Introduction
============

Periodontal disease (PD) is a chronic bacterial and inflammatory disease whose prevalence is reportedly as high as 90% [@B1], [@B2]. PD can routinely injure or damage both oral soft and hard tissues such as gingiva, cementum, periodontal ligament, and supporting alveolar bone, and it is the primary risk factor for tooth loss in adults [@B3], [@B4]. Recent studies have also found PD to be associated with cardiovascular disease, pregnancy disease, chronic respiratory disease, diabetes mellitus, and erectile dysfunction [@B5]-[@B8]. Oral pathologic bacteria are not only a dominant risk factor for PD, but are also a risk factor for major systemic diseases due to several mechanisms involving modulation of the inflammatory pathway and systemic immunity [@B9].

Chronic inflammation caused by bacterial infection has been reported as one of the main factors underlying the development of cancer [@B10]. Cumulative studies have demonstrated the relation between oral bacteria and initiation or progression of systemic cancer [@B11]. In particular, oral pathologic bacteria such as *Aggregatibacter actinomycetemcomitans*,*Fusobacterium nucleatum*,*Tannerella forsythia*,*Treponema denticola*, and *Porphyromonas gingivalis* are significantly positively associated with oral, pancreatic, and gastrointestinal cancers [@B12], [@B13]. Oral pathologic bacteria can either up- or down-regulate proinflammatory cytokines and chemokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and C-reactive protein, resulting in oral pathologic bacteria being able to affect the oral and systemic immune systems of the body and also induce oncogenic responses [@B14], [@B15].

Prostate cancer (PC) is the most-common cancer in men in the United States and Europe. While PC is only the fifth-most-common diagnosed malignancy and the seventh leading cause of cancer death among men in South Korea, the rapid aging of the Korea population and the increasing prevalence of obesity have resulted in a steep increase in the incidence of PC recently. PD is apparently associated with oral bacterial dysbiosis and systemic immune responses, but the role of oral pathogenic bacteria in carcinogenesis remains unclear. Also, a direct correlation between PD and PC is still unclear and controversial. While numerous cross-sectional and cohort studies have identified associations between PD and facial cancers, especially those of the oral and neck, there have been very few well-designed studies with stratified longitudinal cohorts comparing PD and PC [@B16], [@B17]. Our aim was therefore to determine whether there is a connection between PD and PC using data from a South Korean nationwide population based on National Health Insurance Service-Health Examinee Cohort (NHIS-HEC).

Materials and Methods
=====================

Study design and data collection
--------------------------------

In South Korea, patients in the NHIS are required to pay approximately 30% of their total medical expenses when they use medical facilities, with the medical providers submitting claims for the remaining 70%. This has resulted in the NHIS having access to an enormous database (DB) of more than one trillion cases involving the entire Korean population of 51 million covering the past decade. This DB includes information on individual qualification criteria, insurance coverage and payouts, medical and dental records, health checkups, rare incurable diseases, systemic cancer, and long-term-care insurance services. The NHIS program is designed for the heads of households, household members aged 40 years or older, and workplace subscribers who receive regular health checkup examinations once every 2 years, which include a self-reported questionnaire on smoking, alcohol consumption, and exercise.

A random stratified sample of 514,866 South Koreans was collected from the NHIS DB by the NHIS Big Data Steering Department, which is conducting 1,476-stage simple sampling based on sex, age, and household income level for the 12 years from 2002 to 2013. After performing stratified sampling of the data for 2002, the data in the cohort DB are maintained to 2013 except when sample objects become ineligible due to death or emigration. To compensate for the natural reduction in the population data over time, neonatal samples are added yearly in order to maintain the sample size, making this a semidynamic cohort. All personal information and identification numbers were deleted prior to the analysis, and a computer-generated random number was allocated to each sample. This study conformed to the STROBE guidelines for reporting observational studies ([www.strobe-statement.org](http://www.strobe-statement.org)) and was approved by the Institutional Review Board, Daejeon Dental Hospital, Wonkwang University (approval no. W1702/004-001).

Identification of sociodemographic factors
------------------------------------------

Potential confounding sociodemographic factors of PD and PC were assessed from the NHIS-HEC DB: sex, age (4 groups: those aged 40-69 years in 10-year intervals, and those aged ≥70 years), household income (5 groups: 41 groups were divided into 5 quintiles based on the insurance fee imposed on each household, with the Medical Aid Program \[MAP\] group corresponds to the first quintile), insurance status (3 groups: those in the MAP and those in the NHIS, where the NHIS group included both self-employed and employees), and residence area (2 groups: with a cutoff of 50,000 residents).

Identification of comorbidity
-----------------------------

Patients who were diagnosed with hypertension, diabetes mellitus, and cardiovascular disease prior to 2002 were excluded, and the following lifestyle-related comorbidities present from 2002 to 2013 were diagnosed by medical doctors: hypertension (Korean Classification of Diseases, 6th revision \[KCD-6\] codes I10 and I15, corresponding to International Classification of Diseases, 10th revision \[ICD-10-CM\] codes I10 and I15), diabetes mellitus (KCD-6 codes E10-E14, corresponding to ICD-10-CM codes E10-E14), cerebral infarction (KCD-6 codes I63-I66, corresponding to ICD-10-CM codes I63-I66), angina pectoris (KCD-6 codes I20, corresponding to ICD-10-CM codes I20), and myocardial infarction (KCD-6 codes I21 and I22, corresponding to ICD-10-CM codes I21 and I22). To increase the validity of the assessment in comorbidities diagnoses, we only included patients with comorbidities who have been diagnosed at least twice during the period from 2002 to 2013.

Identification of patients with prostate cancer
-----------------------------------------------

97% of cancer patients are fully or partially covered by NHI and all cancer patients are registered in NHIS and Health Insurance Review and Assessment Service (HIRA) DB. Therefore, this study performed that PC diagnostic and prescription codes were obtained based on the NHIS and HIRA. Patients who were diagnosed with cancer prior to 2002 and aged \<40 years were excluded according to self-reported questionnaire information in the NHIS regular health check-up examination. We selected patients who had been newly diagnosed with PC (KCD-6 codes C61; corresponding to ICD-10-CM codes C61) by medical oncologists and urologists from 2002 to 2013 and the date of which cancer was first diagnosed was assigned as the index date.

Identification of patients with periodontal disease
---------------------------------------------------

This study involved adult patients who were aged ≥40 years and diagnosed with acute periodontitis, chronic periodontitis, periodontosis, other PD, and unspecified PD (KCD-6 codes K05.2 to K05.6, corresponding to ICD-10-CM codes K05.2 to K05.6) prior to the index date between 2002 and 2013. The patients who were diagnosed as acute and chronic gingivitis (KCD-6 codes K05.0 to K05.1, corresponding to ICD-10-CM codes K05.0 to K05.1) were excluded as a reversible inflammatory PD. To increase the validity of the assessment of PD diagnoses, we only included patients with PD who have been diagnosed at least twice with a specific PD related-disorder during the period from 2002 through 2013. Clinical and radiographic diagnoses of PD were based on measurements of the amount of bleeding on probing, probing depth (measured between the gingival crest margin and the base of the apical lesion), clinical attachment level (measured between the cementoenamel junction and the base of the apical lesion), gingival inflammation, and radiographic alveolar bone loss according to criteria of the Centers for Disease Control and Prevention/American Academy of Periodontology case definitions by local dentists or periodontists [@B18], [@B19].

Statistical analysis
--------------------

The chi-square test was used to identify significant differences in categorical cohort data for sociodemographic factors (sex, age, household income, insurance status, and residence area), comorbidities (hypertension, diabetes mellitus, cerebral infarction, angina pectoris, and myocardial infarction), self-reported questionnaire (smoking status, day\'s smoking, alcohol intake habits, one-time alcohol intake, and regular exercise), and incidence of PC with PD. We calculated hazard ratios (HRs) with 95% confidence intervals (CIs) using multivariate Cox proportional-hazards regression models while adjusting for sociodemographic factors, comorbidities, smoking status, alcohol intake, and regular exercise. The cumulative incidence of PC with/without PD was estimated using the Kaplan-Meier method and compared with the log-rank test. In all assessments performed in this study, *P* \< 0.05 was considered statistically significant and statistical tests were two-sided. All cohort data used in this study were calculated using the Statistical Analysis System (version 9.2, SAS Institute, Cary, NC, USA) by the Department of Health Insurance Research, Ilsan Hospital, NHIS.

Results
=======

Incidence of prostate cancer patients with periodontal disease
--------------------------------------------------------------

Fig. [1](#F1){ref-type="fig"} shows a cohort flow chart for the inclusion and exclusion of the study participants. Among the total cohort of 514,866 people, 326,932 (Female and cancer history) were excluded from the study and 187,934 (18.3%) males and aged ≥40 years were recruited. We enrolled 934 subjects who were diagnosed with PC, comprising 531 PD patients (56.9%) and 403 Non-PD patients (43.1%). Those aged 60-69 years (*n* = 398) accounted for 42.6% of the surveyed subjects, while 361 (38.7%) were in the fifth quintile of household income, 927 (99.3%) were in the NHIS, and 782 (83.7%) lived in urban areas (Table [1](#T1){ref-type="table"}).

