Uncertainty exists on the prevalence of glucose intolerance in women with a recent diagnosis of gestational diabetes (GDM) based on a two-step screening strategy and the 2013 World Health Organization (WHO) criteria. Our aim was to evaluate the uptake of postpartum screening, the prevalence and the risk factors for glucose intolerance in women with a recent history of GDM.Retrospective analysis of the medical records of women with a recent history of GDM diagnosed in a universal two-step screening strategy with the 2013 WHO criteria. All women with a history of GDM are advised to undergo a 75g oral glucose tolerance test (OGTT) around 12 weeks postpartum. Indices of insulin sensitivity  and an index of beta-cell function, the Insulin Secretion-Sensitivity Index-2 (ISSI-2) were calculated based on the OGTT postpartum. Multivariable logistic regression was used to adjust for confounders such as age, BMI, ethnicity and breastfeeding.Of the 191 women with GDM, 29.3% (56) did not attend the scheduled postpartum OGTT. These women had a higher BMI , were more often from an ethnic minority (EM) background  and smoked more often during pregnancy  than women who attended the OGTT postpartum. Of all women (135) who received an OGTT postpartum, 42.2% (57) had prediabetes  and 1.5% (2) had overt diabetes. Compared to women with a normal OGTT postpartum, women with glucose intolerance were older , were more often obese , were more often from an EM background , less often breastfed  and had more often an abnormal fasting glycaemia at the time of the OGTT in pregnancy . In the multivariable logistic regression, an EM background  and the HbA1c level at the time of the OGTT in pregnancy  remained significant predictors for glucose intolerance postpartum. Women with glucose intolerance postpartum had a similar insulin sensitivity  but a lower beta-cell function compared to women with a normal OGTT postpartum, remaining significant after adjustment for confounders .Glucose intolerance is very frequent in early postpartum in women with GDM based on the 2013 WHO criteria in a two-step screening strategy and these women have an impaired beta-cell function. Nearly one third of women did not attend the scheduled OGTT postpartum and these women have an adverse risk profile. More efforts are needed to engage and stimulate women with GDM to attend the postpartum OGTT. Gestational diabetes (GDM) was historically defined as ‘any degree of glucose intolerance with onset or first recognition during pregnancy’ . The incRetrospective analysis of the electronic medical records of the University Hospital UZ Leuven in Belgium, from 01-03-2014 till 08-02-2016 of women with a recent history of GDM diagnosed with the 2013 WHO criteria. The study was approved by the Institutional Review Board of UZ Leuven (S58329). The study was conducted according to the principles expressed in the Declaration of Helsinki. Due to the retrospective nature of the study there was no need for informed consent from the participants  as in compliance with the Belgian Law of December 8,1992 on the protection of privacy and the Belgian Law of August 22, 2002 on the rights of the patient.Approximately 2400 women are delivered annually at our hospital. The background prevalence of T2DM in Belgium is 6.5% . In the Until March 2014, a 100g 3-h OGTT with the Carpenter and Coustan criteria was used for the diagnosis of GDM in our hospital. Since March 2014, we use a 2-h 75g OGTT with the 2013 WHO criteria for the diagnosis of GDM. However, we continued to use a universal two-step screening strategy for GDM in our hospital with a non-fasting 50g glucose challenge test (GCT)  between 24–28 weeks of pregnancy. Those testing positive [threshold after 1-h ≥ 140 mg/dl (7.8 mmol/l)] had a 2-h 75-g OGTT in the hospital at 24–28 gestational weeks using the 2013 WHO criteria for GDM  . Women wIn our center women with a previous diagnosis of GDM are recommended to receive a 2 hour 75g OGTT three months after the delivery. Women receive an appointment for the postpartum OGTT at the delivery and a reminder is send with a SMS text message using an automatic electronic system within two weeks before the scheduled appointment. If women do not attend the scheduled postpartum OGTT, the diabetes nurse will try to reach women once more by telephone to make a new appointment. As part of the normal routine in our hospital, the OGTT is generally postponed to max. 6 months postpartum If women are still breastfeeding at 3 months postpartum.2), obesity (BMI ≥30 Kg/m2), weight gain , ethnicity, parity, smoking and alcohol habits, family history of diabetes, history of GDM, history of polycystic ovary syndrome, hypertension, dyslipidemia and glucose intolerance before pregnancy. Excessive weight gain was defined according to the most recent Institute of Medicine (IOM) guidelines, except for obese women for whom we use the recommendation to gain ≤ 5Kg during pregnancy , preterm delivery (<37 weeks of gestation) and cesarean section (planned + emergency sections combined). The following neonatal outcomes were recorded: birth weight, macrosomia (birth weight > 4kg), large-for-gestational age infants , small-for-gestational age infants , shoulder dystocia, Apgar score < 7 at five minutes and admission at the neonatal intensive care unit (NICU).To define glucose intolerance postpartum, the criteria of the American Diabetes Association were used: diabetes was defined as FPG ≥126mg/dl (7.0mmol/l) and/or 2-hour glycaemia at the OGTT postpartum ≥200mg/dl (11.1mmol/l)] and prediabetes was defined as impaired fasting glucose (IFG) with a FPG ≥100-125mg/dl (5.5–6.9mmol/l) and/or impaired glucose tolerance (IGT) with a 2-hour glycaemia at the OGTT postpartum ≥140-199mg/dl (7.8–11.0mmol/l)  at the time of the OGTT . In the Compared to women with a normal OGTT postpartum, women with glucose intolerance postpartum had a similar insulin sensitivity but a lower beta-cell function, remaining significant after adjustment for confounders  .Compared to women with IGT postpartum, women with IFG, were more often obese , had less often a history of GDM , had a lower GCT value and a higher fasting but lower 2 hour value at the OGTT in pregnancy . There wThere is uncertainty about how detection of GDM using the 2013 WHO criteria influences the longer term risk of developing T2DM in women. The use of the 2013 WHO criteria for GDM results in a greater proportion of women diagnosed with mild forms of GDM which might presumably lead to a lower proportion at risk for postpartum glucose intolerance. Previous studies have evaluated the risk of postpartum glucose intolerance in women with a diagnosis of GDM based on a universal one-step approach with a 75g OGTT and the 2013 WHO criteria . The useOur study shows much higher rates of glucose intolerance in early postpartum compared to studies in Irish women where a rate of prediabetes and diabetes of 19% was reported in women with GDM based on the 2013 WHO criteria in the first year postpartum and this increased to 25% in the same cohort when re-screened up to 5 years post-delivery . This maOf all women with prediabetes, 57.9% had IGT, 28.1% had IFG and 14.0% had IFG and IGT combined. A FPG alone postpartum would therefore have missed the majority of women with glucose intolerance, confirming the need for an OGTT in early postpartum in our population. This is in contrast with the most recent British guidelines of the ‘National Institute for Health and Care Excellence’ (NICE) stating that a 75g OGTT should not be routinely offered for women with a history of GDM and instead recommending the use of a fasting glucose in early postpartum ,29.The most important risk factors to develop glucose intolerance in early postpartum differ according to the populations studied . The mosWe also show that compared to women with IGT postpartum, women with IFG are more often obese and have a higher FPG at the time of the OGTT in pregnancy. We have previously shown that after a diagnosis of GDM with the Carpenter and Coustan criteria women with IFG in early postpartum, have a lower insulin sensitivity compared to women with IGT and this seems to be largely driven by a higher BMI . In currIn our study nearly one third of women did not attend the scheduled OGTT postpartum. This is higher than the non-attendance rate of 21% we have previously reported in women with GDM based on the Carpenter and Coustan criteria . More efStrengths of the study are the detailed characterization of a relatively large cohort of women with recent GDM based on a two-step screening strategy and the 2013 WHO criteria using a good database. A multivariable regression analysis was used to evaluate independent predictors of glucose intolerance postpartum. Multiple measures of beta-cell function and insulin sensitivity were calculated and adjusted for confounders. A limit of the study is the retrospective nature of the analysis and the lack of longer term data postpartum on the risk for glucose intolerance. We have no data on the HbA1c level postpartum and on weight loss of women postpartum, both possible predictors of glucose intolerance.In conclusion, we show that glucose intolerance is very frequent in early postpartum in women with GDM based on the 2013 WHO criteria in a two-step screening strategy and these women have an impaired beta-cell function. Nearly one third of women did not attend the scheduled OGTT postpartum. More efforts are needed to engage and stimulate women with GDM to attend the postpartum OGTT.


