The diagnosis and treatment of the possible visual sequelae in those with traumatic brain injury (TBI) represents an important area of health care in this special population. One of their most prevalent yet elusive visual symptoms is visual motion sensitivity (VMS). In this review, we present the basic VMS phenomenon and its related symptoms, clinical studies in the area, clinical research investigations using the visual-evoked potential (VEP) as a cortical probe, and possible mechanisms and related neurophysiology that may underlie VMS. Lastly, therapeutic interventions are briefly described, as well as future directions for clinical research and patient care in those with VMS and TBI. The use of binasal occluders (BNO) and bitemporal occluders (BTO) in medicine, in particular ophthalmology, has a long and interesting history for the treatment of strabismus . Mention of such occlusion dates back to at least 600 AD or so  for a re. Thus, sHowever, approximately 30 years ago, the employment of BNO in optometry took a new direction. It began to be used for the treatment of visual motion sensitivity (VMS) in brain-injured patients, typically those with mild traumatic brain injury (mTBI). VMS is a relatively common symptom in these patients, likely occurring in approximately 40% of the patients  Figure , sittingIn this review, we will cover both the clinical and laboratory-based studies involving BNO and VMS, with an emphasis on the objective, visually-evoked potential (VEP) findings. Both the clinical utility, as well as the possible underlying perceptual and neurological mechanisms, will be addressed. Future directions for research will also be considered.The following resources were used for the present review. They included computer searches using PubMed, Yahoo, Google, and Google Scholar. Search terms included “visual motion sensitivity”, “VMS”, “visual motion perception”, “binasal occlusion”, “BNO”, “vision in mTBI/TBI”, “brain injury”, “VEP”, and “magnocellular pathway”. In addition, relevant books, book chapters, and published papers in the area were used other than those found in our formal computer searches. Surprisingly, given the high prevalence and bothersome nature of VMS, there is a relative paucity of clinical studies and case reports in this area of mTBI/TBI, as well as in other forms of acquired brain injury (ABI) and neurological disorders  ,8. UnforOne of the earliest case reports in this area was that of Proctor 2009) in a 46-year-old male patient  in a 46-,4,6. He first patient was a 67-year-old male who was involved in an MVA. His initial visual symptoms included diplopia, pain in his right eye with ocular movement, and balance problems. An ophthalmological vision examination shortly after the MVA revealed diplopia for which base-out prisms were prescribed, but without any relief. Months later, he was examined by his optometrist of many years, and thus all of the MVA-related visual symptoms could be compared against those pre-injury. The patient reported the same visual symptoms as he did earlier to the ophthalmologist, but he now exhibited several new visual signs: reduced distance visual acuity, reduced stereoacuity at near, vertical phoria at near, and large exophoria at near. None of these symptoms and signs were present prior to the MVA. Due to his visual symptoms, especially related to balance, it was decided to try BNO on the patient. Immediately, his symptoms were markedly reduced, especially during ambulation. The BNO, along with the oculomotor-based vision therapy, were prescribed with continued success. The second patient was a young, female administrator who had sustained an mTBI one year earlier. She had hit the top of her head and was temporarily “stunned”. Her symptoms included reading difficulties, light sensitivity, discomfort on the computer, variable ocular focusing, spatial disorientation, and balance problems. All busy environments, including her office, triggered her balance and disorientation problems. The patient’s comprehensive optometric vision examination was unremarkable, and visual acuity was 20/20. However, she was nauseous for several hours after each section of the basic vision test battery, which is not uncommon in these patients. Thus, she elected not to perform any further vision testing and to forgo the tentatively suggested vision therapy. About two years later, the patient returned to the same optometric clinic with the same persistent visual problems. In the interim, she was being treated for anxiety, with some degree of intermittent relief of her visual symptoms. There were now fewer symptoms during the subsequent vision examination, and all testing could be completed. She was recommended a course of vision therapy. In addition, BNO was attempted. The effect of the added BNO was immediate, with about 80% reduction in her symptoms as she ambulated about the doctor’s office. The markedly reduced symptoms of VMS in busy environments, such as the street and her workplace, continued. The combination of the BNO and the vision therapy has allowed her to remain much less symptomatic under all naturalistic conditions over the several months of follow-up.More recently, Gallop (2014) presented two cases of mTBI in which BNO was found to be helpful . The firBased on the above clinical studies, what might be the prevailing mechanism? A most intriguing suggestion was proposed by Gallop 1998) [ [8]. He Once again, and surprisingly, there have been a paucity of studies in clinical laboratories assessing the efficacy of BNO in mTBI/TBI. The first investigation was published nearly 20 years prior to the more recent studies. All three investigations included the VEP as the primary outcome measure.The first investigation of BNO in mTBI/TBI was by Padula et al. (1994) . This hoFollowing a comprehensive optometric vision examination, the VEP  testing commenced. Important details of the methodology are as follows. A central/near retinal periphery field, checkerboard pattern was used as the test stimulus. It had high-contrast with a check size of 30 minutes of arc and a stimulus pattern reversal rate of 1.9 reversals/s. An ABA experimental design was used: A = habitual distance refraction, B = habitual distance refraction plus the vertically-oriented, translucent tape BNO and 2 BI prisms before each eye, and then condition A again to assess for and assure a stable baseline. The primary VEP metric was its peak-to-peak amplitude (N1-P1), a standard VEP parameter. The VEP was completed in all ten normal controls and in nine of the ten individuals with TBI.t-test, p < 0.01). Some patients reported a reduction of symptoms  with the BNO/prism combination incorporated into their distance spectacle prescription. No other related testing was performed, such as formally assessing visuomotor ability or general ambulation with and without the BNO/prism combination. The VEP findings were as follows. In the normals, the VEP amplitude with the combined BNO/prisms increased in 6/10, decreased in 2/10, and remained the same in 2/10, with a range of −14% to +11%, and a mean decrease in amplitude of −0.41 microvolts, as compared to the baseline values. These small, mixed findings suggested a ‘noise’ phenomenon; that is, a lack of any consistent cortically-based physiological effect, as one might predict in this control population. In contrast, in the TBI group, the amplitude increased in 8/9 and decreased in 1/9, with a range of −5% to +54% . There was a statistically significant effect of the BNO/prism combination in the TBI group versus the control group  [ [8], PadThe next and most recent experiments in this area were performed in the Ciuffreda laboratory about 20 years later ,12. Thesfirst investigation by Ciuffreda et al. (2013) [In the . (2013) , the queThe VEP  stimulus and test conditions were as follows. The test target consisted of a 17 H by 15 V degree, alternating , black-and-white, checkerboard pattern  placed one meter away along the subject’s midline. Luminance was 64 cd/m-squared with a contrast of 85%. The test duration was 20 s. Two trials per test condition  were averaged in each subject, and then averaged across each group, for the parameters of amplitude and latency. A schematic representation of a subject with the BNOs in place is presented in decrease with BNO was found in all 10 subjects, which was statistically significantly  . These findings were repeatable 2–7 days later in a subset of subjects in each diagnostic group.The mean amplitude (microvolts) findings are presented in  < 0.01) . In the subjects , which wLatency was also assessed. It was within normal limits, and was not statistically different under any of the test conditions and diagnostic groups.p < 0.01). They reported reduced symptoms . Furthermore, they reported more accurate and comfortable ambulation, fixating of objects in the field, and grasping of objects. They also indicated that the VEP stimulus appeared clearer, brighter, and flickered less. In the other two mTBI/VMS subjects, they disliked the blocked field of the BNO, despite their objective cortical and subjective improvements with the BNO.Lastly, the subjective impressions of both groups were queried. All VN subjects disliked the BNO. The blocked field was annoying and produced a sense of visual discomfort. In contrast, eight of the ten mTBI subjects with VMS liked the BNO (second investigation by Yadav and Ciuffreda (2014) [and/or base-in prisms on adults with chronic mTBI and symptomatic VMS?” There were 15 patients with medically-diagnosed mTBI (mean age 35.2 years) with the symptom of VMS, and there were 20 matched, visually-normal (VN) controls (mean age 25.5 years) without the symptom of VMS, with both groups having 20/20 or better visual acuity in each eye. None had a history of seizures, strabismus, amblyopia, or any ocular, systemic, or degenerative neurological disease. All underwent a comprehensive optometric vision examination prior to laboratory testing.In the a (2014) , the quep < 0.05).The same VEP apparatus as described in detail in their earlier experiment above was used . HoweverVN  : Firstly, the baseline (20.8) and the prism only (20.6) conditions were not significantly different, which suggested that the prisms did not affect the VEP amplitude. Secondly, the BNO only condition (17.1) was significantly less than found in either the baseline (20.8) or the prism only (20.6) conditions. Thirdly, the BNO (17.1) and the BNO plus prism (18.1) conditions were not significantly different. Together, these findings suggested that the BNO condition alone was sufficient to have a significant effect on the VEP amplitude; the incorporation of the base-in prisms had no cortical advantage.mTBI  : Firstly, the baseline (20.9) and the prism only (21.0) conditions were not significantly different. Secondly, the BNO only (23.2) condition was significantly greater than either the baseline (20.9) or prism only (21.0) conditions. Thirdly, the BNO only (23.2) and the BNO plus prism (22.0) conditions were not significantly different. These findings suggested that the BNO condition alone was sufficient to have a significant effect on the VEP amplitude; the incorporation of the base-in prisms had no cortical advantage. mTBI  : For both diagnostic groups, the mean latency was significantly increased for the BNO and/or prism conditions as compared to the baseline by a few milliseconds. This suggests small but significant increases in visual processing time for these optical conditions. However, all were within their laboratory and literature normative values.p < 0.05) showed an increase in amplitude with the BNO only (range −9.7 to +40.6), there was a mixed result (7/15 increased) with the combined BNO plus prism (range −19.4 to +92.3), and again a mixed result (8/15 increased) with the prism only (range −18.8 to +22.7). Again, the mTBI findings revealed the uniqueness and consistent advantage of the BNO alone on cortical activity.The individual amplitude findings  are presented in The VEP findings were repeated three weeks later in two subjects from each diagnostic group. The coefficient of variation (COV) statistic was used to assess this aspect. Values can range from 0.00 to 1.00, with smaller values indicating less variability, and hence better repeatability. Values ranged from 0.00 to 0.07, thus indicating an extremely good repeatability in these subjects.p < 0.05) reported the tasks to be best with the BNO, thus consistent with and confirming the objective VEP findings. Some of their comments are worth noting: most felt more stable, comfortable, and confident ambulating with the BNO; some commented on perceiving less “visual noise” especially in the periphery; and others indicated a sense of reduced sensory overload. Thus, there was a manifold of subjectively-based changes and improvements with the BNO present. Further research in this important area is warranted.Lastly, subjective testing was performed and compared under three visual and/or visuomotor conditions in those with mTBI: (1) viewing of either a blank wall, a stationary checkerboard pattern, or a flickering checkerboard pattern; (2) near grasping of objects; or (3) walking down a long corridor. For all conditions, most subjects . Fourth, testing should be extended to the moderate and severe TBI populations using the VEP and simpler vision and visuomotor testing/tasks. Fifth, in all types of TBI, the impact on quality of life (QOL) needs to be assessed to determine the positive effects on specific visual demands and tasks, both vocational and avocational. Sixth, and lastly, brain imaging should be performed to determine the site(s) of activity unique to the application of BNO in all categories of TBI.BNO is a non-invasive method and is easy to apply in patients with mTBI and VMS.BNO seems to be an effective treatment method in patients with mTBI in reducing the symptoms caused by increased VMS.BNO provides immediate and sustainable effects in patients with mTBI and VMS.BNO seems to have a positive visuo-cortical effect in patients with mTBI and VMS.Using this simple clinical technique would be beneficial in treating these patients and helping them in performing their activities of daily living (ADL).The use of BNO in patients with mTBI should be fruitful in future investigations. The clinical and laboratory data thus far show promise in the mTBI population with symptomatic VMS. While the possible mechanisms remain somewhat unclear and elusive, this should not prevent the clinician from attempting this approach, and related ones, in these patients. The clinician’s goal should be to provide symptom reduction and a better quality of life, which frequently will occur with the prescription of BNO.


