseline accommodative amplitude at 2 hours (63 vs. 38%, P = .01). There were no severe adverse events, with only mild to moderate conjunctival hyperemia that resolved in most patients by 6 hours., Conclusions: Phentolamine mesylate ophthalmic solution at 1% reversed medically induced pupil dilation more rapidly than placebo treatment regardless of which mydriatic was used (adrenergic agonists and cholinergic blockers) with a tolerable safety profile., Competing Interests: Conflict of Interest Disclosure: The publication of this article was sponsored by Ocuphire Pharma, Inc. ARM, MPS, RMJ, KC, JEY, SAK, and ATA are or were employees, directors/officers, or paid consultants for Ocuphire Pharma. PMK, MBM, ESL, JSP, and GDH are on the medical advisory board for Ocuphire Pharma. PMK, SAF, SMM, and SRK were principal investigators for this clinical trial funded by Ocuphire Pharma. CBS serves as Chief Medical Officer of Oculos Development Services, LLC, which was the contract research organization for this clinical trial. The authors indicated that they have no other conflicts of interest with regard to the content of this article. The authors listed report a financial conflict of interest (PMK, SAF, SMM, SRK, CBS, ARM, MPS, RMJ, KC, JEY, SAK, ATA, MBM, GDH, ESL, and JSP). The sponsor (Ocuphire) provided financial and material support and participated in study design, analysis, and interpretation. The authors were responsible for the preparation of this article and the decision to submit this article for publication. Each of the authors had (full/limited) access to the study data and takes full responsibility for their presentation in this article., (Copyright © 2021 American Academy of Optometry.)

Purpose: Phentolamine mesylate ophthalmic solution (PMOS), applied to the eye topically, was shown previously to have beneficial effects in patients with dim light vision disturbances (DLD), including decreased pupil diameter (PD), improved best-corrected distance visual acuity (BCDVA), as well as lower intraocular pressure (IOP). The ORION-1 trial evaluated the long-term safety and efficacy of PMOS in a glaucomatous, presbyopic population., Patients and Methods: In this randomized, double-masked, multi-center, placebo-controlled, multiple-dose Phase 2b trial, 39 patients with elevated IOP were randomized to receive one evening dose of study medication or placebo for 14 days. The primary outcome measure was mean change in diurnal IOP, and the key secondary outcome measures included changes in PD, distance-corrected near visual acuity (DCNVA), and conjunctival hyperemia., Results: Use of 1% PMOS did not lead to a statistically significant decrease in diurnal IOP compared to placebo ( P = 0.89) but trended toward a greater decrease in patients with lower IOP baselines. PMOS produced a statistically significant mean 20% PD reduction under both photopic and mesopic conditions that was sustained for 36 hours post-dosing. A statistically significant number of patients with PMOS compared to placebo demonstrated ≥1 line of improvement in photopic DCNVA at day 8 ( P = 0.0018), day 15 ( P = 0.0072), and day 16 ( P = 0.0163), with a trend for 2- and 3-line improvements at all time points. There was no statistical difference in conjunctival hyperemia compared to placebo., Conclusion: Although mean IOP was not lowered significantly, daily evening dosing of 1% PMOS was found to be well tolerated with no daytime conjunctival redness and demonstrated improvement in DCNVA with sustained PD reduction in a glaucomatous and presbyopic population. Smaller pupil size can have beneficial effects in improving symptoms of presbyopia and DLD, which will be the focus of further studies., Competing Interests: MPS, ARM, RMJ, KC, ATA, JEY, and SAK are or were employees, directors/officers, or paid consultants for Ocuphire Pharma. JSP, ESL, PMK, and MBM are on the medical advisory board for Ocuphire Pharma. PJH, HBD, MAA, RJSM, and SEM were principal investigators for this clinical trial funded by Ocuphire Pharma. CBS serves as Chief Medical Officer of Oculos Development Services, LLC, which was the contract research organization for this clinical trial. Dr Jay S Pepose reportspersonal fees from Acufocus, Kala Pharmaceuticals, Keeler, MG Therapeutics, Mimetogen Pharmaceuticals, Novartis, Ocunexis Therapeutics, Okogen, Stuart Pharmaceuticals, Sun Pharma, Thea Pharma, TearLab, and Ocuphire, outside the submitted work. Dr Robert J Smyth-Medina reports research study site funding from Ora. Dr Sayoko E Moroi reports grants from R01 EY022124 (PI Moroi), R21 EY030363 (MPI Musch + Moroi), R01 EY025752 (PI Komaromy), NSF 1,760,291 (MPI Argento + Moroi), Aerie Pharmaceuticals, Inc, Allergan, Ocuphire, Icare USA, Wolters Kluwer Health: Royalty for Shield’s Textbook of Glaucoma, grants from MCubed, outside the submitted work. Mr Alan R Meyer have patents US 9,789,088 B2, US 9,795,560 B2, and US 10,278,918 assigned to Ocuphire Pharma issued. Dr Konstantinos Charizanis reports a patent PCT/US2019/056324 pending. Dr Marguerite B McDonald reports personal fees from Allergan, Alcon, Bausch and Lomb, Eyevance, Novartis, Tarsus, Visus, Aperta, Ocusoft, OCULUS USA, DOMPE, BioTissue, BlephEx, Akorn, Quidel, ORCA Surgical, TearLab, J&J Vision, TearCare, NuLids, Ocuphire, STROMA, Hellas LTD, Sun Pharma, Avedro, Omeros, Scope, and Sight Sciences, during the conduct of the study; personal fees from Visus and Allergan, outside the submitted work. The authors indicated that they have no other conflicts of interest with regard to the content of this article., (© 2021 Pepose et al.)