Based on the hypothesis that an imbalance in excitatory and inhibitory input is a central mechanism of knee osteoarthritis chronic pain (KOACP), this exploratory study had the following aims: to compare whether the function of the descending inhibitory pain pathway is associated with the state of inhibition in the corticospinal system indexed by the motor-evoked potential (MEP) and the cortical salient period (CSP) in patients with severe osteoarthritis (OA) and healthy controls; and to determine if there is correlation between the measures of intracortical inhibition  with changes on the numerical pain scale (NPS [0–10]) in KOACP during a conditioned pain modulation (CPM)-task considering the effect of self-reported function assessed by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and analgesic use.In a cross-sectional study, we included females (n = 21), with disability by pain or stiffness due to KOACP and healthy controls (n = 10), aged 19 to 75 years. The motor cortex excitability parameters (MEP and CSP) were assessed using the transcranial magnetic stimulation. We assessed the pain and disability by the WOMAC, and change on NPS (0–10) during CPM-task.P = 0.16). The adjusted mean (SD) on the CSP observed in OA patients was 23.43% lower than in healthy subjects (54.54 [16.10] vs 70.94 [22.87]), respectively (P = 0.01). The function of the descending pain modulatory system assessed by change on NPS (0–10) during a CPM-task was negatively correlated with the cortical excitability parameter indexed by the CSP (P = 0.001). Also, the CSP was negatively correlated with the pain and disability assessed by the WOMAC index.A Multivariate analysis of covariance revealed that the adjusted mean (SD) on the MEP amplitude was 13.53% higher in the OA than in healthy subjects (1.33 [0.49] vs 1.15 [0.13]), respectively (These findings support the hypothesis that the change in cortical plasticity in KOACP is associated with less intracortical inhibition, as measured by the CSP. These results show that the neural change in the motor cortex in KOACP is associated with pain and disability levels, and also with decreased activation of the endogenous pain-modulating system by a CPM-task. Convincing evidence exists to support that the descending modulatory systems in chronic pain are disrupted; shifting from a state of inhibition to a maladaptive state of facilitation.11 Also, functional magnetic resonance imaging (fMRI) studies have demonstrated that compared with healthy subjects, patients with OA demonstrate an increased vigilance and a decreased ability to disengage from pain.12 These changes were associated with abnormal activity in the cingulate cortex, the amygdala, the insula, the nucleus accumbens (NAc), and the prefrontal areas.11Although TKR surgery may produce a complete resolution of pain in a large percentage of patients (up to 73%) within the first 2 to 7 years after surgery,14 trigeminal neuralgia,15 phantom pain,16 chronic migraine,17 low back pain18 and myofascial pain syndrome.19 Also, the M1 is a target that allows us to characterize pathophysiological consequences associated with chronic pain at the motor cortex by neurophysiological measurements made by transcranial magnetic stimulation (TMS).20 Among these parameters, the increase in the motor-evoked potential (MEP) is considered a basic index of corticospinal excitability21 and its amplitude has been observed after painful experiences22 and under experimental pain.23 Also, it has been reported that neuropathic pain leads to a disinhibited state indicated by a shortened cortical silent period (CSP).24 TMS protocols using paired pulse suggests that the inhibition in the motor cortex assessed by CSP tap into different mechanisms. Gamma aminobutyric acid (GABA)-B agonists, such as baclofen, was shown to enhance the CSP.25 Indeed, the early part of the CSP relies on spinal inhibition.26In fact, long-term pain induces cortical reorganization involving the primary motor cortex (M1), which has been a target to assess the cortical excitability and to treat chronic pain conditions. Several studies have shown that M1 stimulation improved pain management outcomes in patients with chronic pain, such as patients with fibromyalgia,Considering that the chronic pain associated with OA reduces quality of life and given the expected exponential increase in the number of primary TKA, a better comprehension of the relationship between the intracortical inhibition and the potency of the descending inhibition system could improve future therapeutic approaches in OA. Thus, based on the hypothesis that an imbalance in excitatory and inhibitory inputs plays a role in the central mechanism of knee osteoarthritis chronic pain (KOACP), this exploratory study had the following aims: to compare whether, in patients with severe OA and healthy controls, the function of the descending inhibitory pain pathway is associated with the state of inhibition in the corticospinal system assessed by both the MEP amplitude and the CSP; and to determine if there is correlation between the measures of intracortical inhibition  with changes on the Numerical Pain Scale (NPS [0–10]) in KOACP during a conditioned pain modulation (CPM)-task considering the effect of self-reported function assessed by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and analgesic use.27 The Ethics Committee at the Hospital de Clínicas de Porto Alegre (HCPA) approved the study (Protocol No. 11–0013). According to the Declaration of Helsinki, all patients provided written informed consent to participate.The “Methods” and “Results” sections are reported according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.4 and experiencing moderate or intense pain or stiffness in the knee. Also, they needed to present functional impairments for at least 6 months that were not controlled with medical therapy.28 The baseline interview included the WOMAC, a validated instrument to assess pain, stiffness, and functional limitations related to OA.29 To be eligible, they could report “moderate,” “severe,” or “extreme” pain or stiffness in response to at least 1 of the 5 pain questions . They needed to report a positive answer for 2 stiffness questions , and also reporting whether they experience “moderate,” “severe,” or “extreme” difficulty with at least 1 of the 17 activities. Additionally, the radiographs of all knees were evaluated for the degree of OA by 1 physiatrist with over 10 years of experience in OA rehabilitation. This was conducted using the Kellgren–Lawrence (K-L) grading scale of 3 to 4,30 because it proved to be highly reproducible to grading severity of knee OA.30 The exclusion criteria were as follows: accompanied orthopedic, rheumatic, or neurological pathologies; surgery on the affected areas in the prior 6 months; habitual use of corticosteroids or other uncompensated chronic pathologies. Additionally, patients were excluded if they had a body mass index (BMI) of >35 m/kg2 or if they had contraindications to TMS.31A cross-sectional study was performed at HCPA, Brazil, between March 2014 and December 2014. Patients were recruited from the general population through public postings in different healthcare units and referrals from physicians in the Psychiatry and Chronic Pain Service at HCPA. Eligibility criteria were designed to study a group of patients who were potentially appropriate candidates for unilateral knee arthroplasty. The sample comprised of 21 right-handed women meeting inclusion criteria of being aged 50 years or older31 The sequence of assessments is presented in Figure Healthy right-handed controls were recruited from the general population using public postings. They were asked to complete screening questionnaires, and were excluded if they were experiencing any painful condition (either acute or chronic); used analgesics or corticosteroids; had any rheumatologic, psychiatric, or neurological disorder; had abused alcohol or psychotropic substances during the 6 months previous to the screening; or if they were using medications with known effects on the central nervous system (CNS). In addition, they were excluded if presented contraindications to TMS.The dependent variables of interest were measurements of the intracortical inhibition indexed by CSP and MEP.The left primary motor cortex (M1) parameters were assessed using the TMS MagProX100 stimulator  through a figure-eight coil (MagVenture Company). It was assessed before the pain pressure threshold assessment. Ag-AgCl electrodes were placed over the first dorsal interosseous (FDI) belly muscle and in its corresponding tendon on the distal phalanx of the index finger. The responses to stimuli were recorded from the FDI muscle of the right hand by surface electromyography (EMG).Each patient was seated in a comfortable chair and informed about the TMS procedure, including possible sensations that might be experienced. The amplitudes of the single and paired pulse TMS, and the latency and the measures of the CSP during the experiment were recorded on an Excel spreadsheet. The data were analyzed offline on a personal computer.32 To ensure constant placement of the coil throughout the TMS assessments, the site was marked with a soft-tipped pen. Firstly, the MT was determined using the lowest stimulus to elicit evoked potentials in the resting FDI, with a minimum amplitude of 50 μV peak-to-peak, in at least 5 of 10 (at least 50%) of successive trials).32To identify the motor “hot spot,” the coil was placed over the left M1 tangentially to the scalp at a 45° angle to the sagittal line. The motor threshold (MT) was defined using the lowest stimulus to induce 50% of the evoked potentials of the resting FDI.33 Ten consecutive trials were recorded. The paired-pulse measurements included the short-interval intracortical inhibition (SICI) with interstimulus intervals of 2 milliseconds and the intracortical facilitation (ICF) with interstimulus intervals of 12 milliseconds.34 To define the individual MT, the first subthreshold stimulus was set at 80%, whereas the supra-threshold stimulus was set at 130% of the MT. The intensity of the supra-threshold test stimuli was adjusted to elicit the test stimuli with peak-to-peak amplitude of approximately 1 mV. At the level of the primary motor cortex, the reduction of the test MEP elicited by TMS is considered to reflect inhibition34 and the increase of the test MEP elicited by TMS is considered to reflect facilitation at the level of the primary motor cortex.21 Thirty recordings  were produced in a random order with an interval of approximately 8 seconds between each pulse. The paired-pulse measurements were analyzed by calculating their individual index .35 These parameters were assessed before and after 2 minutes of rest.36Single-pulse measures including the MEP and the CSP were recorded at an intensity of 130% of the MT. The MEP value was the elicited evoked potential with a 1-mV peak-to-peak amplitude. The mean of 10 consecutive trials were recorded. For the CSP, patients were instructed to perform isometric voluntary contractions with approximately 10% of maximal contraction of the FDI. The transient silence during the isometric voluntary EMG activity was elicited in the tonically contracting FDI muscle at approximately 10% of the maximal voluntary contraction, and the CSP was preceded by the MEP.38 The patients’ depressive symptoms were assessed using the Beck Depression Inventory,39 and sleep quality was assessed using the Pittsburgh Sleep Quality Index.40 Pain catastrophizing was assessed using the Brazilian Portuguese Pain Catastrophizing Scale (BP-PCS).41 A standardized questionnaire was used to assess comorbidities and demographic data. The WOMAC was used to evaluate self-report of knee-specific impairment based on symptoms during the preceding 48 hours. The WOMAC assesses the pain, joint stiffness, and perceived disability associated with OA to determine the overall impact on a patient's perceived function. It comprises 24 questions with responses given to each using a Likert scale. The pain subscale consists of 5 possible responses: no (0), mild (1), moderate (2), severe (3), or extreme (4) problems. This 20-point scale (range of 0–20) assesses pain in everyday situations . The stiffness subscale is a 2-item (range 0–8), 10-point scale that assesses perceived knee stiffness after walking and at the end of the day. The disability subscale is a 17-item assessment of perceived physical function in a variety of everyday situations . The WOMAC is a valid, reliable, and responsive instrument that is commonly used to assess pain and disability in studies of knee OA.29 A total WOMAC score (range 0–96) is calculated by summing the items for all 3 subscales.42All psychological tests used in this study were validated for the Brazilian population.2 (lb/cm2). The PPT was recorded at the site of greatest sensitivity where the device had a 1-cm2 hard-rubber probe applied to the myotome and sclerotome structures at the L1-L5 and S1-S2 dermatomes at the knee with greater pain .To measure the pressure pain threshold (PPT), we asked patients to differentiate the perception of pressure versus the perception of “onset of pain.” The patient was instructed to report the perception of pain onset verbally. A trained investigator assessed the pain threshold. An experienced rehabilitation physician (MGT) systematically evaluated superficial and deep hyperalgesia by assessing the PPT using an electronic algometer . Three successive readings taken at intervals of 3 to 5 minutes were used to define the PPT in kgf/cm43 protocol, and the experimental pain stimulus used was in accordance to the guidelines for the cold-pressor task (CPM-task).44 The CPM-task is a strong nociceptive stimulus applied over a large body surface area44 that takes place over a lengthy time span.45 The CPM-task allows us to modify the descendent pain modulatory system.To test the CPM, we used the Tousignant-Laflamme et alTo assess the CPM-task, the patient immersed the nondominant hand in cold water (0–1°C) for 1 minute. During the last 30 seconds of the cold-water immersion, the PPT procedure was administered to the dominant forearm. During the entire experiment, the cold-water temperature was maintained constant. The PPT that elicited pain ratings of 6/10 on the NPS (0–10) (PPT60) was used for the first PPT before the CPM-task (PPT0). After a short break, the PPT0 was applied at the S1-S2 dermatome at the knee of the leg with higher hyperalgesia. After PPT0, the CPM-task was used to trigger the CPM. One minute after the CPM-task, we applied the second PPT (PPT1). To quantify the CPM, the mean pain rating of PPT1 was subtracted from the first PPT0 before the CPM-task (PPT1); negative values indicate inhibitory CPM.Analgesic use was defined by an average of analgesics used per week during the previous month. For data analysis, analgesic use was included as a dichotomous variable . This approach was chosen because patients with chronic pain rescue analgesic use changes each week, depending on their level of pain.To reduce assessment bias, only 1 researcher (MGT) was involved in all of the assessments. The evaluator (MGT) is a practicing physiatrist of the outpatient clinic at HCPA with vast clinical expertise, who is well trained to make the TMS measures. Also, the evaluator was trained to apply clinical scales and PPT assessment, and also in the care of chronic pain patients. In our study, all patients were submitted to a clinical evaluation by the same physician (MGT), who had many years of experience in treating patients with OA, to revise the severity of OA and inclusion criteria. The algometer used to make measurements was manufactured by .f2 = determination coefficient) of 0.4 for the multiple hierarchical regression analysis allowing for 2 predictors .46 A sample of 18 patients was chosen to account for unexpected factors that would decrease the study power such as increased variability of the sample or missing data. A sample of 21 patients would detect an effect size for correlations of 0.4, with a power of 88% at a 0.05 alpha level.The number of patients was estimated based on a type I and type II error of 0.05 and 0.20, respectively, and in anticipation of an effect size  was used to assess the relationship between the dependent variables, the cortical excitability parameters  with the main interest independent variable, and the change on the NPS (0–10) during the CPM-task in patients with OA and healthy subjects. The covariate included in the model was age. We used Bonferroni multiple comparison test to adjust the differences for multiple comparisons.A regression multiple analysis was used to explore the relationship between the change on NPS (0–10) during the CPM-task and cortical excitability parameters (CPS and MEP) in OA patients. This procedure was done to adjust this analysis for potential confounding factors in OA patients, such as analgesic use and disability assessed by the WOMAC index. Also, a regression analysis was used to generate the scatter plot of correlation between CSP and change on NPS (0–10) during the CPM-task. The data were analyzed using SPSS version 22.0 .Twenty-one women with OA participated in this study along with 10 healthy women. The baseline demographics, psychological characteristics, and cortical excitability parameters are shown in Table λ = 0.43, F [4] = 5.08, P < 0.01). The power of this analysis was 0.92%. The adjusted determination coefficient of this model is R2 = 0.47 . This analysis revealed that the function of the descending pain modulatory system, assessed by change on NPS (0–10) during CPM-task, is negatively correlated with the cortical excitability parameter indexed by the CSP (P < 0.05) (Table P > 0.05). The adjusted mean (SD) on the MEP amplitude observed in OA was 13.53% higher than in healthy subjects (1.33 [0.49] vs 1.15 [0.13]), respectively  and their outcomes related to cortical excitability measurements (CSP and MEP amplitude) with the descending pain modulatory system as assessed by the reduction on NPS (0–10) during CPM-task  during the CPM-task Table . Consider) was −0.72  and the coefficient of determination (R2), that is the proportion of the variance explained by the association between the change on NPS (0–10) during CPM-task and the CSP was 52% (R2 = 0.52).An important question is to identify if this result could be explained by other confounding factors. Thus, we run a multiple regression analysis only with the OA patients Table . In thisThis study extends the literature about the correlation between measures of the intracortical disinhibition as indexed by CSP and the dysfunction in the descending control of nociceptive processing as indexed by lower activation of the CPM-task in KOACP. Whereas, a lower CSP indicates that the trigger to induce a membrane potential is lower, thereby, there is less inhibition. A higher value of the change on the NPS (0–10) during the CPM-task indicates that the heterotopic stimulus to induce CPM was less effective. This assignment on descending pain modulatory system may explain the inverse correlation between the CSP with the disability related to pain as assessed by the total WOMAC.47 The intracortical disinhibition state observed in this study is in disagreement with findings of a previous report, which did not find any significant changes in the motor cortical excitability in OA patients compared with healthy subjects.48 It is possible that the severity of OA and the long-term pain level could explain this divergence. Our sample patients were shown to have moderate to severe knee OA, intense chronic pain, and disability during more than 5 years, whereas the above mentioned study noted OA pain in the hands, which carries a mild severity.49Our finding suggests that sustained peripheral inflammation increases the excitability of ascending nociceptive pathways, which induces dysfunction in descending modulatory systems, as assessed by the change on the NPS (0–10) during the CPM-task. At the cortical level, a shorter CSP reflects the decreased excitability of the inhibitory interneurons. This disinhibited state observed in OA, but not in healthy subjects, support the hypothesis that the chronic pain in OA induces a disinhibitory effect at the intracortical level, and also in the descending pain modulatory system. Whereas, the intracortical disinhibition is in agreement with what has previously been described in neuropathic and fibromyalgia pain.50 it is conceivable that sustained pain—in this case, triggered by peripheral inflammation—could lead to a cascade of events resulting in dysfunctional inhibitory function. Part of this defective cortical inhibition could be explained by recent evidence showing decreased gray matter volume associated with chronic pain syndromes, including OA.51 Central reorganization in chronic pain seems to lead to an inhibitory state that might facilitate activation in nonpain networks, such as the primary motor cortex. Thus, disturbances in the GABAergic and glutamatergic intracortical networks might explain the disinhibition found in KOACP.In the present study, higher disability was inversely correlated with the CSP. Given that the CSP index assesses the function of GABAergic transmission,52 This activation of the GABAergic system governs the state of inhibitory interneurons within M1.52 Also, it is known that the balance between inhibitory and excitatory systems is influenced by age. Older subjects present a slower motor response and a decline in the modulation of the corticospinal activity system.53 Whereas this association is consistent in healthy subjects,54 our results suggest that chronic pain leads to changes in favor of excitability, as demonstrated by a shorter CSP. Also, the adjusted analysis shows that age is intrinsically associated with slower intracortical excitability, because the direction of correlation between the CSP and age changed when we analyzed the interaction of age with the group  , a diminished activity of serotonin and norepinephrine.56Thus, changes in cortical plasticity in OA could be explained by a steady pain induced by a peripheral neural lesion, which increases the synaptic efficacy of neural structures involved in pain processing likewise occurring on a neuropathic lesion. The CSP in this study suggests that ongoing nociception from knee-related structures is essential to the chronic nature of this process and the development of sensitization.57In this study, the lower activation of the CPM induced by the CPM-task was correlated negatively with the CSP Figure , which s58; to determine strategies to manage patients with a higher risk of more severe chronic pain after knee arthroplasty, which includes anesthetic and analgesic approaches.4 Also, it improves the understanding of underlying neurophysiological mechanisms of chronic pain in OA, which could give support to plan new neuromodulatory approaches to induce a top down  and bottom-up modulation technique  or pharmacological interventions.The downward negative spiral of pain and central disinhibition has severe clinical consequences, which are as follows: it increases pain and local knee hyperalgesia and it is associated with a loss of cortical inhibitory mechanisms. Although the design of this study prevents determining the deterioration in the central pain modulatory system, it does permit us to better understand the dysfunctional process of disinhibition at cortical and intracortical regions in severe KOACP. Thus, the pieces of evidence these findings possess may hold important clinical implications such as to support an understanding of the bidirectional pathways between peripheral inflammation and central brain changes in OA; to select the best therapeutic approach based on the neurophysiological phase state of each patient, because chronic pain has been associated with unfavorable pain outcomes after knee arthroplasty59 This limitation restricts the possibility of a direct comparison with other studies, but has the advantage of avoiding possible contamination of the data. Because OA is more prevalent in females,60 these results might have greater clinical implications. Although we recruited healthy volunteers to assess the relationship between the cortical inhibitory function and the descendent pain inhibitory system, it is worth noting that our control sample was younger on average.The small sample size is a limitation of this study. The study design is a limitation because it is not possible to determine a causative effect. The use of TMS assesses the neurotransmitter system activity in an indirect manner, and it has been shown to have relatively low specificity. However, TMS is a useful tool for neurophysiological assessment because it induces activity and evaluates the response of the subject. In this study, only females were evaluated, taking into account that sex differences in pain perception and modulation are a controversial topic.These findings support the hypothesis that a change in cortical plasticity in KOACP is associated with less intracortical inhibition, as measured by the CSP. These results show that this neural change in the motor cortex in KOACP is associated with pain and disability levels, and also with decreased activation of the endogenous pain modulating system by the CPM-task.