ANTXR2) located on chromosome 4q21. The main clinical features of both entities include papular and/or nodular skin lesions, gingival hyperplasia, joint contractures and osteolytic bone lesions that appear in the first few years of life, and the syndrome typically progresses with the appearance of new lesions.Hyaline fibromatosis syndrome (HFS) is a recently introduced alternative term for two disorders that were previously known as juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH). These two variants are secondary to mutations in the anthrax toxin receptor 2 gene  was carried out on DNA samples from the proband and his parents.ANTXR2  that yielded a diagnosis of HFS was noted.A mutation in The main goal of this paper is to add to the knowledge related to the clinical and radiographic aspects of HFS in adulthood and to show the importance of Next-Generation Sequencing (NGS) techniques in resolving such puzzling cases. Molluscom fibrosum [Juvenile hyaline fibromatosis  is a rare inherited autosomal recessive disorder  that wasfibrosum . Affectefibrosum , 5, and fibrosum . Most peInfantile systemic hyalinosis , another rarer disorder, shares many similarities with JHF . It is cANTXR2; OMIM # 608041) have been shown to cause both JHF and ISH [ANTXR2 encodes a 55 kDa type I transmembrane (TM) protein which comprises an extracellular N-terminal von Willebrand A (vWA) domain, followed by an immunoglobulin-like domain (Ig-like), a TM domain and a cytosolic tail [Deleterious mutations of Anthrax toxin receptor 2 gene,  is described here. The aim of this report is to augment the findings related to the clinical, radiographic and differential diagnosis of HFS.We identified one Lebanese Shiite family with three branches from a small village in North Lebanon Fig. . All fivAt the time of physical examination, patient VI-3 was 42 years old, patient VI-4 was 36 years old, patient VI-5 was 30 years old, patient VI-10 was 28 years old and patient V-15 was 58 years old. Patients VI-3, VI-4, VI-10 and V-15 presented with an important postural deformity, and had been in wheelchairs since they were 10 years old, whereas patient VI-5 had a severe retardation of physical growth and development that caused movement difficulty.The patients were thin with underdeveloped musculatures. Cognitive development, hearing and eyesight were noted to be normal in all patients.Each patient was found to have recurrent, painless, variable-sized nodules over the scalp, ear, lobules, postauricular folds, forehead, nose, upper lip, shoulder, elbows, thorax, chest, back, fingers, perianal area, knee and feet. Small, pearly papules were limited to the chin and paranasal folds. The nose and ears were deformed and bulbous, secondary to numerous tumors. All patients had severe gingival hypertrophy covering the teeth completely. Patients VI-10 and V-15 had flexion contractures of the elbows, and fingers and hips and knees, which resulted in a frog leg position Fig. . SwellinHematological and biochemical investigations were within normal limits. Only, patient VI-3 reported having persistent diarrhea since the age of 2 years. The clinical features of the present cases and of ISH and JHF, on the basis of the work of Urbina et al. are showA skeletal X-ray of patient VI-10 showed subcutaneous soft tissue calcifications in the pinna of both ears and in the parietal region of the scalp, radial bone bowing, thoraco-lumbar scoliosis with paravertebral calcifications at T10, T11 and T12 levels, deformity of the iliac bones, thinned pubic rami, severe narrowing of the hip joints, acetabular protrusion, erosion of joint spaces, coxo-femoral ankylosis, thinned fibula, amyotrophy and cutaneous calcifications Fig. .Fig. 3X-The patients refused biopsies of their lesions.Genomic DNA was extracted from peripheral blood samples by standard salt-precipitation methods . Whole ghttp://www.broadinstitute.org/ftp/pub/seq/references/Homo_sapiens_assembly19.fasta), using BWA aligner [https://xbrowse.broadinstitute.org/). For the trio, the model of inheritance “autosomal recessive” was selected, with the severity of the variant effect set to ‘moderate to high impact’ , call quality as high  and allele frequency < 1% in 1000 genomes and The Exome Aggregation Consortium (ExAC) v0.3 datasets. The functional consequences of amino acid substitutions were predicted using various tools [Raw data were mapped to the human genome reference, build 19  was obtained from UCSC Genomic Browser on Human. Primers used for PCR amplification were designed using Primer3 software (http://frodo.wi.mit.edu) to amplify the region surrounding the mutation detected by WGS in exon 4. PCR reactions were performed using Taq DNA polymerase . PCR fragments were run on 1% agarose gel. The fragments were purified using « SIGMA-ALDRICHTM» kit and then sequenced using the Big Dye_ Terminator v 1.1 Cycle Sequencing Kit . Sequence reaction was purified on Sephadex G50  and then loaded into an ABI 3100 system after the addition of Hidi formamide. Electropherograms were analyzed using Sequence Analysis Software version 5.2 (Applied Biosystems) and then aligned with the reference sequences using ChromasPro v1.7.6.1 .Genomic sequence of ANTXR2 (NM_058172.5: c.347G > T) and in zinc finger protein 618 (ZNF618) (NM_133374.2: c.832G > T). The ANTXR2 mutation results in a substitution of glycine by valine, and the zinc finger protein 618 (ZNF618) mutation leads to a premature stop codon. Both had damaging effects, according to the majority of the effect predictors tested  and a mutation in ANTXR2 (c.347G > T).Here, we report five adult patients from a consanguineous Lebanese family, who presented with nodular skin lesions, gingival hyperplasia, joint contractures and bone lesions. By WGS, we identified 2 mutations: a mutation in ZNF618, also known as KIAA1952 or NEDD10, is a protein-coding gene located on chromosome 9q32 and is implicated in transcriptional regulation. Association studies have demonstrated that ZNF618 may be involved in the occurrence of cleft lip [left lip , high blleft lip , kidney left lip  and, in left lip , 37. On ANTXR2, also called the capillary morphogenesis protein gene-2 (CMG2) is located on chromosome 4q21 and is implicated in basement membrane matrix and cell morphogenesis [ANTXR2 to be a candidate gene responsible for the phenotype of the patients studied here.ogenesis . MutatioA clinical diagnosis of HFS was missed because of the advanced age and status of the patients, the stage of the disease, the severity of the clinical manifestations, and incomplete knowledge of the syndrome’s pathogenesis. The patients reported here had undergone multiple surgeries in infancy for the resection of cutaneous nodules, but long-term regression was unlikely, and the tumors continued to increase in size and number. Their parents stopped treating the lesions, and no follow-up was performed for economic reasons. Biopsies were refused by the patients for many reasons, including pain and the absence of treatment. WGS allowed us to diagnose the disease, assess the genotype-phenotype correlations and offer genetic counseling and prenatal diagnosis to the people of the village.HFS and inherited systemic hyalinosis represent the same disorder, comprising two variants with severe (ISH) and mild (JHF) forms of the disease.Gilaberte et al.  have proIn contrast, Nofal et al.  have claHFS is a rare genetic disorder, but it has been documented in families of different ethnic backgrounds on several continents . The lifThe diagnosis of this syndrome is based on the clinical features and/or the presence of a molecular diagnosis.The clinical features associated with HFS syndrome consist of multiple subcutaneous skin nodules/papules, gingival hypertrophy and joint contractures and may be accompanied by systemic symptoms.The specific pathogenesis of HFS also remains unclear, but some authors have suggested that it results from an abnormality in type IV or VI  collagen  or defecTo our knowledge, 33 cases of HFS have been reported in adults, of which 14 with X-ray findings. In 13 of these 14 cases, joint contractures, osteolytic destruction of the skull, of the large joints, of the long bones and of the extremities, triangular carpal bones and an isolated cortical erosion of mandibular bone and calcifications in the subcutaneous tumors were noted , 46–52. Magnetic resonance imaging (MRI) of HFS lesions has rarely been described in adults and shows a hypointense, central, radiating scar and heterogeneous signal intensity. After the administration of a gadolinium contrast medium, the lesion showed diffuse enhancement, with the exception of the central scar and discrete enhancement of subcutaneous masses in contrasted phases , 51, 53.Computed tomography (CT) of the head has demonstrated a normal aspect  or an abThe histopathologic features of this disease include a normal epidermis with few inflammatory cells in the dermis and minor pigmentary incontinence. Deposits of an amorphous, homogeneous and eosinophilic, hyaline substance (periodic acid–Schiff positive), can be found in the papillary and reticular dermis, accompanied by a proliferation of spindle cells without atypia , 22.Electron microscopic studies have shown stromal deposits of a fibrillogranular material focally displaying a banding pattern similar to that of type VI collagen and fibroblasts with prominent Golgi complexes, dilated endoplasmic reticulum, multi vesicular bodies and vesicles filled with a fibrillogranular material , 10, 43.The results of biopsy cases from patients with gastrointestinal signs include villous atrophy, edema, lymphangiectasia and hyalinosis. Rapid transit time has been described in real-time upper-gastrointestinal imaging investigations .Deficits of the humoral and cellular branches of the immune system have been observed .Laboratory examination may demonstrate a normal , 57, 58 ANTXR2 is the only gene in which pathogenic variants are known to cause HFS. Mutations of this gene disrupt the formation of basement membranes. This disruption may allow the hyaline material to leak from plasma components through the basement membrane into the perivascular space, thus explaining the histological features of HFS [s of HFS .ANTXR2 mutations have been described. Yan et al. [As shown in Table n et al.  have repn et al.  in an 11n et al. . These dAn autosomal recessive mode of inheritance has been established for HFS. Therefore, the risk for a parental carrier to have an affected offspring is 25%.ANTXR2.Currently, only symptomatic treatments for HFS are available. Early surgical excision of the lesions is recommended for functional and cosmetic improvement , 52. HowWe report a Lebanese family with five adult patients with HFS. WGS showed an ability to establish a diagnosis in such puzzling cases in which the clinical signs are atypical or very severe for the classical phenotype. HFS is still a poorly understood disease with a severity that ranges from being lethal during early childhood to chronic at a later age. More studies to find an effective treatment are essential. An accurate diagnosis of the disease requires an exhaustive analysis of the radiologic and histopathological clinical findings, and genetic studies are required for family planning and counseling.