Background: Severe spasticity may negatively impact functionality and quality of life after spinal cord injury (SCI). Intrathecal baclofen treatment (IBT) is effectively used to manage severe spasticity and reduce comorbidities. However, long-term IBT may have a negative effect on bone mineral content (BMC), bone mineral density (BMD) and body composition (such as percentage fat mass and lean body mass). We demonstrated the negative effects of long-term IBT use in a single case compared with two non-IBT users., Case Summary: A 46-year old Caucasian male Veteran (case) with a 21 year history of complete tetraplegia (complete C6 SCI) was implanted with IBT for 20 years. The case was matched to two participants with different time since injuries [2 (match 1) and 13 (match 2) years] without IBT. Knee BMC and BMD at the epiphysis and metaphysis of the distal femur and proximal tibia were evaluated using dual knee and the dual femur modules of GE Lunar iDXA software. Total and leg body composition assessments were also conducted for the three participants. Potential effect of long-term IBT was demonstrated by changes in BMD, consistent with bone demineralization, at the distal femur and proximal tibia and changes in percentage fat mass and lean mass of legs. The case showed 113% lower BMD at the distal femur, and 78.1% lower at the proximal tibia compared to match 1, moreover the case showed 45% lower BMD at the distal femur, and no observed changes at the proximal tibia compared to match 2. The case had 27.1% and 16.5% greater leg %fat mass compared to match 1 and match 2, respectively. Furthermore, the case had 17.4% and 11.8% lower % leg lean mass compared to match 1 and match 2, respectively., Conclusion: Long-term IBT may impact bone health and body composition parameters in persons with complete SCI. It may be prudent to encourage regular screening of individuals on long-term IBT considering the prevalence of osteoporosis related fractures, cardiovascular diseases, and metabolic disorders in this population., Competing Interests: Conflict-of-interest statement: No conflict of interest., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: Skeletal muscle mitochondrial activity is reduced by ∼ 50-60% after SCI, resulting in impaired energy expenditure, glucose utilization and insulin sensitivity. Near infra-red spectroscopy (NIRS) is a non-invasive tool that can be used to assess mitochondrial capacity. Objectives: (1) Highlight methodological limitations impacting data acquisition and analysis such as subcutaneous adipose tissue (SAT) thickness, movement artifacts, inadequate muscle stimulation, light interference, and ischemic discomfort. (2) Provide technical considerations to improve data acquisition and analysis. This may serve as guidance to other researchers and clinicians using NIRS. Study Design: cross-sectional observational design. Settings: Clinical research medical center. Participants: Sixteen men with 1 > year post motor complete SCI. Methods: NIRS signals were obtained from right vastus lateralis muscle utilizing a portable system. Signals were fit to a mono-exponential curve. Outcome Measures: Rate constant and r 2 values for the fit curve, indirectly measures mitochondrial capacity. Results: Only four participants produced data with accepted rate constants of 0.002-0.013 s -1 and r 2 of 0.71-0.87. Applications of studentized residuals ≥2.5 resulted in sparing data from another four participants with rate constants of 0.010-0.018 s -1 and r 2 values ranging from 0.86-0.99. Conclusions: Several limitations may challenge the use of NIRS to assess mitochondrial capacity after SCI. Acknowledging these limitations and applying additional data processing techniques may overcome the discussed limitations and facilitate data sparing.

Purpose: Dual energy X-ray absorptiometry (DXA) and magnetic resonance imaging (MRI) permits quantification of visceral adipose tissue (VAT). However, DXA has not been validated against MRI in persons with chronic spinal cord injury (SCI). A predictive equation was generated from the measurement of VAT by MRI, a "gold" standard to quantitate VAT, compared to that of DXA, a method with several practical advantages., Method: DXA and MRI scans were performed in 27 participants with SCI. MRI multiaxial images were captured for VAT analysis. DXA-VAT was quantified at the android region (DXA-VAT ANDROID-VOL ) using enCore software. Android regions of DXA and MRI were matched using android height. Volumes of multiaxial MRI-VAT and subcutaneous adipose tissue (SAT) were quantified for the android region (MRI-VAT ANDROID-VOL , MRI-SAT ANDROID-VOL ) and total trunk (MRI-VAT ANDROID-VOL ). Linear regression analysis was used to establish the proposed predication equations. The prediction equations were then applied to an independent sample that consisted of 98 participants with SCI. Bland-Altman analysis was used to determine the limits of agreement., Results: DXA-VAT ANDROID-VOL predicted 92% of the variance in MRI-VAT ANDROID-VOL (SEE = 252.5, p < 0.0005) and 85% of the variance in MRI-VAT TRUNK-VOL (SEE = 1526.9, p < 0.0005). DXA-SAT ANDROID-VOL predicted 81.5% of the variance in MRI-SAT ANDROID-VOL (SEE = 458.2, p < 0.0005). Bland-Altman analysis revealed a high level of agreement between MRI-VAT ANDROID-VOL and DXA-VAT ANDROID-VOL (mean bias = 58.45 cm 3 ). A predicted mean DXA-VAT ANDROID-VOL of 995.2 cm 3 was estimated as the population-specific cut-off point for high levels of VAT., Conclusion: DXA-VAT ANDROID-VOL may accurately predict MRI-VAT ANDROID-VOL in persons with SCI. The ability of DXA to detect VAT changes in longitudinal studies in persons with SCI should be performed., (Copyright © 2018 The International Society for Clinical Densitometry. Published by Elsevier Inc. All rights reserved.)

