In birds, seasonal reproduction is regulated by day length, with long days in the spring activating the hypothalamic-pituitary-gonadal axis and reproductive behaviors. The photoreceptors mediating this process remain unknown, but recently, the premammillary nucleus (PMM) of the hypothalamus has been implicated as the site of photoperiodic signaling in turkeys. We performed electrolytic lesions of the PMM to elucidate its role in the photoactivation and maintenance of egg production in female turkeys. Our results show that ablation of the PMM does not alter the normal lay cycle. No differences were found between lesioned birds and sham controls in the latency to lay following photostimulation, nor in subsequent egg production over a period of 29 weeks. No differences in the incidence of gonadal regression were found, indicating that the PMM is not essential for the termination of breeding. We conclude that any role of the PMM in photoperiodic regulation, if it exists, is redundant with other components of the system. Many avian species breed seasonally, using the annual changes in day length to synchronize reproduction with favorable environmental conditions. Under short-day conditions in winter, most birds are reproductively quiescent and the gonads are in a regressed state. Increasing day length in the spring stimulates the reproductive axis, leading to gonadal growth and reproduction. After continued exposure to long photoperiods, reproductive activity ceases, the gonads regress, and the birds become photorefractory (insensitive to the stimulating effects of long days). Photorefractoriness ensures that reproduction does not occur during unfavorable conditions in winter. After exposure to short days, the animal regains photosensitivity and the cycle starts over. This process is known as photoperiodism (reviewed in ). The phCoturnix japonica). Long days induce the expression of thyroid stimulating hormone (TSH) in the pars tuberalis (PT) of the pituitary, which upregulates the expression of the gene for Type 2 deiodinase (Dio2) in the MBH. Dio2 enzyme converts thyroid prohormone (T4) to its more active form (T3) [3 in the MBH promotes the secretion of gonadotropin releasing hormone (GnRH) from GnRH neuron terminals in the median eminence (ME) by altering neuroglial interactions [In birds, it is well-established that neither the eyes nor the photosensitive pineal gland are required for the perception of photoperiodic information , 6. Rathorm (T3) –9. It apractions , 11. GnRractions . In contractions , 13.Gallus domesticus), lesioning the lateral septal region, which expresses the circadian photopigment melanopsin (OPN4), attenuates photostimulation of the gonads [Meleagris gallopavo), which also expresses melanopsin [Although the neural mechanisms of photoinduction have been well-described, the exact location of the DBPs remain elusive. Recent works characterizing opsin expression in the avian brain have uncovered 4 candidate locations, expressing several distinct opsins, that may serve as DBPs. 1) In chickens  Elece gonads , 17. 3) e gonads , 19, expe gonads . 4) The lanopsin , shows planopsin  and is planopsin , 23. Amoc-fos mRNA [GnRH-1 mRNA in the bed nucleus of the pallial commissure (nCPa) [TH and TPH1 mRNA cycle rhythmically in constant darkness (in opposing phases), indicating that both transcripts are driven by an endogenous circadian clock, and their expression patterns differ under short- and long-day conditions [The present study focuses on the PMM, a small area of the caudal MBH that has been implicated as the site regulating seasonal reproduction in turkeys. Light given during the photoinducible phase  activates neurons in the PMM, as measured by changes in fos mRNA . This ace (nCPa) , 25, a re (nCPa) . In addie (nCPa) . Circadie (nCPa) . Furthere (nCPa) , 22. Some (nCPa) . Dopamine (nCPa) –29, and e (nCPa) , 30. In nditions . TH-immunditions .Taken together, these findings suggest that the turkey PMM may contain a light-sensitive circadian oscillator, comprised of melanopsin photoreceptors and canonical clock gene machinery, coupled to dopamine and melatonin output. This system is sensitive to changes in photoperiod, and may integrate this information with the HPG axis to regulate gonadotropin release and the reproductive cycle . Despitead libitum; custom-formulated starter and grower diets  were used as appropriate. Lighting conditions were changed to 8L:16D at 16 weeks of age to condition the birds for photostimulation. Room doors were light-proofed and light baffles were placed over ventilation fans to ensure the integrity of dark conditions. All procedures were approved by the Institutional Animal Care and Use Committees at Pennsylvania State University and the University of Kentucky .Turkey poults  were obtained on hatch day and housed under standard breeder housing conditions until 26 weeks of age. Poults were initially kept under 24 hours of constant light (incandescent overhead lighting) and infrared brooding lamps. At day 2 of age, overhead lights were changed to a 14L:10D schedule. Starting at one week of age, heat was gradually reduced over 9 weeks from ~29°C to a target temperature of 20–21°C and infrared brooding lamps were removed. Room lighting intensity measured at the level of the birds' heads was maintained at 10 lux for the remainder of the study. This light intensity was used to inhibit aggression and resulted in egg production that met performance targets for the strain . Food anBirds were randomly assigned to one of two groups: birds in the unstimulated (U) group (N = 40) were kept under short-day conditions (8L:16D) and remained unstimulated until after surgery, whereas birds in the stimulated (S) group (N = 42) were stimulated by light (16L:8D) prior to surgery. After surgery, all birds were placed under photostimulating (16L:8D) conditions, where they remained for the duration of the study. The U group allows us to test the hypothesis that the PMM is involved in photostimulation, whereas the S group reveals the role of the PMM in the maintenance and/or cessation of egg-laying following normal photostimulation. Groups of ~4 birds were transferred to photostimulating conditions each day, starting at 26 weeks of age, to create the S group. Pubic bone spacing, squatting behavior, and egg-laying were monitored in photostimulated S group birds prior to surgery. Surgery was performed when photostimulation was confirmed, i.e., within 0–2 days of egg-laying. Concurrently, we also began surgeries on birds in the U group, alternating surgeries between the S and U groups. The resulting age-matched birds ranged between 28–33 weeks (surgery) and 26–32 weeks (photostimulation).Bilateral electrolytic lesions of the PMM were performed under general anesthesia. Food and water were withheld 2 hours prior to surgery. To reduce bleeding and cerebral edema during and after surgery, birds were injected with dexamethasone (1 mg/kg) 20 minutes prior to anesthetic induction. Anesthesia was induced and maintained with isoflurane , delivered via a nose cone for induction, and via tubing inserted in the mouth and nares for maintenance. Anesthetic depth was monitored by examining muscle tone, toe pinch reflex, corneal reflex, and respiratory rate. Anesthetized birds were placed into a stereotaxic instrument  with the head positioned at a 45° angle. Coordinates for the PMM lesion were 0.0 mm lateral-medial, +10.6 mm rostral-caudal, and +1.6 mm dorsal-ventral, relative to the reference .An incision was made through the skin on the top of the head to expose the skull. A surgical drill was used to make a small hole (~1.5mm diameter) in the skull over the target coordinates, drilling ventrally until the dura mater was visible. A modified sterile needle was used to pierce the dura. An electrode  was lowered into the brain according to target dorsoventral coordinates. To make the lesion, positive current (1mA) was applied for 15 seconds using a DC constant current lesion maker (Grass Instruments Model D.C. LM5). Midline lesions with this protocol resulted in bilateral ablation of the PMM. The electrode was allowed to cool for 2 minutes before being withdrawn. Sterile gel foam was applied to the craniotomy and the skin was sutured shut. The bird was monitored closely for ~1 hr and then returned to experimental housing, where it was kept in a separate recovery pen for ~18–24 hrs before being released into the open room with the other birds. Within each experimental group (U and S groups), a subset of birds received sham lesions in which the electrode was lowered into the brain, but current was not applied.2) with photostimulating lighting . Birds from U and S groups were evenly distributed between the two rooms. Food and water were available ad libitum; a custom-formulated breeder diet  was introduced following photostimulation and used for the remainder of the study. To monitor egg-laying in individual birds, each room was equipped with 16 custom-built trap nest boxes (71H X 76D X 61W cm). Upon entering the nest box, the trap door closed, preventing entry by other birds and prohibiting the trapped bird from leaving. Every 2–3 hours, nest boxes were checked: we recorded bird ID and egg presence (yes/no), removed any eggs, removed trapped birds, and re-set the trap doors. Check times were ZT0, 3, 6, 8, 10, 12, 15 . Forced ejection from nest boxes is known to promote continued egg-laying while inhibiting incubation behaviors and is routinely utilized