This report assesses the impact of a series of education sessions delivered to office staff on the delivery of smoking cessation services among patients seeking care at a community-based women’s health center.A quasi-experimental design was used to examine the delivery of smoking cessation services to patients in a medical office before and after office staff attended a series of 3 educational sessions intended to increase their knowledge and self-efficacy to address cessation. Delivery of smoking cessation services was documented through a systematic review of medical records using a structured abstraction form.While nearly all smokers (93%) were asked about smoking status at their last office visit, few smokers at baseline or follow-up were assessed for interest in setting a quit date or offered pharmacotherapy. Referrals to the smokers quit line increased from <1% at baseline to 8% at follow-up (p<0.001) and “any assistance” also showed a modest but significant increase .This evaluation failed to identify clinical meaningful changes in the delivery of smoking cessation services in this women’s health office before and after completion of a series of educational interventions for office staff. It is anticipated that the implementation of patient centered medical homes, and EMR systems, will help to enhance the delivery of smoking cessation services to women seeking medical care. Smoking rates continue to decline with 18.1% of adults age 18 or older identified as currently smoking cigarettes in the United States in 2012, compared to 20.9% of adults who were smokers in 2005 ,4. LifelEducation and income are inversely associated with smoking behaviours. Persons with limited educational attainment  exhibit the highest rates of tobacco use compared to those with associate 17.9%), bachelor (9.1%) and graduate degrees (5.9%). Also, persons living below the poverty level have a smoking rate of 27.9% compared to 17.0% for persons at or above the poverty level [7.9%, bacTobacco cessation counselling increases the likelihood of successfully quitting, more intensive counselling  increases effectiveness by about 2-fold . HoweverIn 2010, 68.8% of US adult smokers reported they would like to completely quit; including 52.4% who made a quit attempt during that year .While all visits provide an occasion to assess smoking status, women’s well visits provide a unique opportunity for physicians and medical office staff to promote smoking cessation since smoking contributes to the persistence of Human Papilloma Virus (HPV) infections which can lead to abnormal pap smears, cervical dysplasia and cervical cancer. The purpose of this report is to assess the impact of a series of education sessions delivered to office staff on the delivery of smoking cessation services among patients seeking care at a community-based women’s health center.This study utilized a quasi-experimental design to examine pre-versus post- intervention assessments of the delivery of smoking cessation services to patients based upon a review of medical records.The study population included staff in the medical office, included 3 advanced practitioners (nurse practitioners and physician assistants) and 5 nursing staff . The intervention consisted of a series of three mandatory educational sessions, delivered monthly over 3 months, to office staff (n=7–8 persons per session) during lunch breaks. The sessions addressed 1) the systematic identification of smokers, 2) use of cessation pharmacotherapy and 3) motivational interviewing/counselling skills; each session last 45–50 minutes. These topics were identified based upon formative research conducted with office staff (unpublished data) as part of on-going community-based collaborative activities.This medical office is located in a medically underserved community within Buffalo, New York. Buffalo ranks as one of poorest cities in the country with a poverty rate of 31.4% compared to a national rate of 14.5% . AfricanBetween 4,500 and 5,000 outpatient visits are completed at this medical office annually. Patients are generally <30 years old (74.8%), non-white , and have Medicaid (49.5%); 8% have no health insurance.Institutional Review Board approval was received for this project. Delivery of smoking cessation services was documented by a systematic review of medical records  was completed in 2012, along with a follow-up chart review in 2013 (n=268) which was approximately 6 months after completion of the educational sessions presented to enhance the knowledge and skills of office staff with regard to smoking cessation. A structured abstraction form was used to collect data on patient demographics, smoking status, and reason for visit. Medical charts for both reviews were available in paper format. Using a random starting point, every 10th chart was reviewed for the baseline review, and every 5th chart was reviewed for the post intervention review. Patients must have been identified as a smoker and have had an office visit within the prior 12 months to be eligible for each review.Chart reviews collected patient demographic information , number of patient visits within the prior 12 months and selected medical data.The main outcome variables included the proportion of smokers with an assessment of smoking status and provision of cessation support including the 5A’s . For smokers who were not yet ready to quit, documentation of the 5Rs was assessed: whether relevance, risks, rewards, roadblocks and/or repetition were used as strategies to encourage smokers to quit.Data analyses was completed using SPSS Version 21 , and included descriptive summaries and a comparison of baseline versus follow-up data using the chi-square statistic. Significance was established at p<0.05 without adjustment for multiple hypothesis testing.As shown in Most smokers had just a single office visit in the past 12 months. A majority of smokers (~94%) were still using tobacco based upon last recorded information. Smoking status was recorded as a vital sign for 75% of smokers at the baseline assessment and for 69% of smokers at the follow-up assessment (p=0.10).Overall, 93% of smokers were asked about smoking status at their last visit based on the baseline and follow-up chart reviews. As shown in This study summarizes results from two medical chart audits performed to assess the impact of a series of 3 educational programs presented to medical office staff to encourage enhanced delivery of cessation services to smokers presenting for care at a women/s health centre. The educational sessions included information on the systematic identification of smokers by assessing smoking status as a vital sign, a review of evidence-based pharmacotherapy to support a quit attempt and use of motivational interviewing techniques to counsel smokers. While office staff reported that the educational sessions were useful, it is possible that additional sessions, including role playing with coaching and feedback, might have served to further reinforce practical approaches and skills for engaging patients in cessation. Also, providing more frequent periodic feedback on the extent of cessation services delivered may help to prioritize this activity.Although the clinical practice guidelines for treating tobacco use and dependence  endorse The medical chart review did identify areas with significant changes between baseline and follow-up: a decrease in the proportion of smokers being advised to quit (from 40% to 22%), an increase in the proportion of smokers assessed for readiness to quit (from 3% to 9%), an increase in the proportion of smokers who were counselled regarding a quit attempt increased (from 12% to 41%) and an increase in referral to the smokers quit line (from <1% to 8%). The decrease in the proportion of smokers advised by a clinician to quit smoking was unanticipated and may reflect limited documentation in the medical charts. The increase in the proportion of smokers assessed for readiness to quit was modest but may serve to establish a foundation for further improvements in the future.The proportion of smokers who were counselled regarding a quit attempt was observed to have increased from 12% to 41%. This observation however, is not consistent with other data points. This change likely reflects a modification in the content of a paper based medical note used to document annual office visits to include a check box if the patient was “counselled to quit smoking”. As such, that check box would appear to be more consistent with providing “advice to quit”, rather than actual counselling support. In this regard, it seems likely that this reflects notations in the medical chart that a smoker was “advised” or “encouraged” to quit smoking rather than action-oriented counselling and/or motivational interviewing, as presented in one of the educational sessions. Also, the provision of pharmacotherapy as part of any quit attempt was quite low. It should be noted that the depth and breadth of counselling support provided to a smoker as part of a quit attempt may be difficulty to fully characterize based upon a chart review.The rate of referral among smokers in this office to the state smokers quit line increased to 8% from a baseline of <1%. While this is a modest actual increase, it is a very large relative increase and is noteworthy as it may reflect a growing awareness of the value of quit lines to work cooperatively with medical offices to promote cessation. State quit lines have been shown to be effective at providing smokers with evidence-based approaches to support quitting including provision of free pharmacotherapy and telephone based counselling support .For smokers who are not yet ready to quit, the Public Health Service guidelines for treating tobacco use and dependence . EncouraCompared to the demographics of the surrounding city and county, patients at this women’s health centre included a disproportionate number of non-whites, mostly African Americans, likely reflecting both the local community demographics and socioeconomic challenges among the patient base who sought medical care at this women’s health center. Smoking rates among Hispanics 12.5%) and Asians 10.7%) are lower than among African-Americans (18.1%), whites 19.7%) and American Indians/Alaska Natives (21.8%) and among persons reporting multiple races (26.1%) [2.5% and .7% and A.7% are lStrengths of this study include the use of a quasi-experimental design to assess intervention impact, and a comprehensive review of a sample of medical records. Limitations includes the reliance upon information as recorded in medical charts; any additional counselling or advice offered to patients that may have occurred and was not recorded on the medical chart was not included. While this office continues to use a paper-based records system conversion to an electronic-based medical records (EMR) system is imminent.Overall, these results suggest a modest impact in the delivery of smoking cessation services to smokers at a women’s health center resulting from a series of 3 education programs presented to office staff. The limited changes may reflect challenges in making changes in process and practice at this office, competing demands of clinical care, a lack of impact from the educational interventions and/or some combination of factors. Implementation of the EMR will likely aid in the systematic identification of smokers as well as enhance the ability to prompt providers to encourage and support quit attempts using the information acquired in the series of educational program.This effort was useful in serving to focus attention on smoking as an important health behaviour and may lead to on-going quality improvement/quality assurance activities on this topic in this office. An unanticipated outcome was the adoption of a smoke-free policy at this medical office. Moreover, it serves as an example of partnership between a community-based medical office and an academic health center coming together to promote smoking cessation.