The use of cannabis for recreational as well as medicinal use is on the rise recently with more states legalizing it. We conducted a review analysis of the literature published on acute respiratory failure from vaping cannabis oil. We have also summarized the clinical details (age, length of stay, mode of ventilation, common clinical findings, and steroid use) along with common laboratory abnormalities. This article aims to educate health care providers on the clinical manifestations and management strategies for vaping-induced acute respiratory failure. We also discussed the different available formulations of cannabis oil and key ingredients responsible for the vaping-associated lung injury.

Background: Non-invasive prenatal testing (NIPT) has been widely adopted for the detection of fetal aneuploidies and microdeletion syndromes, nevertheless, limited clinical utilization has been reported for the non-invasive prenatal screening of monogenic diseases. In this study, we present the development and validation of a single comprehensive NIPT for prenatal screening of chromosomal aneuploidies, microdeletions and 50 autosomal recessive disorders associated with severe or moderate clinical phenotype., Results: We employed a targeted capture enrichment technology powered by custom TArget Capture Sequences (TACS) and multi-engine bioinformatics analysis pipeline to develop and validate a novel NIPT test. This test was validated using 2033 cell-fee DNA (cfDNA) samples from maternal plasma of pregnant women referred for NIPT and paternal genomic DNA. Additionally, 200 amniotic fluid and CVS samples were used for validation purposes. All NIPT samples were correctly classified exhibiting 100% sensitivity (CI 89.7-100%) and 100% specificity (CI 99.8-100%) for chromosomal aneuploidies and microdeletions. Furthermore, 613 targeted causative mutations, of which 87 were unique, corresponding to 21 monogenic diseases, were identified. For the validation of the assay for prenatal diagnosis purposes, all aneuploidies, microdeletions and point mutations were correctly detected in all 200 amniotic fluid and CVS samples., Conclusions: We present a NIPT for aneuploidies, microdeletions, and monogenic disorders. To our knowledge this is the first time that such a comprehensive NIPT is available for clinical implementation., Competing Interests: Competing interestsThe authors, GK, AA, MN, CL, KT, EK, VV, MI and PCP are employed by NIPD Genetics Ltd. The authors CS, GC, PC, ME, IP, DS declare that they have no competing interests. PM was employed by NIPD Genetics Public Company Ltd. at the time of the study but currently has no competing interests., (© The Author(s). 2019.)

Background: Reduced level of physical activity, high-fat diet and skeletal muscle atrophy are key factors that are likely to contribute to deleterious changes in body composition and metabolic following spinal cord injury (SCI). Reduced caloric intake with lowering percentage macronutrients of fat and increasing protein intake may likely to improve body composition parameters and decrease ectopic adiposity after SCI., Aim: To highlight the effects of dietary manipulation and testosterone replacement therapy (TRT) on body composition after SCI., Methods: A 31-year-old male with T5 SCI was administered transdermal TRT daily for 16 wk. Caloric intake and percentage macronutrients were analyzed using dietary recalls. Magnetic resonance imaging and dual-energy x-ray absorptiometry were used to measure changes in body composition., Results: Caloric intake and fat percentage were reduced by 445 kcal/d and 6.5%, respectively. Total body weight decreased by 8%, body fat decreased by 29%, and lean mass increased by 7%. Thigh subcutaneous adipose tissue cross-sectional area was reduced by 31%., Conclusion: Manipulation of caloric intake, fat percentage, and protein percentage may have influenced body composition after SCI.

Objectives: A rapid decline in lean mass (LM), fat-free mass (FFM) and increased intramuscular fat (IMF) predispose persons with spinal cord injury (SCI) to chronic medical conditions including dyslipidemia, insulin resistance, type 2 diabetes mellitus and cardiovascular disease. (1) To determine the relationship between dual energy x ray absorptiometry (DXA) and gold standard magnetic resonance imaging (MRI) LM values; (2) to develop predictive equations based on this relationship for assessing thigh LM in persons with chronic SCI. Study Design: Cross-sectional predicational design. Settings: Clinical research medical center. Participants: Thirty-two men with chronic (>1 y post-injury) motor complete SCI. Methods: Participants completed total body DXA scans to determine thigh LM and were compared to measurements acquired from trans-axial MRI. Outcome measures: MRI was used to measure whole muscle mass (MM MRI-WM ), absolute muscle mass (MM MRI-ABS ) after excluding IMF, and knee extensor muscle mass (MM MRI-KE ). DXA was used to measure thigh LM (LM DXA ) and (FFM DXA ). To predict MM MRI-KE, LM DXA was multiplied by 0.52 and yielded LM DXA-KE . Results: LM DXA predicted MM MRI-WM [ r 2   = 0.90, standard error of the estimate (SEE) = 0.23 kg, P  < 0.0001] and MM MRI-ABS ( r 2  = 0.82, SEE = 0.28 kg, P  < 0.0001). LM DXA-KE predicted MM MRI-KE ( r 2  =  0.78, SEE = 0.16 kg, P  < 0.0001). Conclusion: DXA measurements revealed an acceptable agreement with the gold standard MRI and may be a viable alternative for assessing thigh skeletal muscle mass after SCI.