Results of comorbidity and regular health check-up examination
--------------------------------------------------------------

Although comorbid disease (hypertension, diabetes mellitus, cerebral infarction, angina pectoris, and myocardial infarction) showed higher incidence of PD patients compared with Non-PD patients, only myocardial infarction was statistically significantly higher in PD patients compared with Non-PD patients (*P* \< 0.001). In the questionnaire survey, smoking and exercise status were only statistically significant between PD patients and Non-PD patients (*P* \< 0.001; Table [2](#T2){ref-type="table"}).

Association of prostate cancer with periodontal disease
-------------------------------------------------------

In the multivariate Cox regression analysis with adjustment for sociodemographic factors, comorbidities, smoking status, alcohol intake, and regular exercise, PD was associated with a 14% higher risk of PC (HR = 1.14, 95% CI = 1.01-1.31, *P* = 0.042). In addition, using the adjusted Kaplan-Meier curve with the number of subjects at risk, we found that the incidence of PC with PD was significantly higher than that of PC without PD (*P* \< 0.001; Fig. [2](#F2){ref-type="fig"}). The presence of both PC and PD was significantly positively correlated with current smoking (HR = 1.68, 95% CI = 1.52-1.85, *P* \< 0.001), smoking ≥20 cigarettes a day (HR = 1.20, 95% CI = 1.04-1.40, P = 0.015), consuming alcohol every day (HR = 1.17, 95% CI = 1.09-1.25, *P* \< 0.001), and consuming ≥1 bottle of beer (360 ml) daily (HR = 1.08, 95% CI = 1.03-1.13, *P* \< 0.001; Table [3](#T3){ref-type="table"}).

Discussion
==========

PD is the most-common inflammatory disease, and many of its known risk factors - sex, age, smoking, obesity, hypertension, and diabetes mellitus - are also considered to be risk factors for systemic cancer [@B20]. We observed that the risk of PC was significantly positively correlated with age ≥70 years, lower income, being registered with the MAP, myocardial infarction, heavy smoking, high alcohol consumption, and less exercise. The data was not statistically significant except second quintile, but the lower the household income, the higher the risk for PC. This can be explained by the reduced accessibility to healthcare services and limitations of the current study involving PC patients who only visited clinics for the cancer but not for the PD. We also found that the incidence of PD - which is well known to be related to oral cancer - was also significantly positive associated with PC in a multivariate analysis.

Few studies have investigated the association of PC with PD [@B21]-[@B23]. In one prospective cohort based on the Health Professionals Follow-Up Study it was found that PD was correlated with a slight but significant increase in the risk of systemic cancer, but PD was also inversely correlated with PC (HR = 0.99, 95% CI = 0.61-1.59) and tooth loss (HR = 0.70, 95% CI = 0.50-0.97) [@B21]. Hiraki *et al.* [@B22] conducted a large-scale case-control study of Japanese patients, and reported that increased tooth loss was correlated with a decreased risk of PC after adjusting for potential confounding factors (odds ratio \[OR\] = 0.49, 95% CI = 0.19-1.26, *P* = 0.049). In particular, the risk of PC was higher when more teeth were remaining, and this may have been due not only to the sociodemographic and economic characteristics of the studied subjects, but also to the number of teeth affected by PD. Unlike these studies, the NHANES I Epidemiologic Follow-up Study found that PD was associated with an elevated risk of PC (HR = 1.81, 95% CI = 0.76-4.34) [@B23]. However, that study was hampered by poor statistical power due to the smallness of the sample and also potentially by the smallness of any real effects.

Both the bivariate analysis (HR = 1.60, 95% CI = 1.40-1.83, *P* \< 0.001; data not shown) and multivariate analysis (HR = 1.14, 95% CI = 1.01-1.31, *P* = 0.042) performed in the present study showed that PD was positively and significantly associated with PC. Both diseases produce generalized inflammation and infection in the body, and especially chronic PD has been shown to increase the level of prostate-specific antigen (PSA) that is produced primarily by epithelial prostate cells and is used to diagnose PC [@B24]. The PSA level is significantly higher in chronic prostatitis patients with PD characterized by a gingival clinical attachment level of ≥ 2.7 mm (10.8±7.0 ng/ml) than in those without such PD (5.6±3.7 ng/ml, *P* = 0.05) [@B24]. In addition, Alwithanani *et al.* [@B25] reported that the mean PSA level was significantly higher in PD patients regardless of the presence or absence of PC and the prostate symptom score, and that the PSA level might decrease after treating PD. There is accumulating evidence of proinflammatory cytokines such as IL-6, IL-8, IL-18, TNF-α, and C-reactive protein being associated with the pathogenesis of PC [@B24]. These observations may indicate the similarity of the etiopathogenesis of PD and PC.

A recent systematic review found a significant positive relationship between PD and erectile dysfunction [@B26]. Both conditions share chronic inflammation and systemic endothelial dysfunction, which are also key mechanisms of carcinogenesis [@B27]. The invasion of oral pathological bacteria, especially *Porphyromonas gingivalis*, may induce traumatic injury and irritation of the epithelium and mucosa, and play a role in subsequent cancer progression [@B28], [@B29]. A nationwide population-based cohort study found that the risk of cancer was 1.42-fold higher (95% CI = 1.03-2.09, *P* = 0.039) in patients with erectile dysfunction [@B30]. In our previous study we found a strong association between PD and vasculogenic erectile dysfunction in a nationwide population-based NHIS-HEC (OR = 1.53, 95% CI = 1.41-1.65, *P* \< 0.001) [@B31]. Although the evidence is not definitive, these results suggest that a diagnosis of PD is associated with a higher risk of PC.

Androgen deprivation therapy (ADT) is the most-common standard treatment for advanced PC, and its application is increasing rapidly [@B32]. However, many studies have suggested that ADT is associated with cardiovascular disease, diabetes mellitus, and Alzheimer\'s disease, with in particular it being the main cause of male osteoporosis since it can affect almost any bone structure of the body [@B33], [@B34]. Male osteoporosis can result from various causes, including bone resorption, bone loss, and bone fracture, and several studies have found that PD can be exacerbated by systemic bone loss such as osteoporosis or rheumatoid arthritis [@B35]-[@B37]. Famili *et al.* [@B38] considered that patients with PC receiving ADT are more likely to develop PD or experience worsening of PD than are patients who are not on ADT. Although it is difficult to conclude that ADT for PC can induce or exacerbate PD, this observation should be considered when deciding whether or not to apply ADT to PC patients.

This study was subject to several limitations. Firstly, selection bias may have been present due to its retrospective design and only including patients who visited dental clinics and hospitals and who were diagnosed with PD and PC [@B39]. Secondly, this study did not analyze medical and dental records, which restricted the ability to diagnose the severity and the follow-up period of PD and PC in the DB of this cohort. Therefore, future cohort studies should consider the severity and the follow-up period of PD and PC in the design. Finally, because the data were acquired from a diverse group of medical and dental specialties, inconsistent diagnostic criteria were likely to have been applied. Nevertheless, the results from this nationwide population-based longitudinal cohort study in South Korea are considered to provide evidence for a link between PD and PC. PD is clearly intimately associated with systemic inflammation and immune dysregulation, which are both direct and indirect etiological factors in systemic cancer [@B40], [@B41]. However, the etiological and bidirectional associations between PD and PC remain unclear, and little is known about their underlying mechanisms. Therefore, further investigations of the biological plausibility and the gathering of clinical and epidemiological evidence are needed to both strengthen and understand the association between PD and PC.

Conclusion
==========

To our knowledge, this study is the first to investigate the association of PC with PD using a retrospective analysis of a nationwide population-based NHIS-HEC. In contrast to some previous studies, the present cohort study has shown that patients with PD have a significantly but slightly positive in patients with PC. Further studies are required to strengthen this hypothesis and focus on identifying the mechanisms underlying the links between PD and PC. If confirmed, this observation could have clinical and public health implications given the increasing prevalence of PD worldwide.

This study used a national sample cohort data of the National Health Insurance Service (NHIS-2017-2-377) and was supported by Wonkwang University in 2017. The authors report no conflicts of interest related to this study.

PC

:   prostate cancer

PD

:   periodontal disease

NHIS-HEC

:   National Health Insurance Service-Health Examinee Cohort

IL

:   interleukin

TNF

:   tumor necrosis factor

DB

:   database

HIRA

:   Health Insurance Review and Assessment Service

PSA

:   prostate-specific antigen

ADT

:   Androgen deprivation therapy.

![Flow chart of the inclusion and exclusion of participants in the National Health Insurance Service- Health Examinee Cohort during 2002-2013; PD: periodontal disease.](jcav08p2959g001){#F1}

![Cumulative incidence of prostate cancer with/without periodontal disease (PD) was estimated using the Kaplan-Meier method and compared using the log-rank test. Incidence of prostate cancer patients with PD was higher and significantly different from prostate cancer patients without PD (*P* \< 0.001).](jcav08p2959g002){#F2}

######

Baseline characteristics of the population included in the National Health Insurance Service-Health Examinee Cohort (NHIS-HEC) according to prostate cancer (PC) patients with/without periodontal disease (PD).