Prevalence of thyroid dysfunction and its impact on cognition in older people has been demonstrated, but many points remain unclarified. In order to study the effect of aging on the thyroid gland, we compared the thyroid gland of very old mice with that of younger ones. We have first investigated the changes of thyroid microstructure and the possibility that molecules involved in thyroid function might be associated with structural changes. Results from this study indicate changes in the height of the thyrocytes and in the amplitude of interfollicular spaces, anomalous expression/localization of thyrotropin, thyrotropin receptor, and thyroglobulin aging. Thyrotropin and thyrotropin receptor are upregulated and are distributed inside the colloid while thyroglobulin fills the interfollicular spaces. In an approach aimed at defining the behavior of molecules that change in different physiopathological conditions of thyroid, such as galectin-3 and sphingomyelinase, we then wondered what was their behavior in the thyroid gland in aging. Importantly, in comparison with the thyroid of young animals, we have found a higher expression of galectin-3 and a delocalization of neutral sphingomyelinase in the thyroid of old animals. A possible relationship between galectin-3, neutral sphingomyelinase, and aging has been discussed. Gal-3 ictosides . It can ctosides . Moreovectosides . Recent ctosides , metabolctosides , and canctosides . In humactosides . In ratsctosides .Increasing studies demonstrate that neutral sphingomyelinase (nSMase), also known as sphingomyelin phosphodiesterase, an enzyme that uses sphingomyelin (SM) as a substrate to produce ceramide and phosphocholine, may regulate many cell physiopathology processes . RecentlAt the moment, nothing is known about Gal-3 and nSMase behavior in the thyroid gland during aging and, therefore, the aim of the present study was to investigate this point.Three CD-1 male mice six weeks old and three CD-1 male mice eighteen months old  were used. Mice were kept under a 12-hour light/dark cycle and housed under controlled conditions as reported in Traina et al. . Mice haThe experimental protocol was approved by the Ethical Committee for Animal Experimentation at the University of Perugia, Italy. Animal care was in compliance with Italian regulations , as well as with European Economic Community regulations (O.J. of European Commission 2010/63/UE).Anti-TSH receptor (TSHR), anti-nSMase, and fluorescein isothiocyanate- (FITC-) conjugated secondary antibody were obtained from Santa Cruz Biotechnology Inc. . TSH, thyroglobulin, and Gal-3 antibodies were from Leica Biosystems .μm thick sections were prepared and mounted on silane-coated glass slides, two for a slide at a distance equal to 140 μm. Between 7 and 24 pairs, sections were sampled excluding the first and the last; sections 7, 13, and 19 were used for hematoxylin-eosin staining; 8, 14, and 20 for TSH detection; 9, 15, and 21 for thyroglobulin detection; 10, 16, and 22 for TSHR detection; 11, 17, and 23 for Gal-3 detection; and 12, 18, and 24 for nSMase detection. Tissue sections were deparaffinized and rehydrated with a series of xylene and ethanol washes.The thyroid tissue was fixed in 4% neutral phosphate-buffered formaldehyde solution for 24 h, as previously reported . 4 μm thThe sections were stained by the hematoxylin-eosin  staining method and investigated by using an inverted microscope, EUROMEX FE 2935 , equipped with a CMEX 5000 camera system (40x magnification), as previously reported . The morTo remove paraffin from tissue sections before rehydration and immunostaining on the Bond automated system , Bond Dewax solution was used as previously reported . Immunosμg/ml anti-TSHR or anti-nSMase primary antibodies diluted in a 0.5% solution of bovine serum albumin (BSA) in PBS overnight at 4°C. The slides were washed 3 times with PBS and incubated with fluorochrome-conjugated secondary antibodies for 1 hour at room temperature. Then, after 3 washes with PBS, the slides were mounted with glycerol and coverslips. The samples were examined under a fluorescence microscope (OLYMPUS IX 51) equipped with an OLYMPUS DP 50 camera system and analyzed at 40x magnification.After 3 washes with phosphate-buffered saline (PBS), sections were incubated with 2 t-test was used for the comparison between young and old mice.Three experiments were performed for each analysis. Data are expressed as mean ± SD, and In order to study the effect of aging on the thyroid gland, we have first investigated the changes of the microstructure. Morphometric analysis, performed on the thyroid of young and old mice subjected to hematoxylin-eosin staining, shows differences in length of the major axis and of the minor axis of follicles. Since the size of the follicles is not at all homogeneous, the results are not statistically significant . ChangesIn the human thyroid gland, there is an age-dependent variation of follicular size and expression of iodine transporters . SorrentHowever, what molecule is involved in the structure/function alteration of thyroid in aging remains yet unclear. In this study, we provided strong evidence that Gal-3, known to be overexpressed in thyroid cancer  by leadiOur data show an increase in the volume of follicles not statistically significant, an increase in the TSH expression that fills the colloid, and in thyroglobulin that diffuses in the interfollicular spaces. Moreover, TSHR also appears upregulated. Notably, Gal-3 and nSMase are upregulated and delocalized in comparison with control mice. Taken together, these data suggest that not only the follicle structure and molecules involved in the synthesis of thyroid hormones change in aging, by supporting previous data –22, but


With the widespread adoption of e-Healthcare and telemedicine applications, accurate, intelligent disease diagnosis systems have been profoundly coveted. In recent years, numerous individual machine learning-based classifiers have been proposed and tested, and the fact that a single classifier cannot effectively classify and diagnose all diseases has been almost accorded with. This has seen a number of recent research attempts to arrive at a consensus using ensemble classification techniques. In this paper, a hybrid system is proposed to diagnose ailments using optimizing individual classifier parameters for two classifier techniques, namely, support vector machine (SVM) and multilayer perceptron (MLP) technique. We employ three recent evolutionary algorithms to optimize the parameters of the classifiers above, leading to six alternative hybrid disease diagnosis systems, also referred to as hybrid intelligent systems (HISs). Multiple objectives, namely, prediction accuracy, sensitivity, and specificity, have been considered to assess the efficacy of the proposed hybrid systems with existing ones. The proposed model is evaluated on 11 benchmark datasets, and the obtained results demonstrate that our proposed hybrid diagnosis systems perform better in terms of disease prediction accuracy, sensitivity, and specificity. Pertinent statistical tests were carried out to substantiate the efficacy of the obtained results. The proliferations of computer usage across all aspects of life have resulted in accumulating a large number of systematic and related data. This has necessitated identifying useful patterns from raw datasets as the next logical step forward. Thus, data mining, a broad discipline encompassing classification, clustering, association, prediction, estimation, and visualization tasks , has emeThe application of data mining and knowledge engineering techniques in the medical domain plays a prime role in the diagnosis of diseases and prognostication . It assiOver the years, several supervised machine learning techniques such as classification as well as several unsupervised machine learning techniques like clustering have been applied to available medical information , 11. IndThis paper presents a hybrid system for diagnosis and prediction of numerous diseases using optimized parameters for classifiers. The classifier parameters are optimized using evolutionary algorithms to enhance classification performance. By juxtaposing the proposed parameter optimization step within existing classifier mechanisms, our method provides improved prediction accuracy. In this paper, 16 classifiers are executed in which two basics are with and without resampling, 6 hybrid intelligent systems without resampling and 6 hybrid intelligent systems with resampling technique. In summary, this paper presents a comparative analysis of parameter optimized versions of two classifiers, namely, support vector machine (SVM) and multilayer perceptron (MLP) for medical data. It has been concluded from experimental results presented in this paper that our proposed hybrid system outperforms state of the art (single or ensemble) for classifying medical data. To contrive the parameter optimization, we have employed three popular evolutionary algorithms, namely, particle swarm optimization (PSO), gravitational search algorithm (GSA), and firefly algorithm (FA) for optimizing parameters of SVM and MLP classifiers. Accordingly, we study the performance of six alternative hybrid systems for classifying medical data towards a diagnosis of such diseases. The performance of the proposed hybrid intelligent techniques is compared with the recent literature results (both simple and ensemble classifiers –16). ThiThe rest of this paper is organized as follows: A brief exposition of existing researches has been dealt with in There have been abundant attempts to analyze and diagnose ailments employing machine learning algorithms. This section gives a summary of the efforts in this field to put the contribution of our work in perspective. These researches, however, vary considerably in terms of classifiers applied and nature of systems employed; for example, some are simple and others are hybrid whereas some others present ensemble systems. There are also major varieties in terms of objective functions chosen, single or multiobjective formulation, the number of datasets on which these methods have been applied, performance parameters employed for validating