Introduction: Non-Invasive Prenatal Testing (NIPT) for fetal aneuploidies using cell-free DNA (cfDNA) has been widely adopted in clinical practice due to its improved accuracy. A number of NIPT tests have been developed and validated. The purpose of this study is to evaluate the performance of the Veracity NIPT test for sex chromosome aneuploidy (SCA) detection in singleton pregnancies, autosomal aneuploidy detection in twin pregnancies and evaluation of Veracity clinical performance under routine NIPT conditions in a diverse cohort., Methods: Blinded retrospective study in singleton pregnancies ( n  = 305); blinded retrospective and prospective study in twin pregnancies ( n  = 306) and prospective evaluation of clinical performance in singleton and twin pregnancies ( n  = 10564)., Results: Validation study results for the detection of SCAs in singleton pregnancies exhibited 100% sensitivity and specificity and correctly classified 7 (45,X), 4 (47,XXY), 2 (47,XXX) and 1 (47,XYY) cases. Validation study results for autosomal aneuploidy detection in twin pregnancies exhibited 100% sensitivity and specificity and correctly classified 3 trisomy 21, 1 trisomy 18 and 1 trisomy 13 samples. Clinical performance evaluation of Veracity was performed in 10564 pregnancies with median gestational age of 13 weeks, median maternal age 35 years and median gestational weight of 64 kg. Based on confirmation feedback the PPV for trisomies 21, 18 and 13 was estimated at 100% (95% CI, 92-100%), 100% (95% CI, 69-100%) and 71% (95% CI, 29-96%), respectively. Estimated PPV for Monosomy X was 57% (95%CI, 18-90%), while the NPV for SCA detection was estimated at 100% (95% CI, 99.94-100%)., Conclusion: Veracity NIPT test is based on a very powerful, highly accurate methodology that can be safely applied in the clinical setting., Competing Interests: Competing interestsEK, MI, ET, IN, VV, KT, AA, PP, GK are employed by NIPD Genetics Public Company Ltd. PM was employed by NIPD Genetics Public Company Ltd. at the time of the study but currently has no conflict of interest. SV, AS, FA have no conflict of interest.

Background: Few areas of medicine demonstrate such international divergence as child development screening and surveillance. Many countries have nationally mandated surveillance policies, but the content of programmes and mechanisms for delivery vary enormously. The cost of programmes is substantial but no economic evaluations have been carried out. We have critically examined the history, underlying philosophy, content and delivery of programmes for child development assessment in five countries with comprehensive publicly funded health services (Denmark, Finland, Norway, Scotland and Sweden). The specific focus of this article is on motor, social, emotional, behavioural and global cognitive functioning including language., Findings: Variations in developmental surveillance programmes are substantially explained by historical factors and gradual evolution although Scotland has undergone radical changes in approach. No elements of universal developmental assessment programmes meet World Health Organization screening criteria, although some assessments are configured as screening activities. The roles of doctors and nurses vary greatly by country as do the timing, content and likely costs of programmes. Inter-professional communication presents challenges to all the studied health services. No programme has evidence for improved health outcomes or cost effectiveness., Conclusions: Developmental surveillance programmes vary greatly and their structure appears to be driven by historical factors as much as by evidence. Consensus should be reached about which surveillance activities constitute screening, and the predictive validity of these components needs to be established and judged against World Health Organization screening criteria. Costs and consequences of specific programmes should be assessed, and the issue of inter-professional communication about children at remediable developmental risk should be prioritised.

Magnetic resonance imaging is considered the "gold standard" technique for quantifying thigh muscle and fat cross-sectional area. We have developed a semi-automated technique to segment seven thigh compartments in persons with spinal cord injury. Thigh magnetic resonance images from 18 men (18-50 years old) with traumatic motor-complete spinal cord injury were analyzed in a blinded fashion using the threshold technique. The cross-sectional area values acquired by thresholding were compared to the manual tracing technique. The percentage errors for thigh circumference were (threshold: 170.71 ± 38.67; manual: 169.45 ± 38.27 cm 2 ) 0.74%, subcutaneous adipose tissue (threshold: 65.99±30.79; manual: 62.68 ± 30.22) 5.2%, whole muscle (threshold: 98.18 ± 20.19; manual: 98.20 ± 20.08 cm2) 0.13%, femoral bone (threshold: 6.53 ± 1.09; manual: 6.53 ± 1.09 cm 2 ) 0.64%, bone marrow fat (threshold: 3.12 ± 1.12; manual: 3.1 ± 1.11 cm 2 ) 0.36%, knee extensor (threshold: 43.98 ± 7.66; manual: 44.61 ± 7.81 cm 2 ) 1.78% and % intramuscular fat (threshold: 10.45 ± 4.29; manual: 10.92 ± 8.35%) 0.47%. Collectively, these results suggest that the threshold technique provided a robust accuracy in measuring the seven main thigh compartments, while greatly reducing the analysis time., Competing Interests: None