  Characteristics        PD (n, %)    Non-PD (n, %)   *P*^a^
  ---------------------- ------------ --------------- -------------
  Total                  531          403
  Age group (years)
  40-49                  48 (9.0)     45 (11.2)       **\<0.001**
  50-59                  139 (26.2)   98 (24.3)
  60-69                  246 (46.3)   152 (37.7)
  ≥70                    98 (18.5)    108 (26.8)
  Household income^b^
  First quintile         49 (9.2)     70 (17.4)       **\<0.001**
  Second quintile        66 (12.4)    56 (13.9)
  Third quintile         87 (16.4)    68 (16.9)
  Fourth quintile        110 (20.7)   67 (16.6)
  Fifth quintile         219 (41.3)   142 (35.2)
  Insurance status
  MAP                    4 (0.8)      3 (0.7)         **\<0.001**
  NHIS (self-employed)   293 (55.2)   206 (51.1)
  NHIS (employees)       234 (44.0)   194 (48.2)
  Residence area^c^
  Urban                  466 (87.8)   316 (78.4)      **\<0.001**
  Rural                  65 (12.2)    87 (21.6)

Abbreviations: MAP: Medical Aid Program.

^a^*P* values were calculated using the chi-square test; boldface denotes statistical significance (*P* \< 0.05).

^b^Divided into five quintiles based on the insurance fee imposed on each household, with the MAP group classed into the first quintile.

^c^Classified with a cutoff of 50,000 residents.

######

Results of comorbidity and regular health check-up examination

  Characteristics                   PD (n, %)     Non-PD (n, %)   *P*^a^
  --------------------------------- ------------- --------------- -------------
  **Comorbid disease**
  Hypertension                      329 (62.0%)   246 (61.0%)     0.776
  Diabetes mellitus                 194 (36.5%)   133 (33.0%)     0.262
  Cerebral infarction               102 (19.2%)   61 (15.1%)      0.104
  Angina pectoris                   22 (4.1%)     13 (3.2%)       0.465
  Myocardial infarction             142 (26.7%)   67 (16.6%)      **\<0.001**
  **Self-reported questionnaire**
  Smoking status^b^
  Yes                               371 (69.9%)   133 (33.0%)     **\<0.001**
  No                                160 (30.1%)   270 (67.0%)
  Day\'s smoking
  \<20 piece                        334 (90.0%)   112 (84.2%)     0.072
  ≥20 piece                         37 (10.0%)    21 (15.8%)
  Alcohol intake habits
  No drinking                       259 (48.8%)   207 (51.4%)     0.153
  1-4 times/week                    237 (44.6%)   159 (39.5%)
  Almost every day                  35 (6.6%)     37 (9.1%)
  One-time alcohol intake
  \<1 bottle of beer (360ml)        237 (87.1%)   170 (86.7%)     0.900
  ≥1 bottle of beer (360ml)         35 (12.9%)    26 (13.3%)
  Regular exercise
  No                                244 (46.0%)   229 (56.8%)     **\<0.001**
  1-4 times/week                    209 (39.4%)   108 (26.8%)
  ≥5 times/week                     78 (14.6%)    66 (16.4%)

Abbreviations: PD: periodontal disease

^a^*P* values were calculated using the chi-square test; boldface denotes statistical significance (*P* \< 0.05).

^b^Subjects were currently not smoking, or had smoked \<100 cigarettes were classified as non-smokers, while the other subjects were classified as smokers.

######

Association of PC patients with PD in multivariate Cox proportional-hazard regression analysis with adjustment for confounding factors.

  Characteristics                          HR      95% CI        *P^a^*
  ---------------------------------------- ------- ------------- -------------
  Age group (ref: 40-49 years)
  50-59                                    2.21    1.92-2.55     **\<0.001**
  60-69                                    5.86    5.14-6.68     **\<0.001**
  ≥70                                      14.31   12.57-16.30   **\<0.001**
  Household income (ref: First quintile)
  Second quintile                          1.33    1.11-1.58     **\<0.001**
  Third quintile                           1.16    0.99-1.35     0.066
  Fourth quintile                          1.12    0.99-1.28     0.069
  Fifth quintile                           1.13    0.97-1.31     0.118
  Insurance status (ref: MAP)
  NHIS (self-employed)                     0.42    0.19-0.91     **0.026**
  NHIS (employees)                         0.46    0.21-0.99     **0.045**
  Residence area (ref: Urban)
  Rural                                    0.97    0.80-1.16     0.713
  Smoking status (ref: No)
  Yes                                      1.68    1.52-1.85     **\<0.001**
  Day\'s smoking (ref: \<20 piece)
  ≥20 piece                                1.20    1.04-1.40     **0.015**
  Alcohol intake habits (ref: No)
  1-4 times/week                           0.94    0.91-0.97     **\<0.001**
  Almost every day                         1.17    1.09-1.25     **\<0.001**
  1-time alcohol (ref: \<1 bottle)
  ≥1 bottle (360ml)                        1.08    1.03-1.13     **0.001**
  Regular exercise (ref: No)
  1-4 times/week                           1.00    0.97-1.03     0.858
  ≥5 times/week                            0.83    0.78-0.89     **\<0.001**
  Periodontal disease                      1.14    1.01-1.31     **0.042**

Abbreviations: PD: periodontal disease; HR: hazard ratio; CI: confidence interval.

^a^Boldface denotes statistical significance (*P* \< 0.05).

Multivariate regression analysis adjusted for sex, age, household income, insurance status, residence area, hypertension, diabetes mellitus, cerebral infarction, angina pectoris, myocardial infarction, smoking status, alcohol intake, and regular exercise.

[^1]: \* Jae-Hong Lee and Helen Hye-In Kweon contributed equally to the writing of this work.

[^2]: Competing Interests: The authors have declared that no competing interest exists.


Introduction {#s1}
============

Essential tremor (ET) is a progressive movement disorder found in human adults, with prevalence ranging from 4% in the population of age 40 years or older to 14% in those older than 65 years [@pone.0069199-Liang1]. Despite its prevalence, there is little known concerning the functional pathology of ET [@pone.0069199-Shill1]. Traditionally, ET has been regarded as a mono-motor symptomatic disease with postural or kinetic tremor(s) in the hands and forearms or isolated tremor(s) in the head and/or of the voice. Now, the evolving viewpoint regards ET as a heterogeneous neurologic disorder accompanied by several motor and non-motor symptoms [@pone.0069199-Teive1]. The complicated clinical characteristics directed the ET origin to the central nervous system; most likely, in the cerebellum [@pone.0069199-Raethjen1].

Consistent pathological markers for ET have not been identified because of limited postmortem studies owing to the low autopsy rate in ET patients. Several studies have shown that in patients with ET, 76% had a decrease in the number of cerebellar Purkinje cells and 24% had lower brainstem Lewy bodies [@pone.0069199-Louis1]--[@pone.0069199-Louis3]. However, Rajput and colleagues' recent studies [@pone.0069199-Rajput1]--[@pone.0069199-Rajput3] showed that Purkinje cells and Lewy bodies were not the pathology of ET, which presented an entirely different picture.

Magnetic resonance imaging (MRI) has been employed to investigate ET in the literature. Recent MRI studies concentrated on structural (voxel-based morphometry or VBM) [@pone.0069199-Bagepally1]--[@pone.0069199-Quattrone1] and microstructural (diffusion tensor imaging or DTI) [@pone.0069199-Buijink1]--[@pone.0069199-Saini1] abnormalities. Results from most studies [@pone.0069199-Bagepally1]--[@pone.0069199-Cerasa1], [@pone.0069199-Klein1]--[@pone.0069199-Saini1] have supported the hypothesis that the abnormalities of the cerebello-thalamo-cortical motor pathway and the fronto-parietal circuit are involved in the functional pathological changes of ET. However, several other studies showed that for most ET patients there was no cerebellar structural changes [@pone.0069199-Quattrone1], and that there was no structural changes in above mentioned regions [@pone.0069199-Daniels1], [@pone.0069199-Buijink1]. In addition, these studies were lack of correlation between structural abnormalities and clinical symptom(s).

Task-designed fMRI is more specific to reflect the correlation between the clinical symptom(s) and brain dysfunction(s) [@pone.0069199-Boecker1]. Bucher and colleagues [@pone.0069199-Bucher1] reported abnormal activation of cerebellar networks involved in ET's tremor generation by using imitation hand tremor stimulation. Other studies [@pone.0069199-Passamonti1], [@pone.0069199-Cerasa2] reported that the dysfunction of cerebello-prefrontal network and prefronto-parietal circuit were responsible for the non-motor symptoms in ET patients (through verbal working memory and stroop tasks). However, because ET patients always have difficulty in completion of task experiment, and the tremor by itself could introduce confounding factors (i.e., uncontrolled body motion), task-designed fMRI is not suitable for ET study. In addition, due to the lack of a unified stimulus paradigm, the existent task fMRI studies could not support each other, thus there is relatively little clinical use [@pone.0069199-Fox1].