the efficacy, and so forth.Among the different disease datasets that have been studied in the literature, heart disease diagnosis has been very prominent within medical engineering circles, and a wide variety of machine learning techniques have been explored towards diagnosing the same. References –38 incluF-measure. k-NN produced higher accuracy than other methods. Bhatla and Jyoti .In 1995, Dr. Kennedy and Dr. Eberhart developed a population-based speculative computational optimization procedure called particle swarm optimization based on the social behavior of living organisms like fish schools and bird flocks . In PSO,g scheme  as depicThe following equation updates the new position of the particle:The basic steps of PSO are given in β0, ϒ, α ∈ .The firefly algorithm is a recently proposed bioinspired, evolutionary metaheuristic that mimics the social behavior of firefly species. Fireflies produce short and rhythmic flashes, the pattern of which characterizes particular species. The artificial, firefly-inspired algorithm makes certain assumptions regarding its functioning, such as unisexual fireflies for ensuring that all artificial fireflies attract each other and that the attractiveness is proportional to their brightness to define the potential of relative firefly movements. The brightness of a firefly is defined based on the problem at hand that it needs to optimize. For the minimization problem, the brightness may be the reciprocal of the objective function value. The pseudocode of the basic firefly algorithm as given by Yang in  has beenith firefly is based on a fraction of attractiveness in the distance between fireflies Xi and Xj in an m-dimensional space and also on α, a small random value in the range 0 to 0.2; the equations related to velocity are given in (Each firefly position is updated based on . The velgiven in  and 22)ith firefTwo classification techniques are used, and the basic details of these techniques are discussed in the subsequent sections.Multilayer perceptron (MLP) is the most commonly used supervised feed forward artificial neural network. It is a modification of the linear perceptron algorithm. It consists of many nodes that are arranged in several layers. In general, MLP contains three or more processing layers: one input layer for receiving input features, one or more hidden layers, and an output layer for producing classification results based on the classes .Each node is represented as an artificial neuron which converts the input features into output using a weighted sum of inputs and activation function.V is the weighted sum of input features, W represents weights, X represents the input features, and Θ is the bias based on the classes.The weighted input is given byf(x). The most frequently used activation functions are sigmoids. They are as follows:The activation function is denoted by The multilayer perceptron is trained using back propagation (BP). The weight update equation used in BP is given inη) and momentum (α) are evolved using evolutionary algorithms presented in The parameter's learning rate . A 3-layer neural network has been executed with an input layer, one hidden layer, and an output layer. The size of the input layer is equal to the number features of the data, and also, the size of the output layer is nothing but the number classes. The size of the hidden layer is the average of input and output layer sizes. Moreover, the performance of these three algorithms is compared in the simulation and is discussed in the Results and Discussion of this paper.xi, iy). The SVM classifier finds an optimal hyperplane to separate negative and positive classes, and it is represented by F(x) = wt · x + b = 0.Support vector machine (SVM) is one of the supervised machine learning algorithms, which are often used for binary classifications. It was originally developed by Vapnik in 1979 . The traBased on the class labels, two hyperplanes are formed, which are as follows:F(x) = wt · x + b ≥ 0 for positive instances (yj = +1) and F(x) = wt · x + b ≤ 0 for negative instances (yj = −1), where w is the weight vector, x is input vector, and b is bias. Classifications are made on the hyperplanes thus formed.The optimization problem formed during the development of soft margin classifier is as follows:C) mentioned in ; and parameters of the classifier . In this paper, we focus on optimizing the parameter classifiers using evolutionary algorithms, and thus, our designed system qualifies as a hybrid system. Preprocessing stage handles missing data of a feature by inserting most popular data or interval estimated data for that feature. As a part of preprocessing, features have also been normalized using min–max norm with the goal of reducing training phase time of classifiers, which takes quite some time due to the varied range of the feature values. In step 3, we have employed two classifiers (SVM and MLP). In step 4, the parameters selected for evolving in SVM are COST whereas for MLP, two parameters, namely, learning rate and momentum, have been selected for evolution. The range of these three parameters  has been set as . In step 5, the objective function selected is either a single objective or multiobjective. If the method of optimization is multiobjective optimization, then for the sake of simplicity or uniformity, convert all the objective functions into either maximization or minimization. The multiple objectives considered for multiobjective optimization are given in . In stepThe hybridization process ensures that the population of the evolutionary algorithms is constructed based on the classifier parameters by satisfying parameter bounds. During the execution of evolutionary algorithms, population fitness is computed by substituting the performance parameter values of the classifier executed on the dataset in step 4.Once all the three EAs are executed individually, optimal parameter values for each of the two classifiers (SVM and MLP) are found, and subsequently, these six HISs are compared based on their fitness values. That HIS having the best fitness value for a particular dataset is considered as the proposed HIS for that particular dataset. The objective function and parameter values of the best hybrid intelligent system are treated as final optimal values.By combining the two classifiers and the three evolutionary optimization techniques for optimizing chosen classifier parameters, a number of hybrid intelligent systems have been obtained as possible alternatives. These alternative hybrid intelligent systems (HISs) have been termed as GSA-based SVM (GSVM), FA-based SVM (FSVM), PSO-based SVM (PSVM), GSA-based MLP (GMLP), FA-based MLP (FMLP), and PSO-based MLP (PMLP). These six HISs are tested on all the eleven benchmark datasets considered in this work, once without employing resampling and then using resampling technique. Hence, these HISs produce a set of sixteen results for each of the disease datasets, eight for SVM and eight for MLP. Out of these eight results, one is for the basic classifier (only SVM and only MLP) without data resampling, another for the same with resampling data, and the remaining six are for the three evolutionary algorithms each, once with original data and again with resampling data. The benchmark datasets are tested with ADABOOST version of SVM and MLP. However, on average, the ADABOOST results are not competitive with the instance-based supervised resampling technique in Weka, and the corresponding performances are given in To check the performance of the proposed hybrid system, 11 medical datasets of various diseases are considered. These data have been collected from the UCI repository , and thebase papers for every dataset . References  through nd w3 in  correspoIn this section, we present a number of statistical analyses for the results obtained from our proposed hybrid system. The following subsections provide details about how these analyses are done.H0: median(X) is equal to median(Y). H1: median(X) is not equal to median(Y). The statistical analysis was done using Wilcoxon signed-rank test . It testThe objective values corresponding to FMLP overall disease datasets are tested over rest of the five techniques on all disease datasets, once with and next without resampling for each of the hybrid system alternatives, namely, FSVM, GSVM, PSVM, GMLP, and PMLP.The Wilcoxon signed-rank test was executed with the level of significances 0.01 and 0.05. The Matlab function “signrank” was used to perform the statistical analysis and the conclusions arrived upon has been presented in Tables t-test is used to test whether the sample X derived from a normal distribution can have the mean m without knowing standard deviation [t-test is executed on the three objectives: prediction accuracy (PAC), sensitivity (SEN), and specificity (SPE). Null hypothesis and alternative hypothesis are set as follows:H0: μX = m, H1: μX ≠ m. Student's eviation . We exect-test is performed by using ttest function available in MATLAB. The performance of this test for various parameter values has been summarized in Tables Student's Q and P∗, where Q is the Pareto front found by test algorithm and P∗ is the subset of true Pareto-optimal members. Before computing these metrics, the data in Q is to be normalized since various objective functions will have different ranges.The important distinct goals of multiobjective optimization are (1) finding solutions as close to the Pareto-optimal solutions as possible and (2) finding solutions as diverse as possible in the obtained nondominated front. In this work, to test the first goal is tested using generational distance (GD) and the second target is tested by computing spacing . In the Q and P∗ as follows: GD = (∑i=1Q||dip)p1//|Q|. For p = 2, the parameter id is the Euclidean distance between the members of Q and the nearest member of P∗: fmk)∗: Veldhuizen introduces this metric in the year 1990 . This medi values. It can be calculated as follows: di = mink∈Q∩k≠i∑m=1M|fmi − fmk| and di's. A good algorithm will be having a minimal SP value. The set Q is caught by executing FMLP for 50 iterations with each iteration having 20 agents. In every iteration, the Pareto fronts are stored in external memory. The metrics for GD and SP for all the three objectives with and without resampling are given in Spacing (SP): Schott introduces this metric in the year 1995 . This meThe best values found in all the hybrid systems are discussed as follows.F-measure (85.74%), recall (85.8%), and precision (85.91%) without resampling. On the contrary, with resampling, PMLP shows the best accuracy (94.1%), but for all the other parameters, like sensitivity (93.49%), specificity (94.77%), F-measure (94.05%), recall (94.05%), and precision (94.07%), PMLP  shows best results. A comparison of Cleveland result with the state-of-the-art result is given in The performance of all the 8 techniques (2 basic machine learning and six hybrid systems) over Cleveland dataset is depicted in F-measure, recall, and precision without resampling are achieved by FMLP, PMLP, FMLP, FMLP, FMLP, and FMLP, respectively; best values found have been bolded for easy identification in F-measure, recall, and precision with resampling are achieved by GMLP, GMLP, FMLP, GMLP, GMLP, and GMLP, respectively; best values found have been bolded for easy identification in The performance of all the 8 techniques (2 machine learning and six hybrid systems) over Statlog dataset with and without resampling is given in F-measure, recall, and precision without resampling are achieved by FMLP, FSVM, FMLP, FMLP, FMLP, and FMLP, respectively, with the values 85%, 88.4%, 74.2%, 83.3%, 85%, and 83.9%. The highest accuracy, sensitivity, specificity, F-measure, recall, and precision with resampling are achieved by GMLP (PMLP), GMLP , GMLP (PMLP), PMLP (GMLP), GMLP (PMLP), and GMLP (PMLP), respectively, with the values 89.5%, 91.9%, 77.3%, 89.2%, 89.5%, and 89.1%. A comparison of Spect result with the state-of-the-art result is given in The performance of all the 8 techniques (2 machine learning and six hybrid systems) over Spect dataset with and without resampling is given in F-measure, recall, and precision without resampling are achieved by FMLP, FSVM, FMLP, PSVM, FMLP, and FMLP, respectively, with the values 82.4%, 88%, 83.3%, 80.6%, 82.4%, and 82.6%. The highest accuracy, sensitivity, specificity, F-measure, recall, and precision with resampling are achieved by PMLP, GSVM, PMLP, PMLP, PMLP, and PMLP, respectively; best values found are bolded for easy identification in The performance of all the 8 techniques (2 machine learning and six hybrid systems) over Spectf dataset with and without resampling is given in F-measure (81.02%), and recall (81.34%), whereas GMLP shows better results for sensitivity (88.41%) and precision (82.5%) without resampling. On the contrary, with resampling, GMLP shows best results for parameters like accuracy (91.39%), sensitivity (88.78%), specificity (93.69%), F-measure (91.40%), recall (91.39%), and precision (91.48%). A comparison of ERIC result with the state-of-the-art result is given in The performance of all the 8 techniques (2 basic machine learning and six hybrid systems) over ERIC dataset is depicted in F-measure (97%), and GMLP and PSVM together show the best result for recall (97%) without resampling. On the contrary, with resampling, FMLP shows the best accuracy (98%), but for all the other parameters, like sensitivity (96.61%), F-measure (98%), recall (98%), and precision (98%), PMLP (FMLP) shows best results and PSVM (GSVM and FSVM) shows best results for specificity (99.55%). A comparison of breast cancer result with the state-of-the-art result is given in The performance of all the 8 techniques (2 basic machine learning and six hybrid systems) over breast cancer dataset is depicted in F-measure (86.77%), recall (87.1%), and precision (86.6%), whereas GSVM (PSVM and FSVM) shows better results for the parameters like accuracy (87.1%) and sensitivity (73.08%) without resampling. On the contrary, with resampling, FMLP (PMLP and GMLP) shows best results for parameters like accuracy (92.26%), sensitivity (80.77%), specificity (94.57%), F-measure (92.14%), recall (92.26%), and precision (92.08%). A comparison of Hepatitis result with the state-of-the-art result is given in The performance of all the 8 techniques (2 basic machine learning and six hybrid systems) over Hepatitis dataset is depicted in F-measure, recall, and precision without resampling is achieved by FMLP, FMLP (PMLP), FMLP, FMLP, FMLP (PMLP), and FMLP (PMLP), respectively, best values found have been bolded for easy identification in F-measure, recall, and precision with resampling are achieved by PMLP (fMLP), FMLP (PMLP), PMLP (FMLP), PMLP (FMLP), PMLP (FMLP), and PMLP (FMLP), respectively, with the values 98.6%, 98.2%, 98.74%, 98.6%, 98.6%, and 98.6%. A comparison of thyroid result with the state-of-the-art result is given in The performance of all the 8 techniques (2 machine learning and 6 hybrid systems) over thyroid dataset with and without resampling is given in F-measure, recall, and precision without resampling are achieved by FMLP, FMLP, PSVM , FMLP, FMLP, and FMLP, respectively, with the values 93.8%, 96.6%, 96.2%, 93.9%, 93.8%, and 94%. The highest accuracy, sensitivity, specificity, F-measure, recall, and precision with resampling are achieved by GMLP, GMLP , PSVM , PMLP (GMLP), PMLP (GMLP), and PMLP (GMLP), respectively, with the values 96.9%, 97.4%, 100%, 96.9%, 96.9%, and 96.9%. A comparison of Parkinson result with the state-of-the-art result is given in The performance of all the 8 techniques (2 machine learning and six hybrid systems) over Parkinson dataset with and without resampling is given in F-measure, recall, and precision without resampling are achieved by FMLP, FMLP, PSVM , FMLP, FMLP, and FMLP, respectively; best values found have been bolded for easy identification in F-measure, recall and precision with resampling are achieved by FMLP, GMLP, FMLP, FMLP, FMLP, and FMLP, respectively; best values found have been bolded for easy identification in The performance of all the 8 techniques (2 machine learning and six hybrid systems) over Pima dataset with and without resampling is given in F-measure, recall, and precision without resampling are achieved by GMLP, PSVM, FMLP, FMLP, FMLP, and FMLP, respectively, with the values 73%, 72%, 74.9%, 72.8%, 73%, and 72.8%. The highest accuracy, sensitivity, specificity, F-measure, recall, and precision with resampling are achieved by GMLP, FMLP, GMLP, GMLP, GMLP, and GMLP, respectively, with the values 73.3%, 67.5%, 78.4%, 73.3%, 73.3%, and 73.9%. A comparison of BUPA result with the state-of-the-art result is given in The performance of all the 8 techniques (2 machine learning and six hybrid systems) over BUPA dataset with and without resampling is given in n, new solutions are brought forth. However, in FA in the worst case, each agent develops O(n) new solution by moving towards all other best solutions. Therefore, in the worst case of FA, space is managed more efficiently than the other two algorithms (GSA and PSO). The same is demonstrated over the 11 medical datasets.In GSA updating of an agent, the position is learned from all other agents, whereas in PSO updating of an agent position is based on two parameters called gBEST and pBEST. In each iteration of these two algorithms at most From the previous observations, it is concluded that MLP without resampling shows improvement in all datasets when compared with latest literature results and the same is depicted in h value is zero, then H0 is true otherwise H1 is true. In m” value. Tables t-test for both resampling and without resampling techniques with LOS 0.01 and 0.05. In these tables, h value is zero for all datasets at 0.01 and 0.05 LOS. Hence, null hypothesis is accepted for all datasets.The outcomes of FMLP are taken as “Due to the complex framework of ensemble approach and the moderate performance of the individual classifier, hybrid systems have a lot of promise in the diagnosis and prognosis of diseases. To overcome these, we proposed a disease diagnosis system by juxtaposing three evolutionary algorithms and SVM and MLP classifiers. Three evolutionary algorithms optimize the parameters of the two classifiers and such enhanced classifiers have been used to train and diagnose diseases. Accordingly, six hybrid diagnosis alternatives have been obtained by working out the combinations of classifiers and evolutionary algorithms. Based on results presented in this paper, it can be concluded that our hybridization approach provides high prediction accuracy than other methods in literature across a wide variety of disease datasets. Even among the six alternative parameter optimized classifier systems proposed, FMLP was found to be the relatively best across the majority of the 11 datasets considered. On average, MLP shows 2.2% and 6.814% improvement in prediction accuracy on the 11 datasets with and without resampling. The ranges of improvements shown by MLP in the objective sensitivity are −2.9 to 75.13 and −9.68 to 86.33 without and with resampling, respectively. The ranges of improvement shown by MLP in the objective specificity are −9.68 to 86.33 and −18.93 to 36.33 without and with resampling, respectively. From the experimental results, it is concluded that FMLP shows outperformance than recently developed ensemble classifiers . As a p


Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla of bacterioplankton communities, which varied in composition, and distribution from dry to wet seasons, and exhibited profound seasonal changes. Actinobacteria, Bacteroidetes, and Cyanobacteria seemed to be more associated with seasonality that the relative abundances of Actinobacteria, and Bacteroidetes were significantly higher in the dry season than those in the wet season (p < 0.01), while the relative abundance of Cyanobacteria was about 10-fold higher in the wet season than in the dry season. Temperature and Water quality ranks the most vital criterion for rivers serving as drinking water sources, which periodically changes over seasons. Such fluctuation is believed associated with the state shifts of bacterial community within. To date, seasonality effects on bacterioplankton community patterns in large rivers serving as drinking water sources however, are still poorly understood. Here we investigated the intra-annual bacterial community structure in the Dongjiang River, a drinking water source of Hong Kong, using high-throughput pyrosequencing in concert with geochemical property measurements during dry, and wet seasons. Our results showed that  Microorganisms, the most abundant and diverse organisms on Earth, contribute to many biogeochemical cycles, such as carbon, and nitrogen cycles , and temperature were monitored n et al. . Permang−1), and 9.5 μl distilled H2O. Reactions were cycled with an initial denaturation at 95°C for 2 min; 25 cycles of 94°C for 30 s, 56°C for 25 s, and 72°C for 25 s; with a final extension at 72°C for 5 min. The PCR products were purified by QIAquick Gel extraction kit . Pyrosequencing of the 16S rRNA gene amplicon was performed on a Genome Sequencer FLX Titanium , and 400 bp-long reads on average were produced from the forward direction 5F. The 454 sequence reads have been archived in the NCBI Short Read Archive under accession number SRR5027088.Genomic DNA was extracted from each water sample as described before , species diversity, and