DNA methylation is the most characterized epigenetic process exhibiting stochastic variation across different tissues and individuals. In non-invasive prenatal testing (NIPT) fetal specific methylated regions can potentially be used as biomarkers for the accurate detection of fetal aneuploidies. The aim of this study was the investigation of inter-individual methylation variability of previously reported fetal-specific markers and their implementation towards the development of a novel NIPT assay for the detection of trisomies 13, 18, and 21. Methylated DNA Immunoprecipitation (MeDIP) combined with in-solution targeted enrichment followed by NGS was performed in 29 CVS and 27 female plasma samples to assess inter-individual methylation variability of 331 fetal-specific differentially methylated regions (DMRs). The same approach was implemented for the NIPT of trisomies 13, 18 and 21 using spiked-in (n = 6) and pregnancy samples (n = 44), including one trisomy 13, one trisomy 18 and four trisomy 21. Despite the variability of DMRs, CVS samples showed statistically significant hypermethylation (p<2e-16) compared to plasma samples. Importantly, our assay correctly classified all euploid and aneuploid cases without any false positive results (n = 44). This work provides the starting point for the development of a NIPT assay based on a robust set of fetal specific biomarkers for the detection of fetal aneuploidies. Furthermore, the assay's targeted nature significantly reduces the analysis cost per sample while providing high read depth at regions of interest increasing significantly its accuracy., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: MI, GK, CL, PM, AA, KT, EK, PCP, SK are employed by NIPD Genetics LTD. PCP has filed a PCT patent application for the MeDIP real time qPCR based NIPD approach (PCT Patent Application No. PCT/1B2011/000217). The authors AK, MHD, MN and CS declare that they have no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Purpose: Problem gambling in adolescents has recently emerged as a pressing public health concern. In this context and in light of the pervasive financial crisis in Greece, the present study aimed to explore adolescents' gambling involvement in Athens region to estimate the prevalence of its problematic form and to identify its risk/protective factors., Methods: A total of 2141 students were recruited from a representative sample of 51 schools located in greater Athens area. The presence of problem gambling was assessed through the use of the DSM-IV-MR-J questionnaire. Data were collected in the form of a self-reported questionnaire during one school hour., Results: Results indicate that 1-year prevalence of high severity problem gambling was found to be 5.6%. Regarding the risk factors for problem gambling; male gender, parental engagement with gambling activities, living without the parents, low grades at school, foreign nationality and the referent absence of availability of food in the household, increased the risk of suffering from the disorder., Conclusion: Gambling behavior among adolescents constitutes a problem in Greece and highlights the need for designing and implementing appropriate preventive interventions, especially amid the ongoing financial crisis.

Noninvasive prenatal testing (NIPT) using whole genome and targeted sequencing has become increasingly accepted for clinical detection of Trisomy 21 and sex chromosome aneuploidies. Few studies have shown that sub-chromosomal deletions or duplications associated with genetic syndromes can also be detected in the fetus noninvasively. There are still limitations on these methodologies such as the detection of variants of unknown clinical significance, high number of false positives, and difficulties to detect small aberrations. We utilized a recently developed targeted sequencing approach for the development of a NIPT assay, for large and small size deletions/duplications, which overcomes these existing limitations. Artificial pregnancies with microdeletion/microduplication syndromes were created by spiking DNA from affected samples into cell free DNA (cfDNA) from non-pregnant samples. Unaffected spiked samples and normal pregnancies were used as controls. Target Capture Sequences (TACS) for seven syndromes were designed and utilized for targeted capture enrichment followed by sequencing. Data was analyzed using a statistical pipeline to identify deletions or duplications on targeted regions. Following the assay development a proof of concept study using 33 normal pregnancies, 21 artificial affected and 17 artificial unaffected pregnancies was carried out to test the sensitivity and specificity of the assay. All 21 abnormal spiked-in samples were correctly classified as subchromosomal aneuploidies while the 33 normal pregnancies or 17 normal spiked-in samples resulted in a false positive result. We have developed an NIPT assay for the detection of sub-chromosomal deletions and duplications using the targeted capture enrichment technology. This assay demonstrates high accuracy, high read depth of the genomic region of interest, and can identify deletions/duplications as small as 0.5 Mb. NIPT of fetal microdeletion/microduplication syndromes can be of enormous benefit in the management of pregnancies at risk both for prospective parents and health care providers., Competing Interests: PCP, GK, EK, KT, PM, CL, AA, and MI are employees of NIPD Genetics. PCP, GK, EK, KT, PM, and AA hold stocks in NIPD Genetics. PCP, GK, EK, KT, PM, and AA have filed a patent (Provisional Application No.15/160,981). This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. NIPD Genetics has launched VERACITY, a non-invasive prenatal test for the detection of fetal chromosomal aneuploidies. The authors MCN, AK, and CS declare that they have no competing interests.

DNA methylation is an epigenetic marker that has been shown to vary significantly across different tissues. Taking advantage of the methylation differences between placenta-derived cell-free DNA and maternal blood, several groups employed different approaches for the discovery of fetal-specific biomarkers. The aim of this study was to analyse whole-genome fetal and maternal methylomes in order to identify and confirm the presence of differentially methylated regions (DMRs). We have initially utilized methylated DNA immunoprecipitation (MeDIP) and next-generation sequencing (NGS) to identify genome-wide DMRs between chorionic villus sampling (CVS) and female non-pregnant plasma (PL) and peripheral blood (WBF) samples. Next, using specific criteria, 331 fetal-specific DMRs were selected and confirmed in eight CVS, eight WBF and eight PL samples by combining MeDIP and in-solution targeted enrichment followed by NGS. Results showed higher enrichment in CVS samples as compared to both WBF and PL samples, confirming the distinct methylation levels between fetal and maternal DNA for the selected DMRs. We have successfully implemented a novel approach for the discovery and confirmation of a significant number of fetal-specific DMRs by combining for the first time MeDIP and in-solution targeted enrichment followed by NGS. The implementation of this double-enrichment approach is highly efficient and enables the detailed analysis of multiple DMRs by targeted NGS. Also, this is, to our knowledge, the first reported application of MeDIP on plasma samples, which leverages the implementation of our enrichment methodology in the detection of fetal abnormalities in maternal plasma.