Resting-state fMRI (RS-fMRI) is a non-invasive technique that based on the spontaneous fluctuation of the blood-oxygen-level-dependent (BOLD) signal in fMRI [@pone.0069199-Fox1], [@pone.0069199-Biswal1]. The RS-fMRI observes the brain's functional change without requirement of an overt task performance or stimulation, thus avoids the inherent shortcomings of the task-designed fMRI [@pone.0069199-Fox1], [@pone.0069199-Biswal2]. The RS-fMRI, as well as functional connectivity measurements derived from it, has been applied to the studies of brain dysfunction in movement disorders [@pone.0069199-Hacker1]--[@pone.0069199-Douaud1] and neurodegenerative diseases [@pone.0069199-Zarei1]--[@pone.0069199-Wang1]. Unlike task fMRI which only focuses on single functional system at a time, the RS-fMRI provides brain functional connectivity or functional network information that will be valuable for simultaneously studying the mechanisms of motor and non-motor functional deficits in ET patients. Up to date to the best of our knowledge, only one paper [@pone.0069199-Popa1] reported using independent component analysis (ICA) of RS-fMRI to explore the brain dysfunction in ET. In this study, ICA was applied to detect functional connectivity within the cerebello-thalamo-cortical network, where ET patients showed impaired functional connectivity. Although ICA is commonly used in RS-fMRI studies and has advantages as an exploratory tool over a hypothesis-based one (e.g., seed correlation), it also has inherent drawback of inconsistency problem induced by model order and initial value.

Regional homogeneity (ReHo) measurement of RS-fMRI provides an approach to investigate local functional connectivity based on temporal correlations between a voxel and adjacent voxels [@pone.0069199-Shukla1]--[@pone.0069199-Zang1]. The ReHo analysis method has several advantages. First, it is based on data-driven approach and thus requires no prior knowledge [@pone.0069199-Zuo1], thus, it is more suitable for studying of a disease with unclear pathological mechanism such as ET. Second, the computation of ReHo using a Kendall's coefficient of concordance is relatively simple and has been implemented in REST and DPARSFA software with many successful applications [@pone.0069199-You1]--[@pone.0069199-He1]. The biological meaning of the ReHo measurement has been proven by Zang and colleagues [@pone.0069199-Zang1]. Recently, Zuo et al. [@pone.0069199-Zuo1] reported that the ReHo measurement has good test-retest reliability. Taken together, the ReHo method has been shown to be an excellent metric for quantification and detection of the functional pathological changes in neurologic disorders and neurodegenerative diseases [@pone.0069199-You1]--[@pone.0069199-He1]. However, to our knowledge, no report has applied ReHo in ET study.

The aim of this study was to quantify the abnormalities of local-range functional connectivity using ReHo on RS-fMRI in ET. For this purpose, we performed group comparison on ReHo maps between ET and healthy control (HC) groups. Second, several regions of interests (ROIs) were defined based on the group differences, followed by correlation analyses between the ReHo value in those ROIs and ET clinical assessments. Finally, an abnormal brain model with regards to various functional systems was proposed to link the brain dysfunctions with the ET symptoms in different dimensions.

Materials and Methods {#s2}
=====================

Demographic and Clinical Evaluation {#s2a}
-----------------------------------

The diagnosis of ET was made according to the National Institute of Health Corroborative Genetic Criteria [@pone.0069199-Louis4]. None of the patients had Parkinson's disease (PD) or dystonia. Twenty ET patients (8 females; age: 50.3±14.2 years) and 20 gender- and age-matched HC subjects were included in our study. All subjects were right-handed and cognitively unimpaired without significant difference between the two groups (Mini Mental State Examination [@pone.0069199-Tessitore1], or MMSE *\>*24, *p* = 0.56). The mean age at onset of tremor was 35.3±9.9 years and mean duration was 14.6±7.7 years.

All patients had hand tremor(s), 2 patients with lower limb tremor(s), 5 patients with head tremor and 2 with voice tremor. A family history of tremors was present in 11 patients. For all the ET patients, this study was their initial visit, and before the RS-fMRI examination, all of them had no medication history. Alcohol ingestion history was positive for 2 patients and negative for 5 patients; the remain13 patients had never intoxicated but were unable to answer the question about alcohol to alleviate symptoms. The severity of tremor(s) was mild to severe, the mean TRS-A&B score was 21.1±14.7, and the mean TRS-C score was 6.9±4.9 (see [Table 1](#pone-0069199-t001){ref-type="table"} and [Table S1](#pone.0069199.s002){ref-type="supplementary-material"}).

10.1371/journal.pone.0069199.t001

###### Demographic and clinical features of ET patients and HCs.

![](pone.0069199.t001){#pone-0069199-t001-1}

  Measure                                                     ET patients                Healthy controls   *P*-value
  --------------------------------------------- --------------------------------------- ------------------ -----------
  Demographic
  Age                                                          50.3±14.2                    50.3±14.2         1.00
  Gender (male : female)                                         12∶8                          12∶8           1.00
  Handedness (right : left)                                      20∶0                          20∶0
  Cognitive function
  MMSE                                                         26.0±2.5                      27.0±2.7         0.56
  Clinical                                                                                NA
  Age of onset (years)                                         35.3±9.9                         NA
  Disease duration (years)                                     14.6±7.7                         NA
  Body parts with tremor                                                                        NA
   Upper limb                                                 20 (20/20)                        NA
   Lower limb                                                  2 (2/20)                         NA
   Head                                                        5 (5/20)                         NA
   Voice                                                       2 (2/20)                         NA
  Family history                                              11 (11/20)                        NA
  Medication                                                     None                           NA
  Response to alcohol                            2+ (2/20), 5-- (5/20), 13 CAN (13/20)          NA
  Fahn-Tolosa-Marin Tremor Rating Scale (TRS)                                                   NA
   TRS-A&B                                                     21.1±14.7                        NA
   TRS-C                                                        6.9±4.9                         NA

ET: essential tremor, HCs: healthy controls, NA: not applicable, +: positive, −: negative, CAN: cannot answer.

All subjects recruited into the study gave their written informed consent approved by the Ethics Committee for the First Affiliated Hospital of Chongqing Medical University in accordance with the Declaration of Helsinki.

Clinical Assessment {#s2b}
-------------------

ET patients were evaluated by two movement disorder specialists (O Chen andxWang). Age at onset, disease duration, body parts with tremor, family history and, medication and alcohol usage in relation to alleviate symptoms were recorded. The severity of the tremor was assessed using Fahn-Tolosa-Marin Tremor Rating Scale (TRS) [@pone.0069199-Louis5]. This scale is composed of three parts: TRS part A, B and C. The TRS part A and B were combined to obtain a single score: TRS-A&B. The TRS-A&B was used primarily in evaluation of tremor severity, location and, the drawing and writing function of hand. This was performed and recorded via videotape record and blinded to the RS-fMRI results. The TRS-C was assessed via self-evaluation to evaluate quality of life for ET patient.

Image Acquisition {#s2c}
-----------------

All MR images were acquired using a GE Signa Hdxt 3.0T scanner (General Electric Medical Systems, USA) with a standard 8-channel head coil. Foam padding was used to minimize the head motion and ear plugs were used to reduce scanner noise. Apart from this, no other special methods were employed to prevent head movement. During RS-fMRI acquisition, all subjects were told to relax, and keep still with eyes closed, but to remain awake (confirmed with post-scan debriefing).

RS-fMRI data were acquired using an echo-planar image (EPI) pulse sequence with 33 axial slices, thickness/gap = 4.0/0 mm, matrix = 64 × 64, TR = 2000 ms, TE = 40 ms, flip angle = 90°C, FOV = 240 × 240 mm. A total of 240 time points was obtained in 8 min.

High-resolution 3D-T1 (TR = 8.3 ms, TE = 3.3 ms, flip angle = 15°, thickness/gap = 1.0/0 mm, FOV = 240 × 240 mm, matrix = 256 × 192) and T2-FLAIR-weighted images (TR = 8000 ms, TE = 126 ms, TI = 1500 ms, thickness/gap = 5.0/1.5 mm, FOV = 240 × 240 mm, matrix = 256 × 192) were also acquired. We did not use T2-FLAIR-weighted images for data processing but for clinical evaluation and data quality assessment (see *Quality assurance*).

Image pre-processing {#s2d}
--------------------

Data pre-processing was conducted using the toolboxes of DPARSFA on version 2.1 ([www.restfmri.net](http://www.restfmri.net), [@pone.0069199-ChaoGan1]) and REST on version 1.8 ([www.restfmri.net](http://www.restfmri.net), [@pone.0069199-Song1]). It was mostly consistent with previous studies using ReHo [@pone.0069199-Zang1], [@pone.0069199-Wu1], [@pone.0069199-Zhang1], [@pone.0069199-Tian1] and consisted of following steps briefly listed here: 1) removal of the first 10 time points, 2) slice timing correction, 3) realignment (for detailed discussion on head motion please see *Quality assurance*), 4) unified segmentation using 3D-T1 images and spatial normalization using the deformation parameters and, 5) time course de-trending and band-pass filtering (0.01−0.08 Hz) (see [Text S1](#pone.0069199.s004){ref-type="supplementary-material"} for details). As described in previous studies [@pone.0069199-Zuo1], [@pone.0069199-Zhang1], spatial smoothing artificially enhances the ReHo intensity. This process was not performed during pre-processing. To improve Gaussianity for statistical analyses, the spatial smoothing was carried out after ReHo calculation. For observing small structures that were hypothesized to have abnormality in ET (e.g., ventral intermediate nucleus), we used a smaller smoothing kernel with FWHM of 4×4×4 mm^3^).