in commercial operations to maximize production. In our study, it allowed us to quantify egg production in individual birds. All birds received at least 12 weeks of continuous trap nesting following photostimulation, capturing the onset of lay and peak production in all birds. Afterwards, birds were given access to nest boxes without traps for 4 weeks (eggs were removed from the room daily at ZT0). Thereafter, we intermittently monitored individual egg production by trap nesting one out of every three weeks for the next 13 weeks .Following surgery, birds were placed into one of two identical light-tight rooms  or active . Brains were removed, flash-frozen in 2-methylbutane cooled with dry ice, and stored at -80°C. Brains were cryosectioned (20 μm), thaw-mounted onto gelatin-coated slides , and post-fixed in 4% paraformaldehyde. One set of slides was Nissl stained with thionin to determine the accuracy and extent of the lesions. As others have done , we firs2 goodness-of-fit test, and negative binomial was found to be a good fit for both variables . Therefore, we used statistical procedures for which this distribution could be specified, or that did not make assumptions about the distribution of the data. For dependent variables latency, total12, and eggs/wk, we used the Kruskal-Wallis test. In addition, for latency and total12, we performed negative binomial regression with a generalized linear model, fit with the GENMOD procedure in SAS. A Type III test was used to evaluate the overall effect of lesion category. The fourth variable, egg production curves, involves repeated sampling within individuals  with missing data across time (weeks without trap nesting) that varies among individuals according to photostimulation date. To assess whether or not lesions had an effect on egg production curves, we utilized the GLIMMIX procedure in SAS to fit a generalized linear mixed model to these data. A negative binomial distribution was found to be a good fit for these data , so this distribution was specified for the dependent variable to obtain a negative binomial regression. We modeled the fixed effects of lesion category, time (in weeks), and their interaction; in addition, since the egg curve is not linear, we also modeled time as a 2nd, 3rd, and 4th order polynomial. Repeated sampling for individuals over time was specified as a random residual effect. We used a spatial power structure for the covariance in the repeated-measures effect, since the time intervals of egg sampling were not evenly spaced across the entire study. Hypothesis tests for the significance of main effects were evaluated with a Type III test of fixed effects. Marginal means for lesion category, adjusted for other model effects, were generated using the LSMEANS option; differences between the control group (sham/missed) vs. partial and full lesions were evaluated. The incidence of gonadal regression was compared among lesion categories using Fisher’s exact test.Individual egg data were recorded in a binary format (egg = 1 or 0) according to date. Because photostimulation occurred on different dates across individuals, date was converted into "number of days following photostimulation." Lesions that missed the PMM were grouped together with shams. Within each photostimulation group (U and S), the explanatory variable was lesion category: sham/missed lesions, partial lesions, and full lesions. We analyzed the following dependent variables: 1) "latency," the number of days following photostimulation until the first egg was laid, 2) "total12," total egg production during the first 12 weeks following photostimulation, 3), "eggs/wk, " mean weekly egg production for the duration of the study, and 4) "egg production curves," individual weekly egg production over the duration of the study. All analyses were carried out in SAS . We tested all dependent variables for homogeneity of variance and normality of distribution. Variance was homogenous for all variables, but none of the variables were normally distributed. For the simple count data , we tested goodness-of-fit for Poisson and negative binomial distributions using the Pearson’s χFor the S group (N = 42), 11 birds were determined to have full lesions of the PMM, 19 birds had partial lesions, and 6 birds were classified as missed lesions. Sham surgeries were performed in 6 birds. For the U group (N = 40), 9 birds had full PMM lesions, 22 birds had partial lesions, and 4 birds had missed lesions. Sham surgeries were performed in 5 birds. Examples of full, partial, and sham lesions are shown in p = 0.36, U group p = 0.39). Furthermore, according to the regression analysis  . Fig 2 sanalysis , lesion p = 0.11, U group p = 0.23) nor for mean weekly production overall . Moreover, for total12, regression analysis showed that lesion category was not a significant predictor of egg production, and the predicted model estimates for full and partial lesions did not differ from shams/missed lesions .In the S group, 7 out of 42 birds had regressed gonads. The breakdown according to lesion category is as follows: full lesions, 1 out of 11; partial lesions, 3 out of 19; sham/missed lesions, 3 out of 12. In the U group, 9 out of 40 birds had regressed gonads. By lesion category, the numbers are: full, 1 out of 9; partial, 4 out of 22; sham/missed, 4 out of 9. Fisher’s exact test for a 2X3 contingency table revealed that lesion categories do not differ in the proportion of regressed gonads (S group The photoperiodic breeding cycle can be separated into 3 active stages: the onset of breeding (photoinduction), the maintenance of breeding condition, and the termination of breeding (photorefractoriness). Our study clearly demonstrates that the PMM is not required for photostimulation or maintenance of the reproductive system in turkey hens. Bilateral ablation of the PMM did not alter the onset of lay following photostimulation, nor subsequent egg production under photostimulating conditions. Moreover, it appears that the PMM is not required for photorefractoriness, as indicated by lesioned birds with regressed gonads under photostimulatory conditions. These results contrast with a body of literature suggesting an important role of the PMM in photoperiodic timekeeping , 23, 26.Our study included two different photostimulation treatments, designed to reveal potential roles of the PMM at different stages of the breeding cycle. In the S group, birds were photostimulated prior to PMM lesions. Thus, any effect of PMM lesions in this group would indicate that the PMM contributes to the maintenance of reproductive activity under long-day conditions after normal photoinduction has already occurred. Egg laying onset aligned with expected latency values , indicatIn the U group, birds received PMM lesions prior to photostimulation. This allowed us to investigate the role of the PMM in photostimulation itself. Surprisingly, the onset of lay appeared to occur normally in this group as well. As with the S group, production latency was within the normal range, and PMM lesion had no effect on latency to lay. These results clearly demonstrate that the PMM is not required for early key events during photostimulation: perceiving the photoperiodic light signal, measuring the photoperiod with a circadian clock, or the activation of output signals that stimulate the HPG axis, despite evidence that the turkey PMM contains the machinery for each of these functions , 23. AddPhotostimulation of the HPG axis has been well studied, and the chain of events beginning with long-day induction of TSH in the PT, and culminating in release of gonadotropins from the pituitary and gonadal growth, has been described in detail , 33, 34.GnRH-1 transcription [Sturnus vulgaris), gonads spontaneously regress under stimulating photoperiods, and do not recrudesce even if the photoperiod is lengthened. In species with relative photorefractoriness, such as Japanese quail, declining photoperiod initiates gonadal regression; increasing the photoperiod causes the gonads to recrudesce. Domestic turkeys are thought to fall somewhere in between these two extremes, showing large individual variation in the expression of relative and absolute photorefractoriness [GnRH-1 mRNA following photostimulation [Although the precise neuroendocrine mechanisms are not well-understood, the termination of breeding and photorefractoriness are associated with decreased secretion of GnRH-1 and a reduction in circulating LH. In many songbird species, GnRH-1 protein content in the hypothalamus changes dramatically in response to photoperiod, and these changes are driven by photoperiodic regulation of cription . Thus, icription . The spetoriness , 38. Altmulation , 25, 26,