Pediatric T-ALL patients have a worse outcome compared to BCP-ALL patients and they could benefit from new prognostic marker identification. Alteration of CRLF2 gene, a hallmark correlated with poor outcome in BCP-ALL, has not been reported in T-ALL.We analyzed CRLF2 expression in 212 T-ALL pediatric patients enrolled in AIEOP-BFM ALL2000 study in Italian and German centers.Seventeen out of 120 (14.2%) Italian patients presented CRLF2 mRNA expression 5 times higher than the median (CRLF2-high); they had a significantly inferior event-free survival (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and overall survival (47.1%±12.1 vs. 73.8%±4.3, p=0.009) and an increased cumulative incidence of relapse/resistance (52.9%±12.1 vs. 26.2%±4.3, p=0.007) compared to CRLF2-low patients. The prognostic value of CRLF2 over-expression was validated in the German cohort. Of note, CRLF2 over-expression was associated with poor prognosis in the high risk (HR) subgroup where CRLF2-high patients were more frequently allocated.Interestingly, although in T-ALL CRLF2 protein was localized mainly in the cytoplasm, in CRLF2-high blasts we found a trend towards a stronger TSLP-induced pSTAT5 response, sensitive to the JAK inhibitor Ruxolitinib.In conclusion, CRLF2 over-expression is a poor prognostic marker identifying a subset of HR T-ALL patients that could benefit from alternative therapy, potentially targeting the CRLF2 pathway.

Purpose: Exosomes deliver signals to target cells and could thus be exploited as an innovative therapeutic tool. We investigated the ability of membrane TRAIL-armed exosomes to deliver proapoptotic signals to cancer cells and mediate growth inhibition in different tumor models., Experimental Methods and Results: K562 cells, transduced with lentiviral human membrane TRAIL, were used for the production of TRAIL(+) exosomes, which were studied by nanoparticle tracking analysis, cytofluorimetry, immunoelectronmicroscopy, Western blot, and ELISA. In vitro, TRAIL(+) exosomes induced more pronounced apoptosis (detected by Annexin V/propidium iodide and activated caspase-3) in TRAIL-death receptor (DR)5(+) cells (SUDHL4 lymphoma and INT12 melanoma), with respect to the DR5(-)DR4(+)KMS11 multiple myeloma. Intratumor injection of TRAIL(+) exosomes, but not mock exosomes, induced growth inhibition of SUDHL4 (68%) and INT12 (51%), and necrosis in KMS11 tumors. After rapid blood clearance, systemically administered TRAIL(+) exosomes accumulated in the liver, lungs, and spleen and homed to the tumor site, leading to a significant reduction of tumor growth (58%) in SUDHL4-bearing mice. The treatment of INT12-bearing animals promoted tumor necrosis and a not statistically significant tumor volume reduction. In KMS11-bearing mice, despite massive perivascular necrosis, no significant tumor growth inhibition was detected., Conclusions: TRAIL-armed exosomes can induce apoptosis in cancer cells and control tumor progression in vivo Therapeutic efficacy was particularly evident in intratumor setting, while depended on tumor model upon systemic administration. Thanks to their ability to deliver multiple signals, exosomes thus represent a promising therapeutic tool in cancer. Clin Cancer Res; 22(14); 3499-512. ©2016 AACR., (©2016 American Association for Cancer Research.)

We extend a spatially explicit agent based model (ABM) developed previously to investigate entrainment and control of the emergent behavior of a population of synchronized oscillating cells in a microfluidic chamber. Unlike most of the work in models of control of cellular systems which focus on temporal changes, we model individual cells with spatial dependencies which may contribute to certain behavioral responses. We use the model to investigate the response of both open loop and closed loop strategies, such as proportional control (P-control), proportional-integral control (PI-control) and proportional-integral-derivative control (PID-control), to heterogeinities and growth in the cell population, variations of the control parameters and spatial effects such as diffusion in the spatially explicit setting of a microfluidic chamber setup. We show that, as expected from the theory of phase locking in dynamical systems, open loop control can only entrain the cell population in a subset of forcing periods, with a wide variety of dynamical behaviors obtained outside these regions of entrainment. Closed-loop control is shown instead to guarantee entrainment in a much wider region of control parameter space although presenting limitations when the population size increases over a certain threshold. In silico tracking experiments are also performed to validate the ability of classical control approaches to achieve other reference behaviors such as a desired constant output or a linearly varying one. All simulations are carried out in BSim, an advanced agent-based simulator of microbial population which is here extended ad hoc to include the effects of control strategies acting onto the population.

Background: There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT)., Methods: We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing., Results: Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%-100%) cases of trisomy 21, 16/16 (95% CI, 79.4%-100%) cases of trisomy 18, 5/5 (95% CI, 47.8%-100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%-100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%-100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction., Conclusions: The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT., (© 2016 American Association for Clinical Chemistry.)

Introduction: Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity., Methods: Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood., Results: The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood., Conclusion: Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.