richness estimators , taxonomic assignment, and comparisons of community structure were analyzed by MOTHUR , and environmental classification was then conducted by hierarchical clustering with the Minitab statistical software  using Euclidean distance between samples  were computed to show the difference of phylogenetic composition and structure of microbial communities between the dry and wet seasons . The relationships between the bacterioplankton communities and physiochemical properties of water column were evaluated to elucidate the inter-relationships among environmental factors and bacterioplankton communities. First, the most correlated parameters were selected by BioENV procedure , suggesting seasonal changes of water quality in the Dongjiang River. Moreover, the samples from upstream sites HY (HYW), and GZ (GZW) were separated from the downstream ones  in both seasons . Although, the microbial community richness of the sampled water has not yet reached saturation at the current sequencing depth  were also observed at the phylum level, including Acidobacteria, Actinobacteria, Armatimonadetes, Bacteroidetes, Cyanobacteria, Firmicutes, Planctomycetes, Proteobacteria, Verrucomicrobia (Table P < 0.001), which explained 27.32% of the total variance . Moreover, separations of community structures between wet and dry seasons were observed based on clustering analysis of the Bray-Curtis distance matrix  plot in two-dimensional taxon space based on Bray-Curtis similarities was first used to estimate the overall community resemblance of samples from two seasons. The stress value was 0.08, and the addition of a third dimension did not substantially improve the model. Dry and wet season clusters were distinct when between-sample differences in bacterial community composition were visualized by NMDS ordination Figure . Using Aia Table . In addie Figure . Additiox Figure .Burkholderiales  from Betaproteobacteria were the most dominant order in groups 1, 2, 3, 4, 6, while Actinomycetales (3.3 and 1.3%) from Actinobacteria dominated groups 5 and 8. In addition, Sphingobacteriales (1.4%) from class Sphingobacteria, and Flavobacteriales (2.5%) from class Flavobacteria were also more abundant than other populations in groups 6 and 7, respectively. These results suggest that the overall microbial communities along the Dongjiang River display obviously seasonal alteration and the discrepancy of community composition between different sampling sites may be affected by water quality.Hierarchical clustering analysis of the overall microbial communities showed an obvious separation of two broad groups between dry and wet seasons Figure . MoreoveMost phylotypes of bacterioplankton populations in the Dongjiang River were detected in both dry and wet seasons, with only a few detected at one season Table . The mosProteobacteria (45.72%), Actinobacteria (24.55%), and Bacteroidetes (14.56%). Strikingly, Actinobacteria and Bacteroidetes seemed to be more season-associated, whose relative abundances were obviously higher in dry season (P < 0.01), while that of Proteobacteria was slightly lower in dry season than in wet season .A total of 40 classes were detected with 12 being abundant (relative abundance >1% at the class level at both seasons). The relative abundance of d Figure . ActinobP < 0.05) in the dry season than in the wet season. Additionally, Limnohabitans, Flavobacterium, Polynucleobacter, Arcobacter, Curvibacter, and Methylotenera were the dominant genera with individual relative abundances higher than 1% in at least one season. Moreover, the former three dominated the dry season , followed by Bacteroidetes , and Actinobacteria , and 440 increased significantly (P < 0.05) in the wet season compared with the dry season . These results suggested that significant changes between dry and wet seasons occurred to the most abundant phylotypes, and some specific planktonic bacteria dynamically responded to the seasonal change.To further compare the difference of planktonic bacteria between dry and wet seasons, the phylotypes with at least six OTUs detected among all samples were identified to conduct response ratio analysis based on the relative abundance of all OTUs  for the distribution at the OTU level. These results suggested that the nitrogen nutrients, TOC, and temperature closely correlated with the seasonal change, and were the dominant factors affecting the distribution of bacterioplankton in the river, although some different environmental factors were also identified to significantly explain the community differentiation at other taxonomic levels.Patterns of dynamic changes in bacterioplankton communities along the Dongjiang River would be affected by the fluctuations of physicochemical parameters of water columns. Based on the forward selection of the significant environmental factors and the variance inflation factors (VIF) of the selected factors <20, redundancy analysis (RDA) was conducted to correlate the difference of bacterioplankton communities at different taxonomic levels (from phylum to OTU) with the possibly key environmental factors  correlated with selected chemical properties, Bacteroidetes, and Verrucomicrobia (P < 0.01) with selected physical properties, while Actinobacteria, and Planctomycetes (P < 0.01 and P < 0.05) with both. In addition, such patterns were also discovered in a certain number of unclassified phylotypes at different taxonomic levels, suggesting that taxonomically undetermined bacterioplankton might be as well-shaped by the water physiochemical parameters  and three chemical , classes , and orders  all significantly correlated across the Planctomycetes with Methylophilales with temperature), or a negative one , while TOC and TSS showed negative and positive correlations with Bacteroidia and Actinobacteria, respectively , and water properties were analyzed by Linear regressions using SPSS 17.0. Among the effects of different physicochemical factors  than from particles (11.6%), and Alphaproteobacteria were the most dominant subphylum of Proteobacteria in the free-living samples in large rivers of the Mississippi River Basin , and rivers  than winter months (2.6–3.7 × 103 cells mL−1) using direct epifluorescence enumerations . Our data however, revealed a potential positive correlation between TSS, and the two groups  (Liu et al., JG and MX initiated and designed the research; WS and CX collected samples and performed the research; WS and MX analyzed microbial diversity and performed the statistical analysis; WS and MX analyzed data; WS, MX, JG, and GS wrote the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Osteoarthritis is prominent among the elderly, with symptoms originating from multiple parts of the body. A cross-sectional study of a nationwide survey was performed to describe the prevalence of and identify factors related to symptomatic hip, knee, and spine osteoarthritis.This cross-sectional study collected data from the Fifth Korean National Health and Nutrition Examination Survey . After excluding ineligible subjects, there were 8976 subjects in this study . All subjects reported symptoms and disabilities related to osteoarthritis. Plain radiographs of the spine, hip, and knee were taken in all subjects.P < 0.05). Multiple-joint osteoarthritis was diagnosed in 10.8% of male patients and 22.8% of female patients with osteoarthritis. Several demographic and lifestyle variables were related to osteoarthritis morbidity. Anthropometric and laboratory measurements were also related to osteoarthritis morbidity. In addition, mental distress and quality of life were significantly compromised in osteoarthritis. There were more significant relationships for these factors among women with a higher prevalence of multijoint osteoarthritis.Overall, 9.3% of male participants and 28.5% of female participants were diagnosed with symptomatic osteoarthritis according to survey criteria. Women showed a significantly higher prevalence in all age groups (A significant proportion of the elderly with single- or multiple-joint osteoarthritis had a variety of pain origins that were closely related. Osteoarthritis was also significantly related to several factors, including mental distress and quality of life. The aim of this study was to investigate the prevalence and related factors of multijoint OA using data from the Korean National Health and Nutrition Examination Survey (KNHANES).Advancements in the diagnosis and treatment of disease have led to an increase in the mean age of the population. More people now experience degenerative osteoarthritis (OA), which can occur in several mobile joints of the human body including the hip, knee, and spine. However, symptoms associated with OA may be similar to those due to other origins.22.1 The survey was performed by the Korean Ministry of Health and Welfare, and was designed to assess national health and nutritional levels. A stratified, multistage probability sampling design was used, and sampling units were based on geographical area, age, and sex. Among a total of 31,596 subjects . The KNHANES is a nationwide health and nutrition survey of children, adolescents, and adults that has been regularly conducted since 1998 by the Korean Centers for Disease Control and Prevention.2.2 The presence of radiographic OA was defined as KL grade≥2. If grades given by the 2 radiologists were discrepant by 1 KL grade for the same case, the higher grade was accepted. If grades were discrepant by more than 2 grades, a third radiologist was consulted, and the grade assigned by the third radiologist was accepted. The concordance rate within 1 KL grade of knee joints for the same case was 94.76%. The weighted kappa coefficient for knee x-rays was 0.65, which demonstrated very high inter-rater agreement. All subjects described their current symptoms related to each joint , and symptoms were scored. Subjects who had experienced arthritic pain of each joint for more than 30 days in the past 3 months were asked to report the average intensity of pain, regardless of pain medication, using an 11-point numeric rating scale (NRS) ranging from 0 to 10, with higher scores indicating a higher level of intensity.Radiographic examinations of the knee, hip, and spine were taken using a SD3000 Synchro Stand . Bilateral anteroposterior, weight-bearing anteroposterior, and lateral (30° of flexion) plain radiographs of the knees were obtained. Bilateral anteroposterior and lateral pain radiographs of the hips were obtained. Anteroposterior and lateral plain radiographs of the spine were obtained. Radiographic changes in each joint were then independently assessed by 2 radiologists using the Kellgren/Lawrence (KL) grading system: Grade 0, none: no visible features of