This pathology is very difficult to treat due to the very long period of growth of the child to the moment of biological maturity and the related risk of progression of the curvature. The aim of treatment in young children with idiopathic scoliosis is to maintain for years the curve at low-angle value in order to make the spine growth possible in the best conditions of minimal asymmetry. Thus, the development of the vicious circle of scoliosis progression3Physiotherapy for the stabilization of idiopathic scoliosis angle in growing children remains controversial. So far, little data on the effectiveness of physiotherapy in children with EOIS were published.4 and in 2010.5 The FITS method comprises 3 stages: examination (stage I), facilitation of correction (stage II), and 3-dimensional (3D) correction (stage III). The results in adolescent patients were previously reported.6 This study aims to assess the early results in children who started therapy before the age of 10 years for EOIS.The Functional Individual Therapy for Scoliosis (FITS method) was introduced by Białek and M’hango, described in 2004, then published in 2008The charts of the patients archived in a prospectively collected database were retrospectively reviewed. The inclusion criteria to this study were as follows: diagnosis of EOIS based on clinical examination and spine radiography, age below 10 years at the beginning of treatment, both girls and boys, Cobb angle between 11° and 30°, Risser zero, FITS therapy, no other treatment in the past or during FITS (like nighttime bracing), and follow-up period minimum 2 years from the initiation of the FITS treatment  Figure .The patients presented curve pattern: A1—single thoracic (5 children), A2—single thoracolumbar (22 children), and A3—double thoracic/thoracolumbar (14 children), totally 55 structural curvatures (Table 7 The X-ray in standing position (not older than 6 months) was measured.All children were examined by the author. The history comprising age of diagnosis, family history and developmental history were investigated. The clinical assessment comprised classical orthopedic examination completed with detailed examination performed according to FITS method principles.The classical examination included:distance from plumb line to: anal cleft, apex of primary curve, apex of secondary curve, and left and right scapula edge Figure ;checking scapulas level with scoliometer Figure ; andmeasurement of the angle of trunk rotation at thoracic Figure  and at lexam for the leg length inequality;assessment of the lower limbs in standing and gait;observation of type and location of compensation;assessment of possibilities for scoliosis correction in standing Figures  and 8 anassessment of the length of muscles in lower limbs and pelvis particularly involved in postural asymmetries in young children, namely adductor longus, adductor magnus, and hamstrings Figures –13.The FITS examination included additionally:Although testing possibility of scoliosis correction by making corrective movement, the therapist is able to feel which myofascial structures should be addressed first. In order to indicate the direction of therapy and assess effectiveness of therapeutic procedures, we test corrective movement during each session. Corrective movement at the beginning of therapy can be done only in 1 plane: shift, rotation, or flexion/extension. In further stages of therapy 3D corrective movement should be included.The clinical parameters at study baseline are presented in Table 9 trigger points, and10 joint mobilization.12We start with relaxation of structures restricting scoliosis correction by using physiotherapeutic techniques described elsewhere like: contract-relax technique, passive and active myofascial release,13SBL ,DFL (deep front line),LL ,SL , andSFL .These techniques are addressed in the area of myofascial bands described by Myers:14—the stage II of the FITS method could be omitted and the therapist went directly to stage III (3D correction).In case the examination revealed no soft tissue restriction and especially when the child presented with soft tissue laxity and joint hypermobility15  the minor functional compensation appeared—of less than 3–4 degree of rotation. This compensation concerned only soft tissues, not structures seen on X-rays. At this moment changing of patterns for upper extremities should be done in the direction to elongate both sides.During the course of FITS treatment the children received individual treatment twice a month (45–60 minutes) at the beginning period. This therapy was performed by the author herself. Between the individual therapy meetings, the patients performed adequately selected and prescribed set of exercises at home, once a day (30–45 minutes). In cases the FITS educated physiotherapists were accessible at proximity, the patients received individual treatment at their places of residence. The patients were also educated to sit in a correct position during classes at school and at homework. Twice a year, a 2-week in-patient physiotherapy was offered in the form of a winter or summer rehabilitation camp. Five children out of 41 analyzed in this study did participated in at least 1 2-week rehabilitation camp.Sample exercises for scoliosis Thdex and Th/L sin shows figures below Figures –22.At follow-up, all clinical parameters were reassessed and the patients were subjected to X-ray analysis for the Cobb angle. The patients had the X-rays taken in the place where they lived. Cobb angle was measured by the treating physician. The percentage of children in whom the Cobb angle decreased by more than 5°, the percentage of children in whom the Cobb angle was stable during the observation period (angle ± 5°), and the percentage of children in whom the Cobb angle increased by more than 5° were calculated at follow-up.The minimum follow-up was 2 years after initiation of the FITS treatment, the maximum was 16 years, mean 4.8 years. At follow-up the mean age was 12.5 ± 3.4 years (range 8–20 years). Out of 41 children, 10 passed pubertal growth spurt at the final follow-up while 31 were still immature and continued FITS therapy. Out of 41 children, 27 improved, 13 were stable and 1 progressed. Out of 55 structural curves, 32 improved, 22 were stable and 1 progressed. Seven of 41 children were lost from observation after 2 years of FITS therapy, and no further data concerning their Cobb angle are available. At the moment these 7 children were lost from the follow-up, their mean age was 10 ± 1, range 9 to 12 years. The values of the measured parameters before FITS therapy and at follow-up are presented in Table The percentages of curve correction, stabilization, and progression at follow-up are presented in Table Example X-ray patient—Aleksandra, who started FITS therapy in 2008, at the age of 8 years and follow-up Table –30.Figures Figures This study aimed to analyze the children with idiopathic scoliosis of early onset below the age of 10 years who were managed with FITS method. All children were treated under control of one physiotherapist (author). All presented a mild angle structural idiopathic scoliosis with clinical signs of the disease, namely the trunk rotation and the trunk imbalance. Functional scoliosis due to pelvic obliquity, postural muscles weakness, or posture maintenance insufficiency were excluded. Radiographs were ordered and examined by doctors, specialists in orthopedics.FITS physiotherapy was performed at regular basis of exercises with physiotherapist. The role of parents in assuring regular at home exercises was extremely important. Surprizingly, many young children easily and rapidly learnt exercises, especially stabilizing ones, better than 3D corrective ones. They learnt more easily in supine position having good contact of the back with the ground. Comparing with adolescents the young children got tired earlier and presented less muscular force, on the other hand their concentration revealed sufficient and often better than that in adolescents.In young children under the age of 10 years the onset and development of idiopathic scoliosis takes place simultaneously with the development of the posture of the child. This is why we can observe the signs of scoliosis together with the signs of postural insufficiency or postural faults. For this reason it seems logical to take into consideration postural aspects when treating children for idiopathic scoliosis. Otherwise, the application of complex 3D active self-correction would be difficult or even impossible to achieve because the child lacks trunk stability and lower limbs correct alignment to be capable of developing corrective movements of the vertebral column.In this series, the Cobb angle ranged from 11° to 30°. From the natural history of mild angle EOIS we do know that many of these curves (up to 50%) are not progressive in the immediate course. They require regular diagnostic visit to rule out progression. This implies the risk of overtreatment of children with expensive, time consuming, and potentially unnecessary treatment. Unfortunately, we are unable to predict the future of a particular child with mild scoliosis. On the other hand, once progression is done, we are unable to reduce the Cobb angle with nonsurgical techniques, which can be considered argument for early treatment.It seems that the clinical examination of the young child with EOIS could exceed the regular evaluation of the trunk deformity approved for scoliosis in order to take into consideration additional parameters, which could measure the trunk muscles  strength, the lower trunk stability, the general physical capacity, and the balance capacities. These parameters are important to ensure effective physiotherapy for EOIS and they are often impaired in young children. In such cases, additional program of physiotherapy directed toward postural improvement, muscles strength improvement, and balance improvement can be justified. We use a term of “good/bad feeling of own body” to shortly describe the above-mentioned factors.Owing to lack of published data on physiotherapy as exclusive treatment for EOIS (bracing excluded) it was difficult to compare the results with other authors.FITS physiotherapy was effective in preventing curve progression in children with EOIS. At follow-up the Cobb angle was stable or improved (1 case progressed), the trunk imbalance was less and the trunk rotation diminished. Final postpubertal follow-up data are needed.