By means of morphological and biochemical criteria, we here provide evidence for the localization and function of premature ovarian failure, 1B (POF1B) in desmosomes. In monolayers of Caco-2 intestinal cells and in stratified HaCaT keratinocytes, endogenous POF1B colocalized with desmoplakin at desmosome plaques and in cytoplasmic particles aligned along intermediate filaments (IFs). POF1B predominantly co-fractionated with desmosomes and IF components and exhibited properties characteristic of desmosomes (i.e., detergent insolubility and calcium independence). The role of NH2 and COOH domains in the association of POF1B with desmosomes and IFs was revealed by transient expression of the truncated protein in Caco-2 cells and in cells lacking desmosomes. The function of POF1B in desmosomes was investigated in HaCaT keratinocytes stably downregulated for POF1B expression. Transmission electron microscopy analysis revealed a decrease in desmosome number and size, and desmosomes of the downregulated keratinocytes displayed weak electron-dense plaques. Desmosome alterations were associated with defects in cell adhesion, as revealed by the reduced resistance to mechanical stress in the dispase fragmentation assay. Moreover, desmosome localization of POF1B was restricted to granular layers in human healthy epidermis, whereas it largely increased in hyperproliferative human skin diseases, thus demonstrating the localization of POF1B also in desmosomes of multistratified epithelia.

Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼ 20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies., (© 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Unlabelled: Although the ETV6-RUNX1 fusion is a frequent initiating event in childhood leukemia, its role in leukemogenesis is only partly understood. The main impact of the fusion itself is to generate and sustain a clone of clinically silent preleukemic B-cell progenitors (BCP). Additional oncogenic hits, occurring even several years later, are required for overt disease. The understanding of the features and interactions of ETV6-RUNX1-positive cells during this "latency" period may explain how these silent cells can persist and whether they could be prone to additional genetic changes. In this study, two in vitro murine models were used to investigate whether ETV6-RUNX1 alters the cellular adhesion and migration properties of BCP. ETV6-RUNX1-expressing cells showed a significant defect in the chemotactic response to CXCL12, caused by a block in CXCR4 signaling, as demonstrated by inhibition of CXCL12-associated calcium flux and lack of ERK phosphorylation. Moreover, the induction of ETV6-RUNX1 caused changes in the expression of cell-surface adhesion molecules. The expression of genes regulating the cytoskeleton was also affected, resulting in a block of CDC42 signaling. The abnormalities described here could alter the interaction of ETV6-RUNX1 preleukemic BCP with the microenvironment and contribute to the pathogenesis of the disease., Implications: Alterations in the expression of cytoskeletal regulatory genes and migration properties of BCP represent early events in the evolution of the disease, from the preleukemic phase to the clinical onset, and suggest new strategies for effective eradication of leukemia., (©2014 American Association for Cancer Research.)

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.

Purpose: Maximum tolerated dose, safety, pharmacokinetics, and pharmacodynamics were assessed in this phase 1 study of PNT2258, a BCL-2-targeted liposomal formulation of a 24-base DNA oligonucleotide called PNT100., Methods: Patients with malignant solid tumors were assigned sequentially to one of ten dose-escalation cohorts of PNT2258 at 1, 2, 4, 8, 16, 32, 64, 85, 113, and 150 mg/m(2) administered intravenously on days 1 through 5 of each 21-day cycle. Pharmacokinetics were determined on days 1 and 5 of the first cycle. Lymphocyte and platelets concentrations were measured for evidence of BCL2-targeted effect. CT scans were used to identify radiologic evidence of anti-tumor effect., Results: Twenty-two subjects received PNT2258, and the maximum tolerated dose for PNT2258 was not reached. Doses at or above 32 mg/m(2) resulted in exposure to PNT2258 above the exposure level required for anti-tumor activity in preclinical xenograft testing of 22,377 ng h/ml (PK analysis 2012). Fatigue was the most commonly reported adverse event. Dose-limiting toxicity, manifesting as a transient increase in aspartate aminotransferase, occurred at 150 mg/m(2), the highest dose tested. Four subjects, two each with diagnosis of non-small-cell lung cancer and sarcoma, treated at doses of 64 mg/m(2) or higher, remained on study for 5-8 cycles., Conclusions: PNT2258 was safe and well tolerated at the doses tested up to 150 mg/m(2). Exposure to PNT2258 resulted in clinically manageable decreases in lymphocyte and platelet concentrations.

Renal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is increasing. There are no standard biomarkers currently used in the clinical management of patients with renal cell carcinoma. A promising strategy for new biomarker detection is comparative proteomics of urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, enriched in renal proteins and excluding high-abundance plasmatic proteins, such as albumin. Aim of the work is to establish the protein profile of exosomes isolated from urines of RCC patient compared with control subjects. We enrolled 29 clear cell RCC patients and 23 control healthy subjects (CTRL), age and sex-matched, for urine collection and vesicle isolation by differential centrifugation. Such vesicles were morphologically and biochemically characterized and proved to share exosome properties. Proteomic analysis, performed on 9 urinary exosome (UE) pooled samples by gel based digestion followed by LC-MS/MS, led to the identification of 261 proteins from CTRL subject UE and 186 from RCC patient UE, and demonstrated that most of the identified proteins are membrane associated or cytoplasmic. Moreover, about a half of identified proteins are not shared between RCC and control UE. Starting from these observations, and from the literature, we selected a panel of 10 proteins, whose UE differential content was subjected to immunoblotting validation. Results show for the first time that RCC UE protein content is substantially and reproducibly different from control UE, and that these differences may provide clues for new RCC biomarker discovery.