Mast cell leukemia (MCL) is a very rare form of systemic mastocytosis (SM) and accounts for less than 0.5% of all mastocytosis. The diagnosis of MCL requires the presence of SM criteria, accompanied by leukemic infiltrating of atypical mast cells (MCs) in bone marrow (BM), peripheral blood as well as extracutaneous organs. MCL is a fatal disease that almost always behaves aggressively, and the median survival time is only about six months. Herein, we present a rare case of de novo MCL without CD25 expression and KIT mutations.A previously healthy 13-year-old boy was referred to our hospital due to incidental discovery of an enlarged right tonsil. Diffuse infiltration of medium-sized hematopoietic blasts was found in his right tonsil, BM and multiple lymph nodes. The neoplastic cell population was subsequently revealed to exhibit differentiation towards the mast cell lineage by expressing CD117 and tryptase, but the cell population lacked expression of CD25/CD2 and the activating mutation of the KIT gene. An abnormal karyotype was identified, but no leukemia-associated fusion genes were found. Involvement of peripheral blood, bone and lung was subsequently demonstrated. The most important differential diagnosis included tryptase-positive (T+) acute myeloid leukemia, myelomastocytic leukemia and basophilic leukemia. The morphological characteristics and infiltrating patterns of the abnormal MCs supported the final diagnosis of MCL. Although intensive chemotherapy and allogeneic stem cell transplants were performed on the patient, he died 18 months after initial presentation.Due to its rarity, the diagnosis of MCL without typical immunophenotype and genetic aberrations is particularly challenging. Comprehensive investigation of clinical and pathological features to exclude other T+ myeloid neoplasms is necessary.The online version of this article (10.1186/s13000-018-0691-2) contains supplementary material, which is available to authorized users. Mast cells (MCs) are defined as a type of leukocytes derived from hematopoietic stem cells, which contain abundant cytoplasmic basophilic granules rich in various mediators, such as serine proteases, and play a key role in innate and adaptive immunity . MastocyMast cell leukemia (MCL), one of the rarest types of leukemia, is the leukemic form of SM that accounts for less than 0.5% of all mastocytosis. There are only approximately 70 well-documented cases reported to date –4. MorphA 13-year-old boy with no symptoms was incidently  found to have an enlarged of right tonsil during an orthodontic procedure. The patient was previously healthy, except for a one-year history of psoriasis. Physical examination showed the right tonsil was obviously enlarged with a multinodular appearance and purulent exudate. Mild splenomegaly was found by palpation and computed tomography (CT). The superficial lymph nodes were not palpable. Laboratory examination was unremarkable. Then, a tonsillectomy was performed. Histological examination revealed a diffuse proliferation of monomorphic, medium-sized cells that partially effaced the architecture. The cells resembled hematopoietic blasts, with finely dispersed chromatin, moderate vacuolated cytoplasm, medium-large sized round nuclei and indistinct nucleoli. Mitotic figures were easily found. Neither angiodestruction nor necrosis was observed. Immunohistochemical studies showed positive immunoreactivity of the cells for CD45, CD43, tryptase, CD117, CD56 and negative immunoreactivity for CD34, TdT, CD10, CD20, CD3, PAX5, CD68(KP1), MPO, CD30, CD2, CD7, CD25, TIA1, GranzymeB, CD123, CD235 and CD61  and one minor criterion  for systemic mastocytosis, with up to 16% atypical MCs in peripheral blood smears. The case did not meet other minor criteria including expression of CD25 and/or CD2, an activating point mutation at codon 816 of KIT, and the serum tryptase level could not be assessed due to technical limitations. However, MCs in MCL may lack expression of CD25 and CD2 in a proportion of MCLs, and they have been reported to be double negative for CD2/CD25 in 38% of MCL cases . SequencBased on the unusual manifestations of our case, the most important differential diagnosis is T+ AML. The compact round blast-like immature cells with expression of CD43, CD33, CD117 and CD56, absence of CD25 and CD2 expression, as well as lack of a KIT mutation in this case, would be consistent with an early stage of myeloid lineage differentiation. Although the expression of tryptase is thought to be specific for mast cells, the existence of T+ AML is reported to be not uncommon according to the published studies , 11. T+ MML is another important differential diagnosis of SM, especially when systemic mastocytosis with an associated hematological neoplasm or MCL with a lack of KIT codon 816 mutations is suspected. MML is an extremely rare and underrecognized myeloid neoplasm belonging to the group of T+ myeloid neoplasms and shares overlapping features with MCL, T+ AML and basophilic leukemia (BL) . AlthougAnother disease that should be ruled out is BL. Mast cells share a number of similar antigens and functional properties with basophils. Basophil granules containing histamine and heparin can be seen in either basophils or MCs. Both cells can release histamine upon binding to immunoglobulin E. However, combined CD117 and tryptase staining is a key feature of MCs that distinguishes them from basophils, as well as other mast cell-associated disorders . TryptasMCLs usually show an aggressive clinical course with a median survival time of no more than 6 months, and there is no approved standard therapy, to date. The patient died 18 months after initial diagnosis, even with aggressive chemotherapy and bone marrow transplant. Due to the rarity of MCL, few options are available for treatment, a situation requiring more clinical trials to explore novel regimens.In summary, herein, we present an unusual case of de novo MCL without CD25 expression and KIT mutations, with an aggressive clinical course and poor prognosis. The diagnosis of MCL is challenging when there are a lack of typical immunophenotypes and genetic aberrations, and the serum tryptase level is unavailable due to technical limitations. Under the circumstances, the characteristic morphology of MCL is the most important evidence that led our diagnosis in the right direction. New potential molecular aberrations in such cases need to be further investigated.Additional file 1:Materials and Methods. (DOCX 22 kb)


Endocarditis with Abiotrophia defectiva represents 4.3% to 6% of all streptococcal endocarditis. The article presents diagnosis issues and the complexity of the treatment.We present the case of a female white patient, aged 26 years, who developed infectious endocarditis caused by A defectiva, in the last trimester of pregnancy, a biofilm-related infection associated with the presence of fixed braces.The diagnosis of infectious endocarditis was confirmed by the cardiac ultrasound examination that revealed a voluminous vegetation on the mitral valve, and acute mitral regurgitation caused by chordae tendinae rupture, and also by isolating Abiotrophia defectiva from two positive blood cultures.The decision to undergo surgical intervention was taken, and a mitral valve replacement was performed. Surgical intervention that was associated with board-spectrum antibiotic therapy.A defectiva, remains a rare cause of infective endocarditis, with a reserved prognosis that is motivated by the extensive valvular lesions and the risk of embolism.The use of antibiotics administered in association, in the management of infective endocarditis, is mandatory. It has been involved in ocular infections, osteoarticular infections, prosthetic joint infections, otitis and sinus infections, cerebral abscesses, iatrogenic meningitis, and pancreatic abscesses. Endocarditis with Abiotrophia spp. represents about 4.3% to 6% of all streptococcal endocarditis. We present the first case of endocarditis with A defectiva, in a pregnant woman, associated with premature birth, and other various coinfections.2We present the case of a female white patient, aged 26 years, who denied any pathological diseases, except for repetitive episodes of pharyngitis. She was at the first pregnancy and was brought to the Obstetrics and Gynecology Department at 31 weeks of pregnancy for premature rupture of membranes and lumbosacral pain associated with mild left motor deficit, being suspected of a L5-S1 disc herniation. She delivered a 1550 g male fetus via caesarean section. She had had fixed braces for about a year, often causing gingival inflammation, for which her dentist recommended local antiseptics until delivery, further decisions being postponed. The postpartum clinical state was associated with frequent febrile episodes, apparently motivated by lactation. On the ninth day of postpartum confinement, she suddenly presented severe respiratory failure, an arterial oxygen saturation measured by pulse oximeter of 70% to 80% on simple face mask, dyspnea with tachypnea, disseminated crepitant rales in both lungs, heart rate of 180 to 190 beats/min, and marked alteration of her general condition.The patient showed acute pulmonary edema, cardiogenic shock, requiring orotracheal intubation and mechanical ventilation. At the cardiovascular examination, a grade 3/6 systolic murmur, with left axillary irradiation was heard upon auscultation, which raised the suspicion of mitral valve injury—chordae tendineae rupture or infectious endocarditis.Investigations were completed by a chest radiography that revealed prominent emphasized hilar interstitial drawing. Predominantly infrahilar, faint shaped, and bilateral parahilar micronodular, and subnodular opacities were also present. Bilateral, inferior lobe, and lung inhomogeneity was present being associated with clearing of the diaphragmatic perimeter.A transesophageal echocardiography was also performed and revealed a massive mitral insufficiency with eccentric mitral regurgitation jets, and a 2-cm long, voluminous vegetation that was attached to the anteromiddle (A2) segment of the anterior mitral valve leaf (AMVL), with a ruptured chorda. Cauliflower-shaped hypermobile vegetative formations of 16-mm diameter were