Quality Assurance {#s2e}
-----------------

The T1 and T2-FLAIR images were inspected by an experienced neuroradiologist (F Lv or T Luo). All subjects had no obvious abnormalities in gross brain structure. We noted that the head-motion is an important factor in RS-fMRI studies [@pone.0069199-Zuo1] and, especially, in the ET studies. To control the potential negative effect caused by excessive head motion (which may stronger for ET patients than HC), we adopted a strict criterion for head movements assessment (less than 1.0 mm and 1.0 degree in the *x*, *y* and *z* directions) and compared the maximal absolute head movement between the two groups. Despite having 5 patients with head tremors involved in our study, these tremors appeared as a postural and intentional. They did not resemble another tremor disease, such as Parkinson's disease (PD patients have obvious resting-state tremors). Therefore, the head tremor was not expected to be a serious problem. All subjects' head movement did not exceed the exclusion criteria, and the maximum absolute head movement showed no significant group difference (*p* = 0.35).

ReHo Calculation and Statistical Analyses {#s2f}
-----------------------------------------

Individual ReHo image was generated for each subject by calculating Kendall's coefficient of concordance (KCC) at each voxel between the time series of this voxel and those of its 26 neighboring voxels within a whole-brain mask (this mask is provided by DPARSFA, excluding non-brain areas). Then, each subject's ReHo image was standardized with regard to the global mean by dividing the ReHo value at each voxel by the averaged ReHo across the whole-brain (using the same whole brain mask). This global mean normalization method has been adopted as an ordinary processing procedure in previous ReHo studies [@pone.0069199-Zang1], [@pone.0069199-Wu1], [@pone.0069199-Zhang1], [@pone.0069199-Tian1]. We also compared the global mean ReHo between two groups. No significant difference was found (two-sample *t* test, *p* = 0.82). The other reason we used this normalization method is to avoid spurious negative values that may be introduced by other methods (e.g., *z*-transformation), which are difficult to interpret.

One-sample *t* tests with AlphaSim multiple comparison correction was performed to compute the group-specific ReHo maps for each group, and to determine the brain regions with ReHo value significantly larger than the global mean ReHo. The brain regions with significant *t* values for both groups were combined to form a mask for a between-group comparison.

To detect group difference in ReHo, a two-sample *t*-test with AlphaSim multiple comparison correction was performed within the mask generated by the one-sample *t*-tests. Of note, the mean-corrected ReHo maps (i.e., standardized ReHo), rather than the raw ReHo maps were fed into the two-sample t test. Meanwhile, we also performed a two-sample *t*-test within the whole-brain mask to further explore the potential group difference in a pure data-driven manner.

Clinical Correlation Analyses {#s2g}
-----------------------------

Based on the two-sample *t*-test findings, the abnormal ReHo brain regions of ET were identified as ROIs. Then, the mean ReHo value in each ROI for all the subjects was extracted, and correlated by using Pearson's correlation with the clinical assessment, including the TRS-A&B scores, the TRS-C scores, the age at onset and the disease duration. To improve data normality, the clinical scores were converted to *z* scores before the correlation analyses [@pone.0069199-Tessitore1]. Kolmogorov-Smirnov tests were conducted to assess the normality of the *z* scores, showing good normality (*z*TRS-A&B: *Z* = 0.65, *p* = 0.78; *z*TRS-C: *Z* = 0.97, *p* = 0.30; *z*Age at onset: *Z* = 0.66, *p* = 0.76; *z*Disease duration: *Z* = 0.59, *p* = 0.86).

VBM Analysis {#s2h}
------------

To check out if there was structural difference between ET and HC, the high-resolution 3D-T1 images were subjected to VBM analysis using DPARSFA software. Briefly, the T1 images were segmented into grey matter (GM), whiter matter (WM) and cerebrospinal fluid (CSF) by using unified segmentation, and then normalized to MNI space. After that, two-sample *t*-tests with false discovery rate (FDR) corrections were performed to investigate group difference in GM and WM density.

Results {#s3}
=======

Within-group Analyses {#s3a}
---------------------

The within-group ReHo t-maps for the ET and the HC group were shown in [Figure 1](#pone-0069199-g001){ref-type="fig"}. For both groups, extensive grey matter regions showed significant larger-than-global-mean ReHo values. Those regions include sensorimotor areas, visual areas, auditory areas, executive networks, prefrontal cortex, insula, middle and inferior temporal cortex, medial and lateral parietal cortex, cerebellum, striatum, thalamus and the default mode network. Compared with the HC group, the ET group showed extensive ReHo reductions, primarily located within the cerebellum and the thalamus.

![Results from one-sample *t*-test on ReHo maps for ET group (upper) and HC group (lower).\
Threshold was set to *p*\<0.05 with AlphaSim correction (cluster size \>2295 mm^3^). The underlying structure image is *Ch2* image.](pone.0069199.g001){#pone-0069199-g001}

Group Difference Analyses {#s3b}
-------------------------

[Figure 2](#pone-0069199-g002){ref-type="fig"} shows the group difference in ReHo within one-sample *t*-test mask between the two groups. Significant ReHo decrease was found in extensive cerebellar cortices, including the anterior (lobules III∼V) and posterior (bilateral lobules VIII and left VI) bilateral cerebellar lobes, the posterior insular lobes and the bilateral thalamus which included mediodorsal (MD) and ventral intermediate (VIM) thalamic nucleus. A significant ReHo increase were found in the left primary motor cortex, the left supplementary motor area, the bilateral prefrontal cortices (including dorso- and ventro-lateral prefrontal cortex, orbitofrontal cortex, and the medial prefrontal cortex) and the bilateral parietal lobes (including the angular gyrus, the supramarginal gyrus and the inferior parietal lobule). The details of the peak coordinates, cluster size and Brodmann's areas are listed in [Table 2](#pone-0069199-t002){ref-type="table"}. Meanwhile, the group difference results found within the whole brain mask are shown in [Figure S1](#pone.0069199.s001){ref-type="supplementary-material"} and [Table S2](#pone.0069199.s003){ref-type="supplementary-material"}. The two results with different masks were quite similar.

![Difference in ReHo value between ET and HC groups.\
The result was derived from two-tailed two-sample *t* test within combined one-sample *t*-test mask (*p*\<0.05, AlphaSim corrected with cluster size \>351 mm^3^). Warm color indicates the regions with larger ReHo value in ET than HC, while cold color indicates those with decreased ReHo in ET. The underlying structure image is *Ch2* image.](pone.0069199.g002){#pone-0069199-g002}

10.1371/journal.pone.0069199.t002

###### Differences in ReHo between ET patients and HCs.

![](pone.0069199.t002){#pone-0069199-t002-2}

  Brain region                                MNI coordinates   *t* value   cluster size (voxels)
  ------------------------------------------ ----------------- ----------- ----------------------- -------- ----
  R Cerebellum VIII                                 20            --55              --60            --2.97   83
  L Cerebellum VIII                                --33           --58              --45            --2.78   96
  L Cerebellum VI                                  --30           --50              --30            --3.92   86
  L Cerebellum IV, V                               --24           --41              --30            --3.45   88
  L Cerebellum III                                  --7           --38              --15            --2.91   22
  R Cerebellum IV, V                                14            --43              --15            --3.25   55
  R Cerebellum III                                  10            --38              --15            --2.91   18
  L Brainstem (inferior olivary nucleus)            --3           --40              --45            --3.26   18
  R Brainstem (inferior olivary nucleus)             4            --40              --45            --3.08   21
  R Thalamus (ventral intermediate, VIM)            16            --22                0             --2.86   23
  L Thalamus (VIM)                                 --14           --20                0             --3.12   21
  R Thalamus (mediodorsal, MD)                       6            --12                0             --2.91   24
  L Thalamus (MD)                                   --5           --17                0             --2.45   26
  L Insula                                         --41           --13               15             --3.16   49
  R Insula                                          44             --6                0             --3.23   21
  R Superior frontal gyrus orbital part             28             64                 0              2.42    28
  R Inferior frontal gyrus triangular part          44             35                15              2.80    32
  R Middle frontal gyrus                            38             37                30              4.05    41
  R Superior frontal gyrus                          18             52                30              2.58    27
  L Superior frontal gyrus orbital part            --45            37               --15             3.76    43
  L Middle frontal orbital part                    --40            48                 0              3.38    51
  L Inferior frontal gyrus triangular part         --50            36                 0              3.55    19
  L Middle frontal gyrus                           --40            37                30              4.01    37
  L Supplementary motor area                        --6            14                45              4.09    52
  R Supplementary motor area                         4             18                60              3.12    56
  L Precentral gyrus                               --49            --1               45              3.45    43
  R Supramarginal gyrus                             64            --41               30              2.89    37
  L Supramarginal gyrus                            --61           --40               30              2.89    29
  R Inferior parietal gyrus                         46            --57               45              3.15    19
  L Inferior parietal gyrus                        --46           --50               45              2.34    29
  R Angular gyrus                                   51            --51               30              2.86    21

ET: essential tremor, HCs: healthy controls, R: right, L: left.