Population-level measurements of phenotypic behaviour in biological systems may not necessarily reflect individual cell behaviour. To assess qualitative changes in the behaviour of a single cell, when alone and when part of a community, we developed an agent-based model describing the metabolic states of a population of quorum-coupled cells. The modelling is motivated by published experimental work of a synthetic genetic regulatory network (GRN) used in Escherichia coli cells that exhibit oscillatory behaviour across the population. To decipher the mechanisms underlying oscillations in the system, we investigate the behaviour of the model via numerical simulation and bifurcation analysis. In particular, we study the effect of an increase in population size as well as the spatio-temporal behaviour of the model. Our results demonstrate that oscillations are possible only in the presence of a high concentration of the coupling chemical and are due to a time scale separation in key regulatory components of the system. The model suggests that the population establishes oscillatory behaviour as the system's preferred stable state. This is achieved via an effective increase in coupling across the population. We conclude that population effects in GRN design need to be taken into consideration and be part of the design process. This is important in planning intervention strategies or designing specific cell behaviours.

Background: Periostin acts as an adhesion molecule during bone formation. Knowledge of its expression in kidney injury is scant., Methods: We investigated periostin function and expression in vivo in Sprague-Dawley rats after 5/6 nephrectomy (Nx), in DBA2J mice with streptozotocin-induced diabetic nephropathy (SZ-DN) and unilateral ureteral obstruction (UUO) and in vitro in mouse distal collecting tubular cells (MDCT) and in tissue and urine from chronic kidney disease (CKD) patients., Results: Periostin messenger RNA was increased after 5/6Nx and SZ-DN demonstrating generalizability of the increment in renal injury. Periostin was expressed predominantly in distal tubule (DT) epithelial cell cytoplasm in situ, in cells shed into the lumen, and, in lesser abundance, in glomeruli undergoing obsolescence, arterioles and in the tubulointerstitium in extracellular and intracellular locations. In affected DT after 5/6Nx, periostin expression appeared de novo, E-cadherin became undetectable and tubule cells displayed the mesenchymal marker proteins fibroblast-specific protein-1 (FSP1) and matrix metalloproteinase-9 (MMP9). Periostin overexpression in cultured MDCT cells dramatically induced MMP9 and FSP1 protein and suppressed E-cadherin. Periostin short interfering RNA blocked these changes. Urine periostin excretion increased over time after 5/6Nx, and it was also excreted in the urine of CKD patients. Urine periostin enzyme-linked immunosorbent assay at a cutoff of 32.66 pg/mg creatinine demonstrated sensitivity and specificity for distinguishing patients with CKD from healthy people (92.3 and 95.0%, respectively) comparing favorably with urine neutrophil gelatinase-associated lipocalin., Conclusion: These data demonstrate that periostin is a mediator and marker of tubular dedifferentiation and a promising tissue and urine biomarker for kidney injury in experimental models and in clinical renal disease.

Context: Dopamine agonists (DA) are the first choice treatment of prolactinomas. However, a subset of patients is resistant to DA, due to undefined dopamine D2 receptor (D2R) alterations. Recently, D2R was found to associate with filamin-A (FLNA), a widely expressed cytoskeleton protein with scaffolding properties, in melanoma and neuronal cells., Objective: The aim of the study was to investigate the role of FLNA in D2R expression and signaling in human tumorous lactotrophs and rat MMQ and GH3 cells., Design: We analyzed FLNA expression in a series of prolactinomas by immunohistochemistry and Western blotting. We performed FLNA silencing or transfection experiments in cultured cells from DA-sensitive or -resistant prolactinomas and in MMQ and GH3 cells, followed by analysis of D2R expression and signaling., Results: We demonstrated reduced FLNA and D2R expression in DA-resistant tumors. The crucial role of FLNA on D2R was demonstrated by experiments showing that: 1) FLNA silencing in DA-sensitive prolactinomas resulted in 60% reduction of D2R expression and abrogation of DA-induced inhibition of prolactin release and antiproliferative signals, these results being replicated in MMQ cells that endogenously express FLNA and D2R; and 2) FLNA overexpression in DA-resistant prolactinomas restored D2R expression and prolactin responsiveness to DA, whereas this manipulation was ineffective in GH3 cells that express FLNA but not D2R. No alteration in FLNA promoter methylation was detected, ruling out the occurrence of epigenetic FLNA silencing in DA-resistant prolactinomas., Conclusions: These data indicate that FLNA is crucial for D2R expression and signaling in lactotrophs, suggesting that the impaired response to DA may be related to the reduction of FLNA expression in DA-resistant prolactinomas.

Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness., (Copyright © 2011 Wiley Periodicals, Inc.)

Gonadotropin-releasing hormone (GnRH) mediates control of reproduction. It is secreted in pulses and acts via intracellular effectors to activate gonadotrophin secretion and gene expression. Sub-maximal GnRH pulse frequency can elicit maximal responses, yielding bell-shaped frequency-response curves characteristic of genuine frequency decoders. GnRH frequency decoding is therapeutically important (pulsatile GnRH can drive ovulation in assisted reproduction whereas sustained activation can treat breast and prostate cancers), but the mechanisms are unknown. Here, we consider the possibility that it is due to convergence of distinct pulsatile signals at the transcriptome. We develop a model that mirrors wet-laboratory data for activation and nuclear translocation of GnRH effectors (extracellular signal regulated kinase and nuclear factors of activated T-cells) and incorporates transcription. The model predicts genuine frequency decoding when two transcription factors (TFs) converge at a cooperative gate, and shows how optimal pulse frequency could reflect TF activation kinetics and affinities. Importantly, this behaviour is revealed as an emergent feature of the network, rather than an intrinsic feature of a given protein or pathway, and since such network topology is extremely common, may well be widespread in biological systems.