attached to the posteromedial papillary muscle (PM) of the left ventricle. At the level of the left atrium, hypoechogenic deposits of vegetation could be visualized. Also, the transesophageal echocardiography, revealed a severe pulmonary arterial hypertension with a pulmonary artery systolic pressure (PASP) of 60 + 20 mm Hg on the pulsed wave Doppler mode.The decision to undergo surgical intervention was taken. A mitral valve replacement with a St Jude Medical no. 27 mechanical heart valve fixed with COR-KNOT device, through a minimal invasive approach—right mini-thoracotomy—was performed. The resumption of cardiac activity after declamping was in a third-degree atrioventricular block (AV block) with variable ventricular rate from the absence of ventricular depolarization to 50 to 60 beats/min, aspect which was maintained for 10 days, the reason why it was decided to implant a bicameral cardiac pacemaker in left subclavicular position.A defectiva was identified using the VITEK 2 Compact analyzer . Antibiotic sensitivity testing has not been performed. The most important laboratory studies are presented in Table From the harvested hemocultures and the bacteriological examination of the vegetation, A defectiva from 2 positive blood cultures . Both major Duke criteria were met. Two of the minor criteria were also met (temperature >38.0°C (100.4° F and vascular phenomena).The diagnosis of infectious endocarditis was confirmed by the cardiac ultrasound examination that revealed a voluminous vegetation on the mitral valve, and acute mitral regurgitation caused by chordae tendineae rupture, and also by isolating 2) for about 1 month for which she did not present for medical consultation. During the third week of hospitalization, she had subungual bleeding at the left middle finger.Postoperatively and after resuming anamnesis the patient reported the presence of chills, fever not measured by thermometer, especially during night, fatigue, weight loss  associated with vancomycin 2 g/d, gentamicin 160 mg/d, and caspofungin 50 mg/d. Since the seventh day of hospitalization, ceftriaxone 2 g/d plus gentamicin was continued for 2 weeks. After the surgical intervention, the respiratory evolution was rapidly favorable.Clostridium difficile infection, required the discontinuation of cephalosporin therapy and the continuation with penicillin G 36 MU/d for another 3 weeks in parallel with oral vancomycin 4 × 125 mg/d for 14 days. The C difficile infection was diagnosed according to the guidelines published by the European Society of Clinical Microbiology and Infectious Diseases using 2 highly sensitive tests: GDH EIA (glutamate dehydrogenase enzyme immunoassay) and toxin A/B EIA. Both tests being positive and no further testing was required. Antibiotic therapy was associated with anti-clotting drugs, metoprolol 100 mg/d and perindopril 2 mg/d.The occurrence of a diarrhea episode, confirmed to be related to a 2. The resulting sonication fluid is vortexed, and 50 mL of sonication fluid is centrifuged at 2500 rpm for 5 minutes. The resulting precipitate was inoculated onto Columbia agar with sheep blood , Sabouraud plate, MacConkey agar plate, glucose broth, lactose broth, and thioglycollate broth. The cultures were incubated at 37°C for 14 days and inspected daily for bacterial growth. Isolated bacteria are identified using the VITEK 2 Compact analyzer . The MICs (minimum inhibitory concentrations) are assessed according to the European Committee on Antimicrobial Susceptibility Testing breakpoints. After sonication, Enterobacter cloacae was identified with the following susceptibility. The strain was susceptible to ticarcillin, piperacillin, ceftazidime, aztreonam, meropenem, imipenem, amikacin, gentamicin, ciprofloxacin, and trimethoprim/sulfamethoxazole.Prior to stopping penicillin treatment, it was decided to remove the fixed braces. The retrieved implants were introduced is a sterile container and saline sterile solution was added in this sterile container. The implants were processed within 30 minutes by sonication (1 minute) using an ultrasound bath  at a frequency of 42 kHz and a power density of 0.22 W/cmThe echocardiographic follow-up on discharge was as follows nondilated left ventricle (45 mm), interventricular septum of 8 mm, posterior wall thickness of 9 mm, with preserved systolic function and an ejection fraction (EF) of 50%. Normokinetic, nondilated right ventricle of 31 mm. The echocardiography demonstrated the mechanical prosthesis with normal mobility for its elements as well as no periprosthetic regurgitation associated with maximum diastolic gradient estimated at 13 mm Hg (average gradient of 4 mm Hg) and mild aortic regurgitation as well as a mild tricuspid regurgitation. The systolic pulmonary artery pressure was of 20 + 10 mm Hg: 30 mm Hg, with an inferior vena cava of 16 mm, without inspiratory collapse in the context of mild hypervolemia. No pericardial effusion was revealed.The favorable evolution of the patient was the result of an interdisciplinary collaboration between gynecologist, intensive care specialist, cardiovascular surgeon, cardiologist, infectious disease specialist, and microbiologist, who helped caring this case. The psychomotor development of the infant is according to his age.3A defectiva was included in the genus Abiotrophia, along with Abiotrophia adiacens, from which it differs by the presence of alpha- and beta-galactosidases, respectively, by the absence of beta-glucuronidase. Subsequently, A adiacens, A elegans,A balaenopterae, and A para-adiacens were reclassified as a new genus, Granulicatella gen. They are NVS, included in viridans streptococci, pleomorphic coccobacilli, non-motile, alpha-hemolytic colonies on blood agar, whose development on culture media requires complex enriched medium with vitamin B6 and l-cysteine. A defectiva possess virulence factors, such as the synthesis of exopolysaccharides, a component part of extracellular polymeric substances (EPSs), along with proteins and other constituents , with a major role in biofilm formation,–7 and moderate ability to bind to fibronectin. In vivo, the growth is supported by a variety of Gram-positive and Gram-negative bacteria. A defectiva is found under normal conditions in the oral cavity, gastrointestinal tract, and genitourinary system.A defectiva has been involved in ocular infections—endophthalmitis or keratitis, osteoarticular infections, arthroplasty infections,–14 otitis and sinus infections, cerebral abscesses,,16 iatrogenic meningitis, and pancreatic abscesses.–19 carbapenems, cephalosporins, daptomycin, or macrolides is not certain.,22Compared to beta-hemolytic streptococci (BHS), NVS have different susceptibility to antibiotics. NVS susceptibility to penicillin (8% according to some studies),Abiotrophia and Granulicatella represents about 4.3% to 6% of all streptococcal endocarditis, being recognized by complications such as acute heart failure, septic embolism, or extensive valvular lesions requiring surgical interventions in a larger percentage of cases compared with other non-hemolytic streptococci.–34 Approximately 125 cases have been published in the literature so far of which in 1 published case the evolution has been complicated by hemophagocytic syndrome.Endocarditis with A defectiva, in our case, was the fixed braces, from which we identified, after 6 weeks of treatment, Enterobacter cloaceae, by sonication, a pathogen that possesses the ability to form biofilm on implants in association with other Gram-positive and Gram-negative bacteria. The particularity of our case lies in the fact that it is the first case of endocarditis with A defectiva in a pregnant woman, associated with premature birth, heart failure, pulmonary edema, and extensive valvular lesions requiring urgent surgical intervention. The postoperative evolution of the case has been associated with various coinfections—C difficile and Candida krusei—associations that required adjustments in the therapeutic management of the case in the absence of knowing the antibiotic susceptibility of the isolated strain of A defectiva.The source of A defectiva maintained its susceptibility to these antibiotics. We think that it would be wiser to perform a cardiac evaluation, especially in adults, at least for major oral cavity surgeries to exclude any possible cardiac lesion at risk of infectious endocarditis. Although we did not have confirmation of any previous heart disease in our patient's condition, poor nutritional status could have been the cause of an undiagnosed preexisting cardiac pathology.The favorable evolution of our patient, under high doses of penicillin and gentamicin has shown that the isolated strain of 3.1Written informed consent was obtained from the patient for publication of this case report and any accompanying images. The study was accepted by the Ethics Committee of the hospital and they encouraged publishing the article. A copy of the written consent is available for review by the Editor-in-Chief of this journal.4A defectiva remains a rare cause of infective endocarditis, with a reserved prognosis that is motivated by the extensive valvular lesions and the risk for embolism. Almost 50% of the valvular lesions require surgical intervention of valve replacement. The variable susceptibility to antibiotics requires a close monitoring of the evolution of the cases. The use of antibiotics administered in association, in the management of infective endocarditis is mandatory.