Correlation Analyses {#s3c}
--------------------

There were 24 clusters which showed significantly abnormal ReHo values in ET compared with HC groups, which were further defined by 24 ROIs. Correlation analyses for each ROI revealed 4 ROIs with significant correlations with the *z*TRS-A&B scores in the ET group. For other ROIs and other clinical scores including the TRS-C scores, the age at onset and the disease duration, the correlations were non-significant. Specifically, a significantly negative correlation was found in the bilateral cerebellar lobules III∼V (peak MNI coordinates for the left one: *x* = −12, *y* = −19, *z* = −18, *r* = −0.69, *p*\<0.01; the right one: *x* = +14, *y* = −43, *z* = −18, *r* = −0.57, *p*\<0.01, both uncorrected) and the right cerebellar lobule VIII (*x* = +19, *y* = −54, *z* = −53, *r* = −0.70, *p*\<0.01, uncorrected). Significant positive correlation was observed in the left precentral cortex (*x* = −44, *y* = +2, *z* = +52, *r* = 0.62, *p*\<0.01, uncorrected) (For the location of these ROIs, box and scatter plots, please see [Figure 3](#pone-0069199-g003){ref-type="fig"}.) Please note that, because of the relatively small group size and the exploratory researching methods, we used an uncorrected *p-*value of 0.01 (rather than a stringent multiple comparison correction method, e.g., Bonferroni correction).

![ROIs with abnormal ReHo in ET group compared with HCs and the correlation between mean ReHo values in each ROI and TRS-A&B scores.\
The left panel shows the average ReHo value in each ROI for subjects in both ET and HC groups. The middle panel shows the location of the ROIs. The right panel shows the scattered maps of the mean ReHo against the TRS-A&B scores in regions with significant (*p*\<0.01, uncorrected) correlations. TRS-A&B: the combination between the scores of the Fahn-Tolosa-Marin Tremor Rating Scale (TRS) part A and B.](pone.0069199.g003){#pone-0069199-g003}

VBM Analysis {#s3d}
------------

The VBM analysis did not detect any significant differences in either grey or white matter density between our study groups. This was consistent with part of the previous studies [@pone.0069199-Daniels1], [@pone.0069199-Quattrone1], [@pone.0069199-Klein1]. Therefore, grey or white matter density may not potentially affect the RS-fMRI result, and thus was not taken into account as nuisance covariables in the two-sample *t*-test.

Discussion {#s4}
==========

We used the ReHo metric of RS-fMRI to reveal the local functional connectivity abnormality in ET patients compared with HC. We found that the ReHo abnormality was mostly located in the cerebello-thalamo-cortical motor pathway and non-motor cortices (including prefrontal, parietal and insular lobes). We propose that the disruption of local functional connectivity in the cerebello-thalamo-cortical motor pathway is involved in tremor generation and propagation of ET. Conversely, the abnormal ReHo in the prefrontal, parietal and insular lobes may take responsibility for the non-motor symptoms (e.g., cognitive, psychiatric and sensory deficits). The VBM analysis did not detect significant grey or white matter atrophy between our study groups. Each of those abnormal brain regions we found will be discussed respectively in the following sections.

Cerebellum {#s4a}
----------

There is a constant debate on whether the cerebellum acts as a tremor generation structure or not in ET. On the one hand, there is a bulk of evidence from histopathological [@pone.0069199-Louis1], electrophysiological [@pone.0069199-Hellwig1], neuroimaging [@pone.0069199-Raethjen1] and treatment effect [@pone.0069199-Popa1] which support that the cerebellum plays a crucial role in ET. However, opposing views are constantly reported [@pone.0069199-Rajput1], [@pone.0069199-Rajput3], [@pone.0069199-Buijink1]. Our study, which found that extensive cerebellar ReHo abnormalities exist in ET, further lends support to the evidence and reinforces the point of the major role of the cerebellum in ET.

First, we found that cerebellar ReHo abnormality was not only located within the spino-cerebellum (the bilateral anterior cerebellar lobules III∼V), but also extended to the cerebro-cerebellum (the bilateral posterior cerebellar lobules VIII and the left lobule VI). It appears that the extensive ReHo abnormalities in the cerebellum were not associated merely with ET pathophysiological basis. As mentioned in the *Introduction* section, ET was typically seen as a mono-motor symptomatic disease, with only superior part of the cerebellum was involved. However, the evolving concepts now regard ET as heterogeneous disorders accompanied by a lot of movement and non-motor symptoms [@pone.0069199-Teive1], [@pone.0069199-Louis6], [@pone.0069199-Louis7]. Several lines of evidence from clinical [@pone.0069199-Santangelo1]--[@pone.0069199-Fasano1], neuroimaging [@pone.0069199-Boecker2], [@pone.0069199-Breit1] and functional topography of the cerebellum [@pone.0069199-Stoodley1], [@pone.0069199-Stoodley2] supported our finding that the extensive cerebellar ReHo abnormalities are involved in pathological changes of ET. First, clinical studies supported the cerebellar ReHo abnormalities involved in these movement and non-motor symptoms. Despite this, tremors are the cardinal clinical features of ET. There are multiple-component motor and non-motor dysfunctions in ET. Balance and gait disorder [@pone.0069199-Hoskovcova1], [@pone.0069199-Stolze1], ataxia and eye-blink conditioning deficits [@pone.0069199-Kronenbuerger1], [@pone.0069199-Kster1] was associated with spinocerebellar impairment. Impaired reaching and hand function [@pone.0069199-Avanzino1] suggests spinocerebellar and cerebrocerebellar dysfunctions. Working memory deficits, cognitive disorder, personality disorder and social dysfunction [@pone.0069199-Teive1], [@pone.0069199-Chandran1] are also related to cerebrocerebellar dysfunction. The cerebellar ReHo abnormalities in our study offer a plausible explanation to support the notion that the extensive cerebellar dysfunction is associated with the heterogeneous symptoms in ET. Secondly, neuroimaging studies support the extensive cerebellar dysfunction linked with ET diverse symptoms. Evidence from task-designed fMRI [@pone.0069199-Bucher1]--[@pone.0069199-Cerasa2] and Positron Emission Tomography (PET) [@pone.0069199-Perlmutter1] showed that the abnormal activations in different cerebellar regions while ET patients were performing motor and non-motor tasks. These cerebellar areas were consistent with our result. In contrast to the task-based approach which requires on complex task stimulus, the RS-fMRI method provides an approach to identify simultaneously the cerebellar motor and non-motor networks using the same dataset without any stimulus. Therefore, the RS-fMRI technique is more suitable for observing these network changes in the pathological state of ET in the clinical realm. Finally, evidence from the functional topography of the cerebellum [@pone.0069199-Stoodley1], [@pone.0069199-Stoodley2] supported the finding that the widely cerebellar ReHo abnormalities caused heterogeneous disorders in ET. The different region of the cerebellum is associated with different physiological functions. Motor and non-motor task-designed fMRI studies showed the finger-taping stimuli often activated the anterior and posterior cerebellar lobes [@pone.0069199-Stoodley1], [@pone.0069199-Stoodley2]; and a non-movement stimulus (such as language, spatial processing and working memory tasks) often activated the posterior cerebellar lobe [@pone.0069199-Stoodley1], [@pone.0069199-Stoodley2]. Taken together, we speculated that ReHo abnormalities in these areas may be a hallmark of physiological dysfunction, which later appears as a heterogeneous disorder in ET. In application, our study provided more targets for cerebellar repetitive Transcranial Magnetic Stimulation (rTMS) for ET treatment. We propose that applying rTMS on our derived cerebellar regions with abnormal ReHo values, rather than traditionally only on the anterior or posterior cerebellar lobes, could largely improve patient's quality of life by reducing the tremor symptom while restoring the impaired high-order functions.

Secondly, we found a different result in ET when compared with another tremor disease, PD. Literature [@pone.0069199-Baudrexel1] has reported that ET and PD shared similar clinical manifestations and a tremor-generation central network. Wu and colleagues [@pone.0069199-Wu1] also used the RS-fMRI method and showed that PD patients had increased ReHo values in the very cerebellar regions that we reported, although our ReHo values were decreased. We postulated that such a difference may reflect the pathological difference between the two diseases. It has been observed that the loss of cerebellar Purkinje cells probably involved in the generation of tremors in ET [@pone.0069199-Louis1]. The Purkinje cells are the only efferent neurons in cerebellum, which coordinate the function between the cerebellar cortices and the deep cerebellar nucleus [@pone.0069199-Louis8]. While it was damaged, the local concordance and connectivity would decrease and lead to a deceased ReHo value. However, no pathological lesions in the cerebellum have been found in PD, and the PD tremors were believed to be mainly driven by the dysfunction of the globus pallidus loops [@pone.0069199-Helmich1]. We proposed that, in contrast to cerebellar ReHo increase which may be a compensation mechanism in PD [@pone.0069199-Wu1], the cerebellar ReHo decrease in ET maybe a direct result of the cerebellar pathological damage.