OA; Grade 1, doubtful: questionable osteophytes or questionable joint space narrowing; Grade 2, minimal: definitive small osteophytes, minimal/mild joint space narrowing; Grade 3, moderate: definitive moderate osteophytes, joint space narrowing of at least 50%; Grade 4, severe: severely impaired joint space, subchondral bone cysts and sclerosis.2.3 Accordingly, physical activity levels were classified as low (<600 MET-minutes per week), moderate (600 to 3000 MET-minutes per week), or high (>3000 MET-minutes per week).Demographic variables included age (years), sex, equivalized monthly household income, marital status, current residence, education level, smoking status , alcohol consumption , and physical activity . Equivalized household income was calculated as the total monthly household income divided by the square root of the total number of household members. Average alcoholic beverage consumption was assessed by self-reported questionnaire, and then converted to the amount of pure alcohol consumed per day. Subjects were classified into 3 groups according to the amount of alcohol consumption per day (g/day) during the 1-month period before the interview: nondrinkers, light drinkers (<15 g/day), and moderate-to-heavy drinkers (≥15 g/day). Subjects who had smoked more than 100 cigarettes in their lifetime were classified as ever-smokers. Physical activity was quantified as the metabolic equivalent of task minutes per week (MET-minutes per week), which was calculated using the scoring protocol of the Korean version of the International Physical Activity Questionnaire short form.2.42 (m2). Body weight and height were measured using standard protocols. Waist circumference was measured at the narrowest point, between the lower costal margin and the iliac crest, during exhalation. General obesity was defined as body mass index ≥ 25, and the cutoff points for central obesity were defined as WC ≥ 90 cm for men and WC ≥ 85 cm for women. Systolic blood pressure and diastolic blood pressure were measured 3 separate times at 5-min intervals using a standard mercury sphygmomanometer. After each participant's BP had been manually measured 3 times by well-trained observers using a mercury sphygmomanometer , the average values of the second and third measurements were used in the analysis. Hypertension was defined as blood pressure ≥ 140/90 mm Hg or current use of antihypertensive medication. Blood samples were obtained from the antecubital vein of each participant after at least 8 hours of fasting, and random midstream urine samples were collected from subjects. Samples were processed and immediately refrigerated, then transported in cold storage units to the Central Testing Institute in Seoul, Korea, and analyzed within 24 hours of transportation. Fasting plasma glucose, total cholesterol, and triglyceride levels were measured by enzymatic methods using a Hitachi Automatic Analyzer 7600 . Serum and urine creatinine levels were measured by kinetic colorimetry using a Hitachi Automatic Analyzer 7600. The estimated glomerular filtration rate (GFR) was calculated using the equation from the Modification of Diet in Renal Disease study. Subjects were considered to have diabetes if they had a fasting blood glucose of ≥126 mg/dL or if they were taking antidiabetic medication.Body weight and height were measured, and the body mass index was calculated using the following formula: body weight (kg) / height2.5 Three dimensions within the domains of health status and mental health were measured: stress, depression, and suicidal thoughts and attempts. Subjects reported their level of stress as none, mild, moderate, or severe. Depression was screened using the Korean version of the World Health Organization Composite International Diagnostic Interview-Short Form, which was validated as a cost-effective screening instrument that is easily integrated into health surveys. To assess depression, participants answered “yes” or “no” to one of the following questions about whether they had experienced a depressed mood for 2 or more continuous weeks during the previous year: “In your lifetime, have you ever had 2 weeks or more when nearly every day you felt sad, blue, or depressed?” and “Have there ever been 2 weeks or longer when you lost interest in most things such as work or hobbies or things you usually like to do for fun?” Suicidal ideation was assessed by the question “In the last 12 months, did you think about committing suicide?” A “yes” or “no” response was used to determine whether the subject had suicidal thoughts. Health-related quality of life (HRQOL) was evaluated based on the EQ-5D, a self-reported questionnaire widely used to assess the quality of life of a general population. The Korean EQ-5D was developed according to guidelines from the EQ group. Its reliability and validity have been tested in patients with rheumatism, cancer, and chronic disease.,26 The EQ-5D consists of 5 questions evaluating a respondent's current health status in terms of mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. In each dimension, a respondent can belong to 1 of 3 categories, and these are classified as “no problems” and “problems,” including moderate to severe problems.,23,27To assess the mental health of the study population, surveys provided to the subjects included the same questions as those in the KNHANES surveys.2.6t-tests and 1-way analysis of variance for continuous variables and the chi-squared test for categorical variables. Multivariate logistic regression analyses were conducted to investigate the relationship between parameters. SES variables with P-values <0.15 in univariate analyses were selected for multivariate analyses. A P-value <0.05 was considered statistically significant.Statistical analyses were conducted using SAS survey procedures  in a manner that reflected sampling weights and provided nationally representative estimates. The characteristics of subjects with OA were compared with those of subjects without OA (control group) using 2 independent sample 33.1P < 0.05).OA was defined as “OA in at least 1 joint on plain radiographs with related pain including hip, knee, or spine.” Radiographic changes of each joint were assessed, and the presence of radiographic OA was defined as KL grade ≥2. Subjects who had experienced arthritic pain of each joint for more than 30 days in the past 3 months were included in the OA group. We used the ACR criteria for knee and hip OA with our criteria to confirm the diagnosis. In cross-match analysis, there were significant mismatches of subjective pain and radiographic findings (Table 3.2P < 0.05). Prevalence progressively increased with age and was highest in the older than 80 years group . Multijoint OA was defined as “OA in more than 2 joints with related pain including hip, knee, and spine.” Overall, 89.2% of male and 77.2% of female OA patients were diagnosed with single-joint OA, whereas 10.8% of male and 22.8% of female patients were diagnosed with multiple-joint disease  according to the survey . In addition, rural residence and no spouse were significantly related to OA morbidity, especially in females (P < 0.05). More significant relationships with these factors were found in females with a higher prevalence of multijoint OA. Smoking, drinking status, and activity level were not significantly related to OA morbidity.Age, living situation, marital status, educational status, and income status were significantly related to OA morbidity . Significance levels were higher with these factors in females with a higher prevalence of multijoint OA.Several anthropometric and laboratory measurements were significantly related to OA, especially in the female and multijoint OA group (3.3.3P < 0.05). In addition, EQL was significantly compromised in the OA group. LQ 1, 2, 3, 4, and 5 were significantly higher in the OA group. Significance levels were higher with these factors in females with a higher prevalence of multijoint OA.There was a significantly higher rate of mental stress in the OA group Table . Mental 4,8,14,28 The projected number of older adults with arthritis or other chronic musculoskeletal joint symptoms is expected to nearly double from 21.4 million in 2005 to 41.1 million by 2030 in the United States. In our study, 9.3% of male and 28.5% of female participants were diagnosed with symptomatic degenerative joint OA. There was a higher rate of multijoint OA in the older segment of the population, including hip, knee, and spine joints, which may be comparable to the prevalence of diabetes or hypertension in the elderly. In this study, the prevalence of symptomatic knee OA in males was 4.5%. The female group showed a prevalence of 19% in the knee, which was significantly greater than in males. Muraki et al analyzed 2126 Japanese men and women using a large-scale population-based cohort study. Although they showed a significant difference in the proportion of men and women compared to our study, they reported that the prevalence of symptomatic knee OA was 5.0% in men and 11.3% in women, which is similar to our result. In a European study, Turkiewicz et al reported that the prevalence of frequent knee pain was 25.1%, which was higher in women and similar across age groups. However, they reported that the prevalence of radiographic knee OA was 25.4%, whereas 15.4% had either symptomatic or clinically defined knee OA in a random sample of 10,000 elderly residents of Sweden. Although the incidence in Asian and European studies was similar to that of our study, if we had focused on knee and spine OA, the incidence of multijoint OA may have been even higher.,28,31,32 Furthermore, we found patients who had radiographic arthritis without symptoms, and vice versa. This selection criterion may have significantly decreased the prevalence, which would have been much higher if we had diagnosed the disease differently.,30,33,34Because of improved treatment for chronic diseases and lower mortality from infectious diseases, the worldwide population is aging. Older people are now living longer with common and disabling conditions such as OA.,16,31,37 Degenerative OA may occur in several mobile joints of the human body, including the hip, knee, and spine. Symptoms associated with degenerative OA may be similar to those from different origins or different manifestations with the same origin. Thus, a more comprehensive investigation in this field is needed. We analyzed the prevalence and related factors associated with multiple-joint OA. There appears to be a close mechanical relationship between the knee, hip, and spine. Each joint may support body weight and compensate for abnormal motion. In this study, a significant proportion of the population was diagnosed with single- or multijoint