Novel, low density structures which combine biologically-based fibers with clay aerogels are produced in an environmentally benign manner using water as solvent, and no additional processing chemicals. Three different reinforcing fibers, silk, soy silk, and hemp, are evaluated in combination with poly matrix polymer combined with montmorillonite clay. The mechanical properties of the aerogels are demonstrated to increase with reinforcing fiber length, in each case limited by a critical fiber length, beyond which mechanical properties decline due to maldistribution of filler, and disruption of the aerogel structure. Rather than the classical model for reinforced composite properties, the chemical compatibility of reinforcing fibers with the polymer/clay matrix dominated mechanical performance, along with the tendencies of the fibers to kink under compression. The preparation of inorganic aerogels was first described by Kissler, who slowly exchanged the water in aqueous silica with volatile organic solvents, which, upon removal, produced low density solids [3 [Aerogels are materials that are produced from wet gels by replacement of solvent with air, typically exhibiting bulk densities of 0.1 g/cmy solids . Great py solids ,5. A simy solids , with a y solids ,8. In ouolids [3 . These colids [3 ,14,15,16olids [3 . Such poolids [3 , and canolids [3 , highly olids [3 , or to eolids [3 . Furtherolids [3 , incorpoolids [3 , and reiolids [3 . These aolids [3 . In a prolids [3 . ExploraMn) of 108,000 kD, a polydispersity index (PDI) of ~1.7, and a hydrolyzation of 99.7% from Polysciences, Warrington, PA, USA) was used as received. PVOH Fibers  were used as received. Hemp and silk fibers  and soy silk  were used as received except for cutting to length. Deionized water was prepared using a Barnstead ROpure low-pressure, reverse osmosis system.Sodium Montmorillonite , poly , where high vacuum (3 μbar/25 °C) was applied to sublime the ice and reveal the aerogel structures of the samples. Cylindrical test pieces for compression testing, measuring approximately 20 mm in both height and diameter, were cut from each sample using a band saw. In a 100 mL beaker, 25 mL of a 5 w/v % PVOH solution was slowly added to a 10 w/v % clay gel and mixed thoroughly by hand. Fibers of the appropriate length (2–20 mm) were then added at a 1 w/v % concentration, and mixing was completed with a hand blender. The resulting clay/fiber hydrogel mixtures were poured into four 5 dram cylindrical polystyrene vials (Fisher Scientific). These forms were immediately placed into an ethanol/solid carbon dioxide bath to freeze their contents. Once the samples were completely frozen, they were removed from the ethanol/dry ice bath, and placed in a freezer at −12 °C for storage. Samples were then placed in a VirTis AdVantageCompression testing was conducted on the prepared test pieces (measuring 20 mm height and 20 mm diameter) using an Instron model 5565 universal testing machine fitted with a 1 kN load cell. These tests were performed at a constant strain rate of 1 mm/min and were stopped when the 1 kN load limit (machine limit) was reached; three samples from three separate batches were tested for each composition of interest, resulting in nine total test samples. All of the samples in this study were sufficiently robust to be handled during testing. Density values were calculated from the masses and initial dimensions of compression test pieces. For each polymer loading, density values were averaged, and the standard deviation and the standard error were calculated. The load-displacement data from each compression test were converted into stress-strain data, and a modulus was calculated from the linear-elastic region of the stress strain curve. For each fiber length, modulus values were averaged, and the standard deviation was calculated. Freeze drying of aqueous clay/PVOH/fiber samples readily produced low density, fiber-reinforced aerogel composite materials, as we have reported previously . In that3/g at 2 mm fiber length, exceeded those of silk-reinforced, 7200 kPa·cm3/g at 2 mm fiber length, and especially those of the hemp-reinforced samples, 5570 kPa·cm3/g at 2 mm fiber length, produced in this study. Poor adhesion between the matrix and the hemp fiber is likely a major reason for the reduced performance of this reinforcing fiber in the present system. The stress-strain curves for a typical hemp-containing sample  which relies upon the fiber ultimate stress, the shear stress at the fiber-matrix interface, τi, , and the fiber diameter, df [Fiber length appears to strongly affect the mechanical properties of the aerogel composites. A sharp decrease in mechanical properties can be observed with reinforcing fiber lengths over 6 mm. The trend with hemp was less well-defined, though it is directionally similar with a maximum value perhaps at 14 mm. Examining the distribution of fibers within the aerogel composite matrix, eter, df . Making It was shown by Thomason that the composite strength is impacted negatively with increasing fiber diameter, and these effects are increased further when there is a large distribution of fiber diameters as opposed to one single diameter . In addiVf is the fiber volume fraction, Pm is the property of the matrix, ξ is the geometry factor, η is the efficiency factor, νf is the volume fraction of filler, νm is the volume fraction of matrix, P is the engineering tensile stiffness parallel (E11) or perpendicular (E22) to the fiber direction or the in-plane shear stiffness (G12), and all subscripts f refer to fiber and m refer to matrix. Pf refers to either the modulus of the fiber (Ef) or the shear stiffness of the fiber (Gf). Pf can also be replaced by Pm which can be equal to either the modulus of the matrix (Em) or the shear stiffness of the modulus (Gm). Reliable estimates of the factor are:l, w, and t are the length, width, and thickness of the reinforcement of the composite. For a circular fiber reinforcement l is the length of the fiber and t and w are both equivalent to the diameter of the fiber. Therefore, is always equal to 1 for all fiber reinforcement and the factor can be rewritten in terms of the aspect ratio (l/d) [Mechanical property data for the three reinforcing fiber materials examined in this study, along with estimated properties of polymer/clay aerogels under shear conditions, are given in io (l/d) :(6)ζE11cu, as calculated using Equations (2)–(5) and values from Comparing all three fiber sets, soy silk has the largest densities, moduli, and strengths in the 2–10 mm fiber length range while hemp has the smallest values . HoweverThe theory also predicts the silk samples are the stiffest, followed by hemp and soy silk, but this is also not in agreement with the collected data. This can likely be attributed to the Halpin-Tsai equations only taking into account the modulus and diameter of the fiber and matrix, and the assumption of perfect adhesion between the fiber and the matrix. However as stated previously, hemp does not have strong adhesion in the matrix and in addition, both silk and hemp are highly susceptible to moisture absorption and kink band defects. Moisture absorption could lead to a changing fiber diameter, which could result in fiber delamination from the matrix, and kink band defects can lead to mirobuckles. None of these properties are accounted for in the Halpin Tsai calculations, and as such the collected data does not agree with the theoretical values. Calculations of the compressive strength were also performed to determine if the main mechanism of failure was via the extensional mode or the shear mode. A compressive strength of 494 kPa for hemp, 380 kPa for silk, and 230 kPa for soy silk were calculated using Equation (8) for the extensional mode, whereas a compressive strength of 1.95 MPa was calculated for all composites using Equation (7) for the shear mode. This is because the shear failure mode is only dependent upon the shear modulus of the matrix, not the filler. Shear Mode:Extentional Mode, Hemp:Both equations overestimate the measured compressive strengths of these composites, provided in Novel, low density structures which combine biologically-based fibers with clay aerogels were produced in an environmentally benign manner using water as solvent, and no additional processing chemicals. A comparison of three different reinforcing fibers, silk, soy silk, and hemp, showed that mechanical properties of the aerogels increase with reinforcing fiber length, as expected, however each material exhibits a critical fiber length, beyond which mechanical properties decline due to maldistribution of filler, and disruption of the aerogel structure. This critical fiber length agreed well with a theoretical prediction for silk and soy silk fibers, and is believed to be inaccurate for hemp due to the large and varying diameter size. In addition, the Halpin Tsai equations were shown to be poor predictors of mechanical properties for these materials, as the chemical compatibility of reinforcing fibers with the polymer/clay matrix dominated mechanical performance, along with the tendencies of the fibers to kink under compression. The knowledge gained from this work will be valuable going forward as such low density aerogel materials find applications in packaging and reinforcement, allowing optimized reinforcement of such products.