Prefrontal/Parietal/Insular Cortices {#s4b}
------------------------------------

Interestingly, our study found that extensive ReHo abnormalities in the non-motor cortices, including the prefrontal, parietal cortices and the posterior insular lobes. These brain regions did not involve in the tremor symptom [@pone.0069199-Raethjen1], but were included in the cognitive control network, default mode network and affective networks [@pone.0069199-Sheline1]. Evidences from task fMRI [@pone.0069199-Coste1] and RS-fMRI [@pone.0069199-Sheline2] have shown that dysfunctions in these networks may result in various non-motor disorders, including cognitive deficits, dementia, anxiety, depression and sleeping deficit. These may explain why ET patients have heterogeneous non-motor symptoms. Meanwhile, epidemiological research [@pone.0069199-BermejoPareja1] has revealed that the risk of developing dementia in patients with ET was significantly higher. This indicated that ET patients may share the similar cognitive impairment with AD patients. Recent RS-fMRI studies have revealed that ReHo was decreased in extensive brain areas in AD patients, including prefrontal, parietal cortices and default mode network. We also observed ReHo changed in the similar areas, but our findings were increased ReHo values, and ET patients in our study had no dementia, as confirmed by MMSE scores [@pone.0069199-Galvin1]. Therefore, we proposed that ReHo abnormalities in these brain areas may serve as a compensatory mechanism which prevents ET patients from apparent dementia symptoms. At the same time, ET patients already have potential cognitive dysfunction.

Thalamus {#s4c}
--------

Our study found that two thalamus regions, VIM and MD, showed abnormal ReHo values. To the best of our knowledge, this is the first report that the spontaneous brain activity abnormality existed in the thalamus of ET patients. Studies [@pone.0069199-Baudrexel1], [@pone.0069199-Molnar1] have shown that there are functional and structural connections between thalamus, cerebellum and the cortices. The VIM (local maxima of group difference was at \[−14, −20, 0\] in MNI space for the left, and \[+16, −22, 0\] for the right) connects cerebello-VIM-motor pathway, through which the cerebellum modulates motor function. This pathway has been proposed to be responsible for tremor propagation in ET patients [@pone.0069199-Baudrexel1]. The MD (local maxima was at \[−5, −17, 0\] for the left, and \[+6, −12, 0\] for the right) is another large nucleus in thalamus, and connects the prefrontal cortices, limbic system and cerebellum, through which the cerebellum modulates non-motor function [@pone.0069199-Lenglet1]. Our study indicated that thalamus is not only involves in the motor dysfunction but also in the non-motor dysfunctions. In future, specific cognitive evaluation will help to understand its mechanism.

Motor Cortex {#s4d}
------------

The motor cortex is involved in generation of pathological tremor in ET, PD and cortical myoclonus patients [@pone.0069199-Shibasaki1]. However, the way of its involvement differs between the cortical myoclonus and ET or PD. Cortical myoclonus originates directly from primary motor cortex (M1) and is related to the hyper-excitability of the primary motor areas. In contrast, tremors in ET or PD arise from subcortical structures, i.e., the cerebello-thalamic circuit [@pone.0069199-Helmich1]. The motor cortex is considered to only serve as a mediated cortex of the oscillations. Therefore, the motor cortex should not be regarded as a substantial tremor generation site. As a result, we proposed the ReHo abnormality in the motor cortex might just be a direct consequence of the functional abnormality in the cerebello-thalamic pathway.

Inferior Olivary Nucleus {#s4e}
------------------------

Animal studies have demonstrated the inferior olivary nucleus is involved in tremor generation for ET [@pone.0069199-Hellwig2]. Our finding of decreased ReHo in the inferior olivary nucleus was consistent with previous reports [@pone.0069199-Hellwig2]. It must be noted that the spatial resolution of the fMRI in our experiment was low, and abnormality in such a small brain structure should be interpreted with caution.

Correlation Analyses {#s4f}
--------------------

Our study showed the ReHo abnormalities in the cerebellum and left primary motor cortex correlated with the tremor severity score in ET patients. Previous studies [@pone.0069199-Shibasaki1], [@pone.0069199-Muthuraman1] showed that these areas are involved in voluntary and involuntary body movement, and dysfunction in these areas will lead to pathological tremors. Therefore, our correlation analyses provide additional evidence that abnormal ReHo in cerebellum and motor-related cortices are involved in the tremor generation and propagation for ET. Meanwhile, no significant correlation was found between the mean abnormal ReHo values in other brain areas and clinical assessment, including TRS-C score, age at onset and disease duration. Perhaps an explanation for this was the reliability of the TRS-C score was low (based on self report), or the ReHo metric was not sensitive to these clinical assessments.

Limitations {#s4g}
-----------

We found extensive brain dysfunctions reflected by altered ReHo, which indicated pathological changes of ET. However, we could not conclude that ET is a neurodegenerative disease merely based on present findings due to this study is cross-sectional by nature and the absence of correlation with disease duration. In future, a longitudinal or follow-up study with more homogenous samples would help to fully understand this feature. Meanwhile, despite abnormal ReHo found in the cerebello-thalamo-cortical motor pathway and non-motor cortices, the relationship between these nodes cannot be revealed using the current method. The two regions may have some relationships. However, such relationships might be caused by a third region or other reasons. The correlation between the ReHo changes in frontal lobe and those in cerebellum should be interpreted with caution. For example, we cannot decide whether the frontal lobe dysfunction was originated from the malfunctioned cerebellum or was just part of the extensive dysfunctions in ET. A long-range functional connectivity or a DTI study should be done in future.

Methodological/Reliability Concerns {#s4h}
-----------------------------------

The pre-processing (and post-processing) procedure is an important methodological issue for RS-fMRI studies. No consensus has been obtained in the literature [@pone.0069199-Zuo1]. However, this factor plays an important role in the correctness and reliability of the results. Few studies have investigated the test-retest (TRT) reliability of different derivatives from resting-state fMRI, such as seed-based functional connectivity [@pone.0069199-Shehzad1], ICA [@pone.0069199-Zuo2] and amplitude of low frequency fluctuations (ALFF) [@pone.0069199-Zuo3]. Meanwhile, there is already a pioneering study [@pone.0069199-Zuo1] systemically investigating these factors and their contributions to the TRT reliability of ReHo. The study recommended regressing out head motion, white matter and CSF signals, but not removal of the global signal in ReHo computation [@pone.0069199-Zuo1]. The study also found that the spatial smoothing will significantly influent the result reliability [@pone.0069199-Zuo1]. The study [@pone.0069199-Zuo1] also compared different head motion correction methods and found there was no significant difference on the TRT reliability between a traditional 6-parameter rigid transformation and newly proposed micro-head motion correction [@pone.0069199-Satterthwaite1]--[@pone.0069199-Power2]. Since our study mainly deals with a brain disease, we decided not to systematically compare the results between various pre-processing methods, which include nuisance regressing, micro-head motion and the size of the spatial smoothing kernel. Another reason we did not do this is because we wanted our results to be compared with previous PD study [@pone.0069199-Wu1] using the same data processing method.

Conclusions {#s4i}
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Using ReHo of RS-fMRI as a metric, we revealed the local functional connectivity abnormality in ET patients compared with HCs. These abnormalities were mostly located in the cerebello-thalamo-cortical motor pathway and non-motor cortices, including the prefrontal, parietal and insular lobes. Our analyses indicated that the ReHo abnormalities in different regions were associated specifically with motor and non-motor symptoms of ET. We demonstrated that our approach can be used to detect and quantify the functional pathological changes in ET.

Supporting Information {#s5}
======================

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**Difference in ReHo value between ET and HC groups within a whole brain mask.** Threshold was set to be *p*\<0.05 with AlphaSim correction. Warm color indicates the regions with larger ReHo value in ET than HC, while cold color indicates those with decreased ReHo in ET. The underlying structure image is *Ch2* image.

(TIF)

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Click here for additional data file.

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**Detailed demographic and clinical features of ET patients.**

(DOCX)

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Click here for additional data file.

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**The brain areas with group differences in ReHo value between ET and HC groups within a whole brain mask.**

(DOCX)

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Click here for additional data file.

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**The detailed data preprocessing steps.**

(DOCX)

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Click here for additional data file.

[^1]: **Competing Interests:**The authors have declared that no competing interests exist.

[^2]: Conceived and designed the experiments: WF TL HZ. Performed the experiments: WF. Analyzed the data: WF. Contributed reagents/materials/analysis tools: FL OC KS YH FW XW JL. Wrote the paper: WF HZ. Revised the manuscript: WL QXY TL HZ.