OA, including 10.8% of male patients and 22.8% of female patients. However, the prevalence of OA may be underestimated due to the rare incidence of hip joint OA. Problems in 1 joint may affect other joints and induce multijoint OA that should be treated simultaneously. In addition, physicians can misdiagnose the origin of symptoms due to the high prevalence of multijoint OA with similar symptoms. In this study, prevalence progressively increased with age and was the highest in patients more than 80 years old . In addition, female patients had a higher prevalence of multijoint OA than males, and several related factors were more closely associated in female patients. It appears that women are more susceptible to multijoint OA than men for several reasons, –38 including hormonal changes and osteoporosis, which may accelerate degenerative changes in multiple joints.There are several studies on the prevalence and incidence of single-joint  arthritis.,40 In particular, we found more significant relationships with these factors and a higher prevalence of OA in the female group. In this study, living in a rural setting was related to OA. It is possible that participants who live in rural areas may engage in harder labor , which may increase disease risk. The low education level was also associated with OA. Body composition factors had significant relationships to OA, with female dominance. As anticipated, the OA group had a higher BMI with higher blood sugar and lipid levels, which may be the cause or result of generalized OA. Importantly, participants with multijoint OA experienced compromised mental status and psychological dissociation significantly more often. This suggests that in addition to physical pain, mental distress should be treated in this population. Patients in the OA group suffered from significant mental distress and were prone to suicidal thoughts. In terms of life quality and mental health, there were significantly higher rates of mental stress in the disease group. In addition, EQL was significantly decreased in the OA group. In the EQ-5D survey, problems in the domains of daily living activities  and pain/discomfort were significantly more prevalent in persons with OA. Considering that quality of life and mental status are significantly affected by OA, understanding the prevalence and etiology may be important in disease treatment.,6,13,16In this study, age, living situation, marital status, educational status, and income status were significantly related to OA morbidity, as were BMI and body composition factors. Previous studies have reported that joint pain is associated with several socio-demographic factors such as sex, old age, low education level, smoking, and occupation.There are several limitations to this study. First, the cross-sectional study design prevented determination of cause-and-effect relationships. Therefore, future prospective studies are required to better elucidate causal relationships. Second, use of a single 11-point NRS did not allow evaluation of the intensity of acute and chronic pain, including functional impairment. In addition, more sophisticated diagnostic tools (such as MRI and CT) may be needed to evaluate the precise status of joints. Third, the results cannot be confirmed or generalized because of ethnic differences. The prevalence or etiology of OA may be influenced by ethnic or environmental factors, which may decrease the significance of our study. Despite these limitations, this study included a large cross-sectional population with sophisticated statistical methods. A higher number of patients with multiple-joint OA were identified than expected. Our results will be helpful for physicians treating OA.In conclusion, a significant proportion of patients with OA in a large cross-sectional population had a variety of pain origins that were closely related to each other. OA was significantly related to several factors, including mental distress and quality of life. To prevent misdiagnosis and malpractice, physicians should be aware of the relationships and factors involved in multiple-joint disease, particularly in an elderly population.The authors used data from the Fifth Korean National Health and Nutrition Examination Survey (KNHANES-V: 2010–2012). The KNHANES is a nationwide health and nutrition survey of children, adolescents, and adults that has been regularly conducted since 1998 by the Korean Centers for Disease Control and Prevention. They acknowledge the Korean Centers for Disease Control and Prevention data collectors.


This is the report of the clinical case of a patient who presents the association of a JAK-2 positive chronic myeloproliferative neoplasia to a subsequent 5q- myelodysplastic syndrome, developed after about 14 years from the first diagnosis. Patient’s symptoms had rapidly worsened, and she became transfusion-dependent. Therapy with low-dose Lenalidomide quickly reduced the splenomegaly and completely brought white cells counts, haemoglobin, and platelets back to normal. After more than one year from the start, blood cell count is still normal. As far as we know, this is the first case of an effective treatment with Lenalidomide reported in this clinical setting. The JAK-2 V617F mutation has been found in almost all patients with polycythaemia vera, and in nearly 50% of those with primary myelofibrosis and with essential thrombocythaemia. However, the JAK-2 mutation may also occur in other nosological entities, such as neoplasms with overlapping dysplastic and myeloproliferative features. The concomitant presence of both the JAK-2 mutation and 5q- MDS has occasionally been reported, but little is known about disease characteristics and response to treatment and even less about the timing of these genetic abnormalities. Sokol et al. found that the two alterations were acquired by two different and independent clones.1Ingram et al. analysed 97 MDS patients from 6 European countries for the JAK-2 V617F mutation and they detected its presence in 6/97 (6.2%) of the samples, thus identifying a cohort of patients with del(5q) syndrome and hypercellular marrow.2Small subsets of del5q) MDS were thus reported to have also developed the JAK-2 mutation.q MDS wer10Herein we report the case of a 71 year old Caucasian female, whose the only known comorbidities were arterial hypertension and a beta-thalassemia trait. In 1998, at the age of 53, the patient first presented fatigue and diffuse pruritus; a complete blood count was performed which showed an increase in white blood cells, red cells and platelets. Polycythaemia Vera was then diagnosed in another hospital by a bone marrow biopsy during the same year and the patient received Hydroxyurea as first-line treatment. She first came to our Haematology Unit about 10 years later, i.e., in April 2008; at that time she was still taking hydroxyurea 500 mg daily, five days per week and acetylsalicylic acid 100 mg per day, together with bisoprolol; her blood count was stable, and kidney, liver, and thyroid functions were normal. Electrolytes, glycaemia, and cholesterol were all within normal ranges. On physical examination, we observed no enlargement of the superficial lymphnodes, of the liver or of the spleen.3, platelets were 605,000/mm3, while haemoglobin was slightly decreased (11.3g/dL). In May 2012, on physical examination, her spleen and liver were respectively 2 and 1 cm below the costal margin. A few days later, haemoglobin levels decreased to 9.5g/dL, and the patient referred palpitations as the primary symptom. Four months later haemoglobin further dropped to 8.8 g/dL; LDH rose to 556, but white cells and platelets were stable. The patient underwent an abdominal CT scan which showed normal liver diameter and enlarged spleen . The bone marrow biopsy was repeated in October 2012: histopathological examination showed an evolution from Polycythaemia Vera to a Myelodysplastic/ Myeloproliferative syndrome, with a myeloblast percentage of 4% . Moreover, there was also an increase in the need for transfusions (3 monthly units of RBCs), therefore, a first 21-day course of lenalidomide (Revlimid) 10 mg daily was started on 9th February 2013. After the first lenalidomide course, the patient reported an improvement in her general conditions, despite mild oedema of the lower limbs; blood count showed that WBCs had dropped to 22,640/mm3, platelets were 266,000/mm3, and Hb was 7.7 g/dL thus requiring RBC transfusion. Hydroxyurea was discontinued, and low-dose hydrochlorothiazide and amiloride were associated to reduce the swelling of the legs. After the second course of lenalidomide, her blood count showed a dramatic improvement: WBCs dropped to 6,000/mm3 and haemoglobin rose to 9.9 g/dL, while platelets were stable . Erythropoietin treatment was discontinued. The spleen was palpable 1 cm above the transverse umbilical line and was significantly reduced in volume. Three months after the start of lenalidomide therapy, the patient’s blood count completely returned to normal with a haemoglobin level of 14 g/dL, while platelets and WBCs were steadily normal. Moreover, the spleen size reduced to 2 cm below the costal margin. After six courses, the patient reported recurrent abdominal pain and vomiting, together with diarrhoea, leading to hypotension and bradycardia, and she was admitted to the hospital. These GI tract symptoms remained for approximately three months, after which the therapy prescribed by the gastroenterlogist  resulted effective. However, we decided to reduce the Lenalidomide dosage to 5 mg per day, after completing 10 courses. Once the dose of lenalidomide was lowered to 5 mg (November 2013), the GI symptoms disappeared, and the haematological response remained stable. To date, the patient has undergone eighteen cycles of lenalidomide. Her general conditions are fine, and she no longer has abdominal pain or diarrhoea. Her most recent blood test  showed a WBC count of 5,600/mm3 , haemoglobin 12.4 g/dl, platelets 158,000/mm3  clone.In our opinion, this is an interesting clinical case in which monotherapy with low dose lenalidomide proved to be effective in achieving sustainable clinical, haematological and molecular remission. Regular, non-dysplastic hemopoiesis was restored, our patient was no longer transfusion-dependent, splenomegaly decreased and the del(5q) mutation disappeared in the peripheral blood. More studies are needed to clarify the biological effects of lenalidomide on patients showing myelodysplastic/myeloproliferative features. It is important to consider lenalidomide as an option for treating selected patients with 5q- syndrome and proliferative bone marrow since it proved, in some cases, to be effective as a single agent and to have quite good subjective tolerance.