Loranthus micranthus with the aid of ball-milling, high speed stirring, and high-pressure homogenization techniques. The milled nanoparticles were characterized using laser diffraction analysis, photon correlation spectroscopy analysis, and light microscopy. The average size of leaf nanoparticles was around 245 nm and that of stem nanoparticles was around 180 nm. The nanoparticles were tested for their antimicrobial and nematicidal properties against a Gram-negative bacterium Escherichia coli, a Gram-positive bacterium Staphylococcus carnosus, fungi Candida albicans and Saccharomyces cerevisiae, and a nematode Steinernemafeltiae. The results show significant activities for both leaf and (particularly) stem nanoparticles of Loranthus micranthus on all organisms tested, even at a particle concentration as low as 0.01% (w/w). The results observed indicate that nanoparticles  of Loranthus micranthus could serve as novel antimicrobial agents with wide-ranging biomedical applications.Nanosizing represents a straight forward technique to unlock the biological activity of complex plant materials. The aim of this study was to develop herbal nanoparticles with medicinal value from dried leaves and stems of  Recent advances in the highly multidisciplinary field of nanotechnology have also stimulated innovative ideas in drug design and delivery, eventually leading to the development of novel nanosized materials for biomedical, pharmaceutical, and agricultural applications ,2. TheseLoranthus micranthus), an obligate semi-parasitic plant of the family Loranthaceae, is common in the rain forest region of Nigeria and other parts of Africa, Europe, and America, where it grows primarily on Kola accuminata, Baphianitida, Citrus limon, and Pentacle thramacrophylla  to more realistic modern-day applications in herbal remedies and in the developing world ,4. Indeehemicals . The biohemicals . Not surWe have therefore considered nanosizing as a possible alternative, as this method requires only a few steps from raw to final product. It can be applied globally and, by milling entire plants, maintains the chemical composition of the original material without adding any artificial ingredients or wasting any material ,8.L. micranthus were harvested in the forests near Federal University, Ndufu-Alike Ikwo, Abakaliki, Ebonyi State, the federal republic of Nigeria. The plants were identified botanically at the Department of Biology, Federal University, Ndufu-Alike Ikwo, Abakaliki, Ebonyi State, the federal republic of Nigeria, where a specimen of this plant had already been deposited prior to this study. The plants were separated into stem and leaves, and each plant part was dried immediately, in shade, at room temperature to reduce the moisture content for prolonged storage. The dried plant parts were blended into powder with a simple mechanical blender (household coffee grinder). The nanosizing of samples was conducted as described previously [® 2000 UP  as 1% w/w coarse suspension. Each sample was then subjected to high speed stirring (HSS) with a Polytron® PT2100  attached to a Polytron® PT-DA-2112/EC aggregate. Three 1 min cycles of high speed stirring (HSS) were performed at 11,000 rpm. Subsequently, a high-pressure homogenizer (HPH) was employed as the so-called “premilling stage” in an APV Gaulin LAB40 high pressure homogenizer  with three consecutive cycles at 200, 500, and 1000 bar pressure and a final homogenisation cycle at 1500 bar pressure (1200 bar pressure was used in the case of the dried leaves).The plants of eviously . BrieflyAll steps for nanosizing were accompanied by adequate characterization procedures, according to a previous report (8). Laser Diffraction (LD) analysis with a Mastersizer 3000  and Photon Correlation Spectroscopy (PCS) with a Zetasizer Nano ZS  were explored. This kind of analysis allows researchers to monitor the efficacy of each step and also to assess the response of the material to the different milling technologies employed. Furthermore, visual confirmation of the acquired nanosized material was determined by Light Microscopy (LM) using an Olympus BX53. Each analysis was repeated in triplicate. Results were represented as mean ± standard deviation (SD).Escherichia coli and Gram-positive Staphylococcus carnosus, the pathogenic fungus Candida albicans, the yeast Saccharomyces cerevisiae, and a nematode with agricultural relevance, namely Steinernema feltiae.The antimicrobial and nematicidal activities of the nanosized suspensions were estimated in bacteria, including Gram-negative w/w. Nutrient Luria-Bertanibroth (LB), bacterial basic media, Sabouraud Dextrose (SD), and Yeast Peptone Dextrose (YPD) were used as media for E. coli, S. carnosus, C. albicans, and S. cerevisiae, respectively. The positive control was composed of a mixture of penicillin, streptomycin, and amphotericin B  for S. carnosus and E. coli, while ketoconazole was used as the positive control for C. albicans and S. cerevisiae. The growth culture with medium for each organism was used as the negative control. The stabilizer 1% Plantacare® stock was used as an additional, negative solvent control. All experiments were carried out in triplicate and on three different occasions (n = 9).Bacterial and fungal cell densities were transiently determined by measuring the optical density (OD) at 570 nm at 0 h (first measurement immediately after incubation), 4 h, and 24 h after incubation at 37 °C according to the method described by Zgoda and Porter  and usinS. feltiae were purchased from Sautter and Stepper GmbH  and stored at 4 °C in the dark. Before each experiment, fresh samples of nematodes were purchased and stored in the freezer upon arrival and used for a maximum of six days. To perform the assay, 200 mg of nematodes was reconstituted in 50 mL of phosphate buffered saline (PBS) (pH = 7.4) to form a homogenous suspension to revive the nematodes. Afterwards, the individual suspensions were allowed to rest at room temperature. The sedimentation was avoided with occasional shaking in moderate light and at room temperature (25 °C) for 30 min. Viability of the nematodes was determined by counting the live and dead nematodes under a light microscope  at four-fold magnification. An initial viability value of more than 90 percent was considered necessary to perform the experiments.The soil nematodes w/w of L. micranthus. Afterwards, 10 µL of nematode suspension was added to each well and the final volume in each well was adjusted to 100 µL with phosphate buffered saline (PBS) (pH = 7.4). PBS was used in the negative control and ethanol in the positive control. Viability was determined at 0 h and 24 h by counting the live and dead nematodes, whereby 50 µL of hot water (50 °C) was added to each well to stimulate the nematodes for the 24 h reading [n = 9).Nanoparticle suspensions of the samples were added into the wells of a 96-well plate to achieve final concentrations of 0.0125%, 0.025%, 0.05%, and 0.1% p < 0.05, ** p < 0.01, and *** p < 0.001.All data  were expressed as the standard error of the mean (±SEM). For nematicidal activity, data comparisons were performed using One-way analysis of variance (ANOVA), and post hoc analysis was carried out by the Student Newman-Keuls (SNK) test. For antimicrobial and antifungal activities, data were analysed by two-way ANOVA, Bonferroni post hoc test. GraphPad Prism  was used for data analysis and to generate charts. Statistical significance was set at * Loranthus micranthus leaves (LML) sample, consisting of larger particles, at the end of the milling procedure, initially showed a better size reduction response when compared to the Loranthus micranthus stem (LMS) sample. This response unfortunately, can only be seen during the high speed stirring (HSS) and initial homogenization steps, whereas it was difficult to break the leaf sample into smaller particles of less than 245 nm in diameter employing the high-pressure homogenization (HPH), most probably because the leaf particles were rather elastic when compared to the stem particles. The smallest size achieved for the leaf particles was 245 nm  employed as the positive control, which inhibited the growth of S. carnosus by 94% , the African mistletoe is a ubiquitous weed which can be accessed locally, easily, in large quantities, and without