Male and female mice from reciprocal consomic strains originating from the C57BL/6ByJ and 129P3/J strains were bred and measured for body weight, length, and adiposity. Chromosomes 2, 7, and 9 were selected for substitution because previous F2 intercross studies revealed body composition QTLs on these chromosomes. We considered a QTL confirmed if one or both sexes of one or both reciprocal consomic strains differed significantly from the host strain in the expected direction after correction for multiple testing. Using these criteria, we confirmed two of two QTLs for body weight (Bwq5-6), three of three QTLs for body length (Bdln3-5), and three of three QTLs for adiposity . Overall, this study shows that despite the biological complexity of body size and composition, most QTLs for these traits are preserved when transferred to consomic strains; in addition, studying reciprocal consomic strains of both sexes is useful in assessing the robustness of a particular QTL.Genetic variation contributes to individual differences in obesity, but defining the exact relationships between naturally occurring genotypes and their effects on fatness remains elusive. As a step toward positional cloning of previously identified body composition quantitative trait loci (QTLs) from F The rationale for this project was born from challenges in mapping obesity genes. Spontaneously occurring mutations in a single gene are sufficient to radically alter body size, body length, and obesity , 2. HoweA way to study naturally occurring variation is by using inbred mouse strains, which differ in adiposity indices, like gonadal fat depot weight, up to 20-fold , 8. ThesOne way to perform this check is to create chromosome substitution (consomic) strains, whereby one chromosome from a progenitor of the original intercross is transferred by breeding into the genome the other progenitor strain  , 12. TheBwq5-6) [Bdln3-5)[Adip20 , Adip26 and Adip27 [Bdln4-5 and Adip27); none were female-specific.The goal of our research program is to genetically dissect a form of mouse obesity that is mild, does not fully manifest until middle age, and is not dependent on a high-fat diet –22. In pBwq5-6) , three f[Bdln3-5), and thrd Adip27 . Three onot previously detected in intercross populations. Mouse husbandry and phenotyping procedures were similar in the original intercross studies and the current consomic study, e.g., mice were fed a low-fat chow diet and were housed in the same animal facility.These eight QTLs are contained on three chromosomes  so we bred six reciprocal consomic strains for Chr 2, 7 or 9 derived from the B6 and 129 inbred strains. . We also bred a consomic strain from Chr 1 to determine how often new QTLs can be detected in consomic strains that were Thus, the specific objective of the current study was to determine if QTLs mapped in the intercross mice are replicated in consomic mice. We considered a QTL replicated if it has met the following criteria: (i) if there was significant difference from the inbred partner strain in at least one sex and at least one reciprocal strain, and (ii) if the direction of allelic effect was the same in the consomic strain as in the original intercross population. We did not require that the sex-specific pattern be discernable in the consomics for a QTL to be considered confirmed, but the consistency of sex effects are considered and discussed separately.http://dx.doi.org/10.5061/dryad.b3bn2.The Institutional Animal Care and Use Committee of the Monell Chemical Senses Center approved all procedures. All body composition data from this study are available through the Mouse Phenome Database at phenome.jax.org (dataset: Reed2)  and throC57BL/6ByJ (B6) and 129P3/J (129) inbred mice were purchased from the Jackson Laboratory  and used as founders of consomic mice and parents of inbred mice used for body composition analyses. Consomic strains were bred by serial backcrossing onto B6 or 129 host inbred strains to make the reciprocal strains. We also created and tested a consomic strain with a donor chromosome (Chr 1) for which no body composition QTL was previously reported in crosses between these specific B6 and 129 strains. In total we bred seven consomic strains: two reciprocal strains from each of three chromosomes  and one non-reciprocal strain (Chr 1). The breeding strategy to produce these strains is summarized immediately below and reported in detail in a separate manuscript currently in preparation.1 hybrids between the B6 and 129 progenitor inbred strains were backcrossed to males from a host strain, B6 or 129 (which resulted in elimination of the Y chromosome from the donor strain) to generate the N2 backcross generation. In the N2 and subsequent backcross generations, male offspring with full-length (non-recombinant) target chromosomes derived from the donor strain were identified and backcrossed to females from the host inbred strain, B6 or 129. To facilitate elimination of the donor genome in non-target chromosomes using a "speed consomic" approach [2 and N3 backcross generations. Heterosomic males (those with one full-length donor chromosome) and the smallest proportion of residual donor genome were used as breeders of the next generation. From the N4 generation onward, markers in the donor chromosome were genotyped, and breeders were chosen if they retained the full-length donor chromosome . After reaching backcross generation N6—N8, heterosomic males and females were intercrossed, and their homosomic progeny  was used to propagate the homozygous consomic strains. These strains were genotyped with a panel of markers to test for residual heterozygosity; the outcomes of these tests are in S2 Table. We had planned to phenotype 20 mice per strain and these target numbers were attained for all 6 strains included in the analyses, except in one case (129.B6-Chr2), which was not included in analyses.Female Fapproach , 25 we cAll inbred and consomic mice used for body composition analyses were born and reared in the Monell Center Animal Facility. Pups were weaned at 21–30 days of age and reared in same-sex groups until they were 6 months of age, when they were euthanized and used for body composition analyses. All mice were housed in temperature-controlled rooms (23°C) with a 12:12 light cycle and had free access to water and pelleted Teklad Rodent Diet 8604.Mice were euthanized by carbon dioxide affixation, weighed to the nearest 0.1 gram, measured for body length to the nearest tenth of a centimeter, and the gonadal fat depot was removed and weighed. Body length was initially measured with a ruler (65% of mice), which was later replaced with electronic calipers .Each group of consomic mice was compared with the appropriate host strain and the expected significant differences between consomic and inbred partner strains were interpreted as the presence of a QTL. These comparisons were made by one-tailed t-test for body weight and body length, and by general linear models for adiposity (with strain as factor and body weight as covariate). We used a one-tailed t-test because the expected direction of effect was known in advance based on the original QTL. To match earlier methods –21, the 2 mapping studies to facilitate interpretation of the new consomic results. Eight QTLs which are the focus of study here were previously identified in an F2 intercross between the B6 and 129 strains (Table 1). Five the QTLs  were named in the original manuscript and those are the names we use in this report, i.e., Bwq5-6 and Bdln3-5. One QTL for adiposity was originally named Adip5 but this name was later changed to Adip20 (the name we use here) by the International Committee on Standardized Genetic Nomenclature for Mice. The two remaining QTLs were not named in the original reports but were retroactively named by this committee (Adip26 and Adip27).We begin by summarizing the previous QTL results from FAdip20 and Adip26. Some of the QTLs had overlapping confidence intervals which suggest they may arise from the same underlying variant, e.g., body length (Bdln3) and body weight (Bwq5) on chromosome 2. The amount of phenotypical variance in F2 accounted for by these QTLs ranged from 2.5 to 10.2%. Five of the QTLs were observed in both sexes, but three were not: Bdln4, Bdln5 and Adip27 were specific to males.These original QTLs differed in the direction and strength of their allelic effects. In six of eight QTLs, the B6 allele increased the trait, the exceptions being S1 Table for list of markers). We were also successful at breeding mice with low residual heterozygosity using the ‘speed method’. The residual heterozygosity for each strain ranged from 0.87% to 0.0%  for congenic and consomic strains [Table 2. Our original goal was to breed 20 mice per strain and with the exception of the 129.B6-Chr2 strain, our breeding goals were met. By intention, mice were six months of age on average when measured for body composition, which previous work suggests is a time of maximal difference among the parent strains [We now turn to outcome of the breeding plan to produce the consomic mice. We tried to breed six reciprocal and one non-reciprocal strains for a total of seven strains. We were successful at breeding mice that had a full length donor chromosome and all mice tested in each strain had the expected donor-region genotype at all markers tested . The results of the statistical tests by each QTL, sex and strain are shown in Table 3 and Fig 1. Twenty five of the consomic-host strain comparisons are at least nominally significant  and 19 comparisons are significant when the Bonferroni correction was applied (marked by a ‘#’).The goal of this project was to determine whether QTLs are detectable when they are transferred to consomic strains and our method of detection is to compare the consomic to host strain for body weight, body length and adiposity. We expect these traits to be higher or lower than the host strain based on the direction of allelic effect of the original QTL. These differences in consomic-host strain means were evaluated by a t-test (for body weight and body length) and by a general linear model for adiposity. . The matches between QTLs detected in F2 and consomic mice are shown visually on Fig 1, where appropriate bars are annotated with symbols for QTLs detected in the F2. As an example of specific expectations, for Bwq6 we expect that the consomic strain with a B6 derived version of chr 9 (129.B6-Chr9) would be heavier (on average) than the host (129) strain and/or that the reciprocal strain (B6.129-Chr9) would be lighter than the host (B6) strain. Using this example, this prediction was correct, i.e., mice of the 129.B6-Chr9 strain weighed on average significantly more than did mice from 129 host strain; conversely, mice from the B6.129-Chr9 strain were lighter on average than were mice from the B6 host strain. In this example, both reciprocal strains and sexes differed in the expected direction, although we only required one of the two reciprocal strains or only one of the two sexes to differ to consider the QTL confirmed. Applying these criteria to the other statistical results in Table 3, each of the 8 QTL was confirmed (Table 4).Next we considered how well these patterns of strain differences meet our expectations based on the original QTL. For each, we expected that one or both sexes from one or both of the reciprocal strain pairs would be different from the host strain in the appropriate allelic direction at the corrected threshold . True to expectations there was a difference between consomic and donor strain in body length for males  but not in females (See Table 3 for test statistics and Table 5 for a summary of these sex-specific results). However, the sex-specific results from Adip27 were not consistent with expectations. These expectations come from two studies of F2 populations that identify this QTL, and in both cases, the B6 allele in males but not females was associated with increased adiposity (see Table 1 for references). However, here not only both male reciprocal consomic strains but also both female reciprocal consomic strains differed from the host either at a nominal  or adjusted threshold (129.B6-Chr7) in expected direction. Thus, the preservation of sex-specific QTLs from F2 to consomic was mixed, two were consistent and one was not. The detection of Adip27 effect in consomic females but not F2 females is probably due to higher statistical power in consomics.We also evaluated whether sex-specific effects were consistent from the FTable 3). This expectation of no-effect was the same one we had for adiposity, but here the expectation was not met. Instead, there was a female-specific QTL, with female consomic mice (129.B6-Chr1) being leaner than the females of the host strain.In addition to verifying QTLs in these consomic strains, we also examined a consomic strain (donor chr 1) which had a substituted chromosome for which no body composition QTLs were reported from prior mapping studies. Thus we did not expect any difference in average body weight or length in the consomic strain compared to the host strain and the data confirmed this expectation  on chromosome 7 but not a body weight or length QTL. But the host and consomic strains did differ in body weight and length .Similarly, there were two new QTLs on chromosome 7, for body weight and length. In the original FAdip26 QTL was confirmed according to the criteria specified above , B6.129-Chr2 males differed from B6 males in the opposite direction (they were leaner). This deviation from expectation is marked in There was another unexpected result. While the There are several methods to pursue QTL identification once they have been identified in intercrossed populations, and the use of congenic strains is a common one, e.g., . HoweverBwq6). These eight QTLs would be logical targets of future positional cloning studies using the congenic approach, and it would also be logical to pick the host-donor combination that yielded the largest mean differences between strains in the hopes that these differences would be make certain aspects of congenic breeding easier.To decide whether a QTL was confirmed, we had to establish criteria we could use to make this decision. We required the following two conditions be met: (i) there was a significant difference between mice from the consomic and inbred partner strains in one or more sexes and in at least one reciprocal strain, and (ii) the same direction of allelic effect in the consomic strain and in the original intercross population. . Overall we found that all eight original QTLs were detected in at least one sex or reciprocal strain, and several were detectable in both sexes and reciprocal strains . The second case was for Adip27 –we expected there would be no difference in female mice between host and consomic groups yet there was. Several factors could explain these unexpected results. The polygenic nature of body composition increases the chance that more than one QTL (with opposing effects) maps to the same chromosome. For example, mouse Chr2 has many previously reported QTLs for adiposity including Adip26 [There were two cases where the results were not what we predicted. g Adip26 . Epistatg Adip26 , 36, 37,g Adip26 , 39. Fing Adip26 –21 and ig Adip26 , 41.2 studies but missed the statistical thresholds and thus were not reported. Alternatively, it could be that the homozygous genetic background of the consomic strains allow for more sensitive detection of some types of QTLs, including these. The increased sensitivity of consomic strains to detect body composition QTLs has been noted previously [New QTLs were detected in the consomic strains that were not detected in the original intercross (on Chr 1 for adiposity and on Chr 7 for body weight and length). It may be that these QTLs were present in the earlier Feviously .2 populations of mice using the same breeding strategies and parental strains [The main finding of this study was that eight of eight body size and composition QTLs originally detected in the intercrosses between B6 and 129 strains were confirmed in the consomic strains. This result agrees with a previously reported 88% of body composition QTLs replicated in new F strains . We and  strains , 46. The strains , 44, the strains –50.S1 TableThe chromosome genotyped (Chr), SNP marker name by rs number, and location in bp based on GRCm38.(XLSX)Click here for additional data file.S2 TableThe symbol of the strain tested and its catalog number , number of markers tested, number of mice tested and the observed percent host genome in the non-target chromosomes.(XLSX)Click here for additional data file.


Genomic, proteomic and high-throughput gene expression data, when integrated, can be used to map the interaction networks between genes and proteins. Different approaches have been used to analyze these networks, especially in cancer, where mutations in biologically related genes that encode mutually interacting proteins are believed to be involved. This system of integrated networks as a whole exhibits emergent biological properties that are not obvious at the individual network level. We analyze the system in terms of modules, namely a set of densely interconnected nodes that can be further divided into submodules that are expected to participate in multiple biological activities in coordinated manner.In the present work we construct two layers of the breast cancer network: the gene layer, where the correlation network of breast cancer genes is analyzed to identify gene modules, and the protein layer, where each gene module is extended to map out the network of expressed proteins and their interactions in order to identify submodules. Each module and its associated submodules are analyzed to test the robustness of their topological distribution. The constituent biological phenomena are explored through the use of the Gene Ontology. We thus construct a “network of networks”, and demonstrate that both the gene and protein interaction networks are modular in nature. By focusing on the ontological classification, we are able to determine the entire GO profiles that are distributed at different levels of hierarchy. Within each submodule most of the proteins are biologically correlated, and participate in groups of distinct biological activities.The present approach is an effective method for discovering coherent gene modules and protein submodules. We show that this also provides a means of determining biological pathways (both novel and as well those that have been reported previously) that are related, in the present instance, to breast cancer. Similar strategies are likely to be useful in the analysis of other diseases as well. Advances in genomic, proteomic and high-throughput gene expression data, when integrated, can be used to map the interaction networks between genes and proteins, as well as their association with specific biological activities. It has also become increasingly clear that an integrated analysis of these extensive components is crucial, especially in the case of cancers = maxj [GO hji ]GO-Hni is the number of proteins in the group i that have any GO annotations, and nji the number of proteins that have a specific GO term j. GOhji represents fraction of protein contained in group j. After assigning nji proteins to a GO term j, ni- nji proteins still remain and these may participate in other groups. This is done iteratively for the complete list as shown in Figure where Unannotated proteins introduce errors when included in GO-H analysis, and are therefore removed from the list. In order to identify the domain cluster represented by a group of GO terms we use OntoVisT which asAs indicated in the Materials section we combined 9 different cancer resources to get 956 breast cancer marker genes, of which 610 and 840 genes were found to be present in Sets 1 and 2 respectively. These are then used to construct the gene layer networks for detailed analysis.Qmax, for the optimal partitions.We construct a partial correlation based gene network using the GGM technique. For each edge the partial correlation (pcor), p-value , FDR  and corresponding posterior probabilities (prob) are calculated, and the FDR criterion is employed to assess its significance. Table For Set 1 a total of 4619 edges are found with a significance level of 30% FDR among the 513 genes  with the results for the total sample set. The number of modules for ER- is slightly larger and may indicate an increased heterogeneity of the subtype, but the modules themselves are quite similar.In Set 2, stratification again gives results that are fairly consistent, with the number of genes being nearly constant across the subtypes. The number of modules varies although the similarity measure again gives a high level of concordance between the modules obtained by considering the entire sample set and the subtypes .Cross validation results (LOO-CV followed by majority voting) indicate that overall, 78% of the modular structure remains intact for Set 1 (71% for the ER + subtype and 60% for ER-) while only 57% is preserved for Set 2 . See Table Modules that are identified in the gene layer for entire sample set are mapped to their expressed proteins. This gives the primary protein network, and the secondary network is then constructed by examining all proteins that interact with the primary network. Both networks when combined give the protein module that corresponds to each gene module. Together this forms the protein layer, as explained in Figure max. varies from 0.632 for module 3 which has a large number of proteins and correspondingly the most interactions, to 0.798 for module 6, a sparse graph of relatively fewer proteins. Qmax is inversely correlated to the number of protein-protein interactions. Module 7 has only 7 genes associated with a sparse graph of 113 interactions among 114 proteins. Redoing our analysis using LOO-CV suggests that more than 86% of the sub-modular structure is robust. The smallest modules, 6 and 7 give the same submodule structure regardless of which nodes are left out. These modules are very sparse and do not contain any hubs. In the larger modules where there are hubs, removal of such a node can give very different submodules although the average Qmax value does not change significantly, indicating that modularity is a robust property.Our results for Set 1 are summarized in Table max value shows larger variation, from 0.62 to 0.87, and the number of submodules vary from 10 to 40 with considerable inter-module linkage. In other aspects such as modularity robustness, this dataset is similar to Set 1.Set 2 when similarly analysed  that form so—called party hubs that interact primarily with other proteins within the same submodule, and bridge proteins (B) which are the “date” hubs that interact with proteins in two or more submodules. Their associated domain properties are obtained by first mapping them to the GOA followedBoth datasets are studied in order to infer the biological activity for different hub classes of each submodule. The three GO categories BP, MF, and CC are separately analyzed. The detailed analysis of the main cluster of each GO category in Set 1 is presented here, and results for the other clusters of both sets are provided in the supplementary information.The two layer network deduced from the functional analysis of breast cancer genes found in Set 1 is shown in Figure ++) increases cell proliferation of breast cancer cells[For instance, thioredoxin, a redox protein with growth factor activity  is indicated as being associated with breast cancer. In panel C12, the focus is on protein phosphatase regulator activity  and protein phosphatase inhibitor activity. In panel C13, GTPase regulator activity instances are clubbed together, including guanyl-nucleotide exchange and Rho guanyl-nucleotide exchange factor activity. These might provide potential candidates for target discovery and therapeutics. Other functional clusters and terms included therein can be explored for relevance, as well as for detailed insight of specific molecular functions; see Additional fileGO:BP analysis for Set 1 suggests that 73.5% of all proteins in this study organize themselves in 539 different process groups that participate in 205 GO:BP terms of which only 70 have hitherto been reported in the literature related to breast cancer studies  Signal transduction (P2).2) Cell adhesion (P3).3) Cell proliferation (P4).4) Apoptosis (P6).5) MAPK signaling pathway via the G-protein-coupled receptor (GPCR) (P7).6) Notch signaling pathway (P8).7) Estrogen induced cell proliferation (P5).8) Cell death (P22).9) Inhibition of epidermal growth factor receptor (EGFR) (P15) transcription.10) Anti-apopotsis (P14).11) Retinoblastoma cell apoptosis (P19).In addition to these terms, cluster analysis also suggests eleven additional specific novel process terms that have not been explored so far in case of breast cancer. It includes small GTPase mediated signal transduction (P9), transmembrane receptor protein tyrosine kinase signaling pathway (P11), phosphatidylinositol-mediated signaling (P12), regulation of r-protein signal transduction (P13), transforming growth factor beta receptor signaling pathway (P16), regulation of JNK cascade (P17), negative regulation of transforming growth factor beta receptor signaling pathway (P18), G-protein signaling, coupled to cAMP nucleotide second messenger (P20), negative regulation of apoptotic process (P10), Positive regulation of transcription, DNA dependent (P21) and positive regulation of proteasomal ubiquitin-dependent protein catabolic process (P23). All these processes should be explored further to get additional insight into the regulation of cellular process in breast cancer.A new candidate that has become evident through the present analysis is the Abraxas gene that hasThe distributions of proteins at various cellular locations in all seven modules of Set 1 are shown in Figure The largest cluster corresponds to the intracellular component (C8) Figure , panel bCluster analysis also indicates more specific intracellular locations in the cytosol, mitochondrion, Golgi apparatus, sarcoplasmic reticulum and endoplasmic reticulum membrane as sites wherein breast cancer activity occurs. A recent study reports Caspase-2 releases from nucleus to cytosol, which is partially required for the apoptosis induction by taxanes in breast cancer cells. SimilarOur analysis of Set 2 for all three GO categories is included in Additional filei validate domains with relevant activity in breast cancer (as reported in literature).ii identify new domain terms to examine details of specific biological activity, andiii determine important domain clusters as the site of major biological activity.In the past few years, the integrated analysis of gene expression data in context of PPI has received considerable attention. One objective has been to derive biologically interesting subnetworks or modules of interpretable size from large scale PPI data. SubnetwThere are, however, caveats. Noise in the available microarray or annotation data as well as incomplete annotations can make the network reconstruction ambiguous, and the complexity of the underlying regulatory mechanism, in particular the combinatorics involved makes it difficult to draw direct inferences. We have considered all currently available annotations for the given set of proteins, but since many proteins are as yet unannotated, this will affect the fraction of proteins in the largest domains and thus can alter the GO-H score for any selected set of proteins. The protocol can be modified to include a predictive aspect by using the principle of guilt-by association in orderIn summary, we have construct the two-layer “network of networks” of breast cancer genes and associated proteins. The breast cancer bilayer network has a hierarchical modular structure, with sub-modules inside modules.This structure is robust: the general topology is unaltered even when some specific proteins are removed (as seen when the LOO-CV protocol is implemented). Alternate methods that test the stability of a control network in comparison to networks formed from a random collection of genes or proteins are available, but these are not applicable here since there is extensive work on breast cancer genes and networks. Furthermore, subgroup analysis for small subgroups is unlikely to be effective and randThe property of submodularity has advantages. Submodules are smaller units of biological organization that allow network abstraction at specific levels and thus helps determine domain activity through a small number of interacting submodular units rather than through the hundreds or thousands of proteins in a complex network. The different hub proteins within the submodules perform specific domain activities in a coordinated manner. We performed a complete ontological analysis to determine the GO profile that is involved in breast cancer. Some of these protein groups are involved in multiple biological activities and also form clusters of specific domain activities associated with various GO terms. This division into smaller groups helps to identify the set of proteins that participate in specific domain activities, and can be used to examine the organization and coordination of complex domain activities. We have also shown that specific submodules contain more conserved domain groups and these are assumed to be preserved during evolution.. The analysis predicts new domain clusters which has not been studied in the context of breast cancer, and identifies Q6UWZ7, a protein expressed by a newly reported breast cancer marker gene Abraxas through the secondary network. In addition to identifying novel domain activities, this protocol can thus also point to new biomarkers.The two-layer protocol allows not only the catalogue of the (well-studied) domain activities in breast cancer but also permits the prediction of more specific domain activities at a lower level of the ontological hierarchyGiven the ever-increasing amount of interaction data available, we expect that the two-layer approach described herein will prove useful in ongoing efforts to explore the protein interaction universe and understand how domain building blocks are assembled together to perform or alter normal activity, not only in breast cancer, but in other complex diseases as well.The authors declare that they have no competing interests.Conceived and design the protocol: RR, AS. Generate the results: AS, SK. Analyze the results: RR, AS, SK. Wrote the manuscript: RR, AS. Read and Approved: RR, AS, SK. All authors read and approved the final manuscript.Gene extracted from different data sources.Click here for fileGene network analysis for Set 1.Click here for fileGene network analysis for Set 2.Click here for fileSummary of topological and biological analysis of gene module and their corresponding protein module for Set 2.Click here for fileMolecular function analysis of modular network of Set 1.Click here for fileBiological process analysis of modular network of Set 1.Click here for fileCellular component analysis of modular network of Set 1.Click here for fileMolecular function analysis of modular network of Set 2.Click here for fileBiological process analysis of modular network of Set 2.Click here for fileCellular component analysis of modular network of Set 2.Click here for file