THE PT BLOG

Here is Tim Galamaga crushing a sled push in this video. Tim is captain of Bedford High’s football team and is 3 months post-op after a complete knee reconstruction. His hard work and dedication will get him back on the field and reaching all his athletic and academic goals.

Post surgical rehab success! Moe had a great outcome with his rehab following rotator cuff repair. He has returned to his active lifestyle with no limitations. Here at AITC we strive for the best possible outcomes in the shortest time possible.

Greg is seen here pulling a sled as part of his physical rehabilitation program after his 6th knee surgery. We are using a functional movement to strengthen his quad while also working on full body strength and conditioning. We treat all of our clients like athletes to their fullest capabilities for their age and condition. […]

Erin came to AITC with progressive knee pain including a history of back pain. She had been diagnosed with Illiotibial band syndrome. This illiotibial band pain was reproduced with a test for sciatica. Because there seemed to be a back component to her symptoms, we addressed this to help resolve her knee pain. Erin worked […]

Christopher came in with a history of 3 years of chronic back pain. His main findings were lumbar joint hyper mobility (loose joints) and weakness from sciatic nerve irritation. His pain ranged from 2/10 to 10/10 and his self-reported disability was at 72% limitation. On his last visit, he had no pain and 0% self-reported […]

This patient presents with chronic right shoulder pain with limited mobility. The first picture is before today’s treatment. The second picture is post treatment after 5 minutes of active release technique. You can clearly see a dramatic change in range of motion with no pain […]

What is anterior pelvic tilt? APT is a change in posture that happens when the front of the pelvis drops forward, and the back of the pelvis rises. The condition pulls the abdominal muscles into a stretched position, which over time leads to a drastically weakened core. The glutes and hamstrings also suffer from being […]

Metabolic Resistance Training: Burn Fat and Build Muscle in Less Time! In about an hour, with lecture learn how to: Maximize Fat Burning and Muscle Building With a Short Session How MRT Enhances Muscles and Fat Loss, as well as Overall Health How to Set up Your Own MRT Training Sessions—Short Sweet and Highly Effective […]


George Grljusich

George Ned Grljusich (15 January 1939 – 6 November 2007) was an Australian sports journalist, commentator and former Australian rules footballer. Born in Wiluna, Western Australia, he played 12 games of football for the South Fremantle Football Club in the Western Australian National Football League in 1960, before quitting football to pursue a media career. Grljusich later became a radio broadcaster, commentating for Perth-based radio stations 720 ABC Perth and 6PR. He died in 2007 at the age of 68, from lung cancer.

Education
Grljusich studied Law at the University of Western Australia, where he graduated in the same year as the future Chief Justice David Malcolm, Crown Solicitor (WA), Peter Panegyres and Governor of Western Australia, Malcolm McCusker, but decided to remain in the media rather than practice law. In 1977 Grljusich stood unsuccessfully for the state Seat of Cockburn.

Playing career
Grljusich made his West Australian National Football League debut for South Fremantle in 1960 on the same day as his brother Tom. While Tom went on to play over 200 matches for the Bulldogs, George quit football at the end of the 1960 season to pursue a media career. Two more of his brothers also played at WANFL league level - Don for South Fremantle and John for East Fremantle.

Media career
His journalistic career focussed on radio sports commentary including a long running stay as the main sports announcer at 720 ABC Perth. From 1996 he worked at commercial radio station 6PR. In radio he commentated mainly on harness racing, soccer and football, as well as hosting weekly sports talkback radio, where his abrasive style often polarised his listeners.

Grljusich provided ABC coverage of six Olympic Games: 1980 at Moscow, 1984 at Los Angeles, 1988 at Seoul, 1992 at Barcelona, 1996 at Atlanta and 2000 at Sydney, as well as ten Commonwealth Games. In 2004, the media box at the Fremantle Oval was officially renamed the George Grljusich Media Box in honour of the veteran broadcaster.

In October 2007, Grljusich retired from broadcasting after being diagnosed with terminal lung cancer. He died at St John of God Hospital in Murdoch on 6 November 2007, aged 68.

In November 2007, Radio 6PR announced the establishment of The George Grljusich Sport Broadcasting Award for students of the WAAPA Broadcasting course.

References

External links
 Obituary

Category:1939 births
Category:2007 deaths
Category:Australian people of Croatian descent
Category:Australian radio personalities
Category:Australian rules football commentators
Category:Australian rules footballers from Western Australia
Category:Deaths from lung cancer
Category:People from Wiluna, Western Australia
Category:South Fremantle Football Club players
Category:West Australian Football Hall of Fame inductees


Q:

How can I calculate the uncertainty of two combined uncertainties?

If uncertainty can be calculated as half the range, and percentage uncertainty is the uncertainty over a mean all multiplied by 100, how can I find what the uncertainty is of A^2 * B^3 when A = 21.3 and the uncertainty of A is +-0.14, and B = 4 and the uncertainty is +-0.01?
Am I right in thinking you just do +-0.14*2 added to +-0.01*3?

A:

If we assume that $A_\text{min} \leq A \leq A_\text{max}$ and $B_\text{min} \leq B \leq B_\text{max}$, and wish to work out $\min{A^2B^3}$ and $\max{A^2B^3}$, the following is useful. (See Mark's answer for discussions on probability, spread, independence etc). This is meant more to be a "calculate rough bounds for school physics" level answer.
The best way to do these questions is to work out the extremes of values and plug them in - though this might not be how you have been taught.
The lowest value is taking: $A = A_\text{min} = 21.3 - 0.14 = 21.16$, $B = B_\text{min} = 4 - 0.01 = 3.99$, thus min answer $ = A^2 B^3 = 28441.33736$
Similarly, $A_\text{max} = 21.3 + 0.14 = 21.44$, $B_\text{max} = 4.01$, max answer $= 29640.3058$.
Actual calculated answer $ = 21.3^2 \ast 4^3 = 29036.16$
So we have:
\begin{align*}
A^2 B^3 = 29036.16 \begin{cases} + & 604.145796 \\ - & 594.82264 \end{cases}
\end{align*}
(Round as appropriate)
Of course...
You may well have been taught "if you multiply or divide, add the percentage errors". This gives approximately the right answer in most cases, in the above, we get A%Error = 0.6572769953...%, B%Error = 0.25...%, so total percentage error is: $2 \ast 0.6572769953...\% + 3 \ast 0.25\% = 2.064553991...\%$
This gives a rough absolute final error of $\pm 29036.16 * 2.064553991\% = \pm 599.472 \approx \pm 600$.
Note that this is conceptually more confusing, almost more work than our previous working, and we get a less accurate answer. (Here ends my rant about education of calculating errors).


'Murderabilia' Web site posts items from accused killer of 11http://toledoblade.com/apps/pbcs.dll/article?AID=/20100124/NEWS24/1240316

Serialkillersink.net selling letters from Cleveland man

CLEVELAND - An Internet site devoted to selling memorabilia from serial killers has posted envelopes, letters, and a Christmas card sent by a Cleveland man accused of killing 11 women and hiding their remains in and around his home.

The Web site Serialkillersink.net had the envelopes, letters, and card for sale recently on the site.

The letters and card, priced at $200, and envelopes, priced at $100, were sent by Anthony Sowell to employees at the Web site.

In one, Sowell, a registered sex offender, tells a California woman that he is available to correspond with her. "So if you need someone to talk to I am here for you," Sowell wrote. "So tell me what do you want to know about me? I know what I want to know about you, what type of woman are you? Do you have a man in your life?"

Sowell has pleaded not guilty to charges of murder, rape, assault, and corpse abuse and is being held in the Cuyahoga County jail.

Sowell writes that he is being treated well in the jail. He mentions his ex-wife who died in 1998.He writes that he can receive money orders but that cash should not be sent.

"I am in need of just about anything. So anything you can do to help me out is a blessing," he writes.

On Friday, the site had one Sowell letter for sale and a Christmas card posted for viewing.

The card, with the preprinted message, "May every road you travel this season remind you that God's gift of Jesus is with you wherever you go," is signed "Tony Sowell."

The Ohio attorney general's office said inmates are not allowed to make money from crimes by selling their stories to book publishers or filmmakers.

Eric Gein, who owns the Los Angeles-based Internet company, said inmates do not get paid for the letters.

He said his biggest customers are criminology professors who use the letters and artwork to teach. The site also sells personal items from inmates.

Eight states have banned inmates from sending items to companies who sell them, said Andy Kahan, director of Crime Victims Assistance for the mayor's office in Houston.

Mr. Kahan, who has led a national movement to end the practice of selling inmates' items, said Ohio is not among those states.

"This is the beginning of the merchandising and marketing of Anthony Sowell," he said.

Mr. Gein said he has been corresponding with inmates for 15 years and started his business four years ago.

Cuyahoga County Jail warden Kevin McDonough said Sowell has received more mail than most inmates and has received small amounts of money and several small deposits to his jail commissary account.

"A lot want to see his soul," Mr. McDonough said. "Many people want to be his friend or pen pal."

_________________We come to love not by finding a perfect person, but by learning to see an imperfect person perfectly