causing any damage to diversity or the environment [S. feltiae has therefore been investigated  is ongoing in our laboratory and this will therefore be of public importance. As for practical applications, the medium- and long-term stability of the nanosuspensions, possible aggregation, degradation, or fouling of the materials and alternatives, such as freeze-drying and resuspension as part of the so-called NaLyRe sequence, need to be investigated [As discussed already, it is likely that the multitude of natural compounds like alkaloids, polyphenols, and phenolics glycosides found in the stems and leaves of the mistletoe is responsible for this pronounced toxicity. These active ingredients, especially alkaloids, possess antimicrobial properties which explain the enhanced activity of stigated . Once suw/w), compete well with those of established antibiotics and fungicides. This study has provided an insight into the antifungal and antibiotics efficacy of herbal nanosized materials with an emphasis on L. micranthus. Our studies with nanosized leaves and stems of the African mistletoe have shown that such nanosuspensions can be prepared easily and exhibit pronounced antimicrobial and nematicidal activities, which at higher particle concentrations of 0.1% (


While Cognitive impairment (CI) has been identified as an independent risk factors for mortality in patients undergoing peritoneal dialysis (PD), it is inadequately assessed. We evaluated the applicability of the Modified Mini-Mental State Examination (3MS) in assessing specific cognitive function and compared it to a detailed neuropsychological test battery as the reference standard.In this multicentric cross-sectional study, we enrolled 445 clinically stable patients from five PD units, who were undergoing PD for at least 3 months. The 3MS was evaluated for general cognitive function. A detailed neuropsychological battery including domains of immediate memory, delayed memory, executive function, language, and visuospatial ability were evaluated as reference standards. Sensitivity and specificity of the 3MS was determined by using receiver operating characteristic (ROC) analysis.The CI prevalence evaluated by 3MS was 23.6%. PD patients with CI performed worse in all cognitive domains. The 3MS correlated well with specific cognitive domains. However, 18.5%, 57.4%, 12.6%, 8.8%, and 41.2% of patients whom were idendified as normal by 3MS still showed executive dysfunction, immediate memory impairment, delayed memory impairment, and language-ability and visuospatial-ability impairment, respectively. The 3MS identified patients having specific cognitive dysfunction with varied extent of diagnostic value, with 0.50, 0.42, 0.35, 0.34, and 0.26 of Youden index in executive function, delayed memory, language ability, immediate memory, and visuospatial ability, respectively.The 3MS is not a comprehensive instrument for major cognitive domains in PD patients. It could, however, be used for executive dysfunction and delayed memory impairment screening. Peritoneal dialysis (PD) as home-care therapy requires patients to self-monitor and self-manage their treatment  and, theThe Mini-Mental State Examination (MMSE) is a well-established tool for screening the CI in general population and in patients with chronic kidney disease , 11. MorIn this study, we aimed to evaluate the applicability of 3MS compared to a detailed neuropsychological test battery as reference for assessing specific cognitive function based on a multicentric cross-sectional dataset. The ability to distinguish between PD patients with and without specific CI across the major cognitive domains was examined by assessing the sensitivity and specificity and the Youden index of the 3MS.Table 1). As for major cognitive domains, the highest prevalence was 64.5% for immediate memory impairment, followed by visuospatial-ability impairment (47%), executive dysfunction (29.9%), delayed memory impairment (20.7%), and language-ability impairment (14.2%).A total of 667 patients were eligible for the study and 495 (74.2%) gave consent, with 445 (89.8%) completing all laboratory analysis and detailed cognitive testing. The mean age, gender, dialysis duration and primary kidney diseases were not significantly different between participants included and excluded. The basic data of our participants was in accordance with the general characteristics of the PD population in China . The meap < 0.05 for all) (Table 1).As compared to patients with normal cognitive function, those with CI tended to be older, of female gender, and less educated. They also had a greater burden of DM, CVD, and CVD risk factors such as higher body mass index, increased cholesterol, and inflammation reflected by higher CRP and lower serum albumin levels, but were more likely to have lower diastolic blood pressure . Furthermore, Spearman correlation analysis indicated that the scores of 3MS were significantly negatively associated with completion time on Trail A and Trail B, but positively associated with scores of immediate and delayed memory, language ability, and visuospatial ability .We further divided all subjects into two groups according to the presence or absence of CI. Patients in the CI group had significantly longer completion time on Trail A and Trail B, and lower scores of immediate and delayed memory, and language and visuospatial ability as assessed by the detailed neuropsychological test battery .However, when major cognitive domains were considered as the reference standards, 33.7%, 12.5%, 52.9%, 68.3%, and 34.6% of patients with CI were not found to have executive dysfunction, immediate memory impairment, delayed memory impairment, and language-ability and visuospatial-ability impairment, respectively, based on 3MS . By contrast, 18.5%, 57.4%, 12.6%, 8.8%, and 41.2% of patients without CI  had executive dysfunction, immediate memory impairment, delayed memory impairment, and language-ability and visuospatial-ability impairment, respectively (Table 4). The ROC analysis displayed an optimal cut-off of the 3MS at ≤82 points for executive dysfunction, ≤88 for immediate memory impairment, ≤82 for delayed memory impairment, ≤80 for language-ablity impairment and ≤89 for visuospatial ability impairment. The sensitivity, specificity and an AUC of each optimal cut-off value were also shown in The ROC curves illustrated different capacities of 3MS, displaying its individual sensitivity, specificity, and AUC, while major cognitive domains were considered as reference methods, respectively . The ROCIn this large-scale, multicentric cohort of Chinese PD patients, we found that the prevalence of general CI diagnosed by 3MS was 23.6%. As compared to previous data, the overal CI prevalence in our PD subjects was very close to that in their HD counterparts as diagnosed by the MMSE , 17, 18 The main goal of this study was to determine the discriminating value of 3MS in the PD population across major cognitive domains. From the previous studies, the reliability of 3MS has been shown to be higher than the MMSE in individuals with different educational backgrounds, normal community elders, geriatric rehabilitation patients, and dementia cases. , 14. HowTaking into account that the detailed neuropsychological test battery is a complicated and time-consuming tool in clinical practice, our findings also revealed that specific cut-off scores for different domains of cognitive function. The optimal cut-off scores based on the maximal sensitivity and specificity using the ROC analysis were 82, 88, 82, 80, and 89 of the 3MS in discriminating executive dysfunction, immediate memory, delayed memory, and language- and visuospatial-ability impairment. Combined with above findings, we recommended 3MS to be a screening tool of executive dysfunction and delayed memory with the cut-off value of 82, due to the relatively good sensitivity and specificity in these two domains. However, our findings are not conclusive regarding a score higher than 80, the present criterion for CI by 3MS, as being normal for immediate memory and visuospatial ability in PD patients.Taken together, our findings indicate that the 3MS could not be considered as a comprehensive tool for assessment of specific cognitive function, especially on immediate memory and visuospatial impairment. Notably, the process of PD therapy itself would be influenced by cognitive dysfunction in various aspects. Executive and memory functions broadly encompass processes responsible for diet management, fluid restrictions, blood pressure and glucose self-monitoring, and compliance to complex medication regimens. Language ability is related to communication with the PD staff, and helps to make decisions on critical medical issues. Visuospatial ability plays an important role in bag exchange, exit-site care, and catheter protection. All these domains are critical to the quality of PD therapy. Therefore, the best option in clinical practice is to simultaneously examine the general and specific cognitive function in order to identify underlying CI. Moreover, we also need to explore whether impairment in specific cognitive domains result in inverse clinical outcomes such as CVD events, PD-related infection, psychological problems, and poor quality of life.Several strengths of this study should be noted. First, this is one of the largest multicentric studies to examine the performance of comprehensive cognitive domains in PD patients. Secondly, we further suggested the discriminating values of 3MS, and the optimal cut-off score of 3MS used for assessment of special cognition function by using a comprehensive, multidimensional spectrum of cognitive neuropsychological tests as the reference. This information would be helpful in screening specific cognitive impairment, especially on executive and delayed memory function. In addition, all assessments of cognitive function were performed in a separate room with one medical staff to one patient, and relatively less variability in cognitive function suspected for the stable PD treatment, ensure the reliability of our findings.Our study also has some limitations. First, although the domains of cognition based on neuropsychological battery were applied, it is possible that it was not adequately sensitive and comprehensive to measure subtle cognitive changes in patients with milder CI. However, the RBANS is the best reference tool for cognitive function for our population, as its reliability and validity have been already proved in the Shanghai and Beijing Chinese populations , 26. SecIn conclusion, 3MS is a limited tool for detecting comprehensive cognitive performance. Our results alert clinicians to highlight the special domains of cognitive function despite a normal general test like the 3MS. However, the 3MS could be applied for screening the executive dysfunction and delayed memory impairment owing to its relatively good discriminating values for these two domains. On the other hand, as detailed neuropsychological test battery while being a gold standard might still be quite impractical in most settings. More practical, comprehensive and well-validated screening tools for CI also need to be developed with respect to clinical and academic applications.This is the affiliated study of a multi-center cross-sectional survy on cognitive funciton in PD. The detailed study design was published elsewhere. Five PDThis study enrolled prevalent PD patients between March 2013 and March 2014. Inclusion criteria for participants were as follows: patients aged ≥ 18 years; undergoing PD for ≥3 months and clinically stable; able to undergo all measurements and respond to questionnaires as required. Patients were excluded if they had a systemic infection, acute cardiovascular events, active hepatitis or cancer, surgery or trauma in the month prior to the study, and all other study-obstructive conditions such as severe eyesight loss, language incompatibility, illiteracy, mental disturbance  and upper-limb disability. We defined the preexisting dementia, confusion and any other mental disorders according to ICD-10 criterioDemographics and comorbidities were recorded, including age, gender, education level, durations of PD, body mass index, systolic and diastolic blood pressure, primary kidney disease, the presence of diabetes mellitus (DM), and history of cardiovascular disease (CVD). Mean arterial pressure was calculated. Level of education was recorded as the highest school level at which a diploma was received, that is, elementary school or lower; middle school; high school; or above high school. CVD was recorded if one of the following conditions was present: angina; class III–IV congestive heart failure (NYHA guidelines); transient ischemic attack; and history of myocardial infarction, cerebrovascular accident, and peripheral arterial disease . CerebroAfter overnight fasting while continuing PD therapy, venous blood samples were drawn from all patients for biochemical measurements. Serum levels of sodium, albumin, calcium, phosphate, triglycerides, total cholesterol, high-sensitivity C-reactive protein (hsCRP), and hemoglobin were calculated as the mean of measurements taken over the preceding 3 months. Biochemical profiles were investigated using an automatic Hitachi chemistry analyzer. Residual renal function (RRF) was defined as the mean of residual creatinine and urea clearance from a 24-h urine collection. Dialysis adequacy was defined as total Kt/V and creatinine clearance.The 3MS  was applA detailed neuropsychological test battery was evaluated as the reference gold standard. Executive function including decision-making and processing speed was assessed by Trail making tests A (Trail A) and B (Trail B) . In addiAssessments of cognitive function were performed in a separate room with one medical staff to one patient. Totally four medical staff participated in this study as observers, and they all completed a training program of the methods employed to ensure the integrity and accuracy of the assessment.Continuous data were presented as means ± SD. Durations of PD, Charlson Comorbidity Index, RRF, and hsCRP were presented as medians with interquartile ranges, owing to high data skewness. Categorical variables were presented as proportions. One-way analysis of variance, Kruskal–Wallis, or the Chi-square tests were used to compare the intergroup differences in demographic and biochemistry data.Correlations of scores of 3MS and completion time of Trail A and Trail B, and scores of immediate memory, delayed memory, language, and visuospatial ability were examined by using Spearman’s rank correlation coefficient analysis. The ability of the 3MS to screen for specific CI in PD patients was evaluated through the comparison to the abovementioned major cognitive domains. To establish the discriminative validity of the 3MS in identifying patients with and without specific cognitive dysfunction, sensitivity and specificity levels for 3MS scores at varying cut-off values were evaluated using the receiver operating curve (ROC) analysis. Area under the curve (AUC) was calculated to ascertain the quality of 3MS scores as a diagnostic tool for specific cognitive function. An AUC of 0.5 is no better than expected by chance, whereas a value of 1.0 signifies a perfect tool. The optimal timepoint of 3MS cut-off value was defined as the earliest point that provided significant discrimination between patients with and with without specific CI on a cognitive domain with optimal sensitivity, specificity, and Youden index.All probabilities were two-tailed, and the level of significance was set at 0.05. Odds ratios and 95% confidence intervals were calculated. Statistical analysis was performed by using SPSS for Windows, software version 20.0 , and Medcalc for Windows software version 9.2.1.0 .S1 File(DOCX)Click here for additional data file.


The recent and exciting images of the 2016 Olympic and Paralympic Games in Rio will beforever imprinted in our minds. Hundreds of world, Olympic, continental and nationalrecords were conquered with a combination of discipline, resilience, competitiveness andovercoming challenges. Overcoming them in every sense - a result of thousands of hoursof training that ensure exceptional aerobic performance from athletes with powerful andextremely healthy hearts.The theme of aerobic exercise and the heart has never been so in evidence. Thus, in theperspective of this Olympic legacy, it is very appropriate to address the relationbetween aerobic exercise and the heart in the Brazilian Archives of Cardiology. Morespecifically, it is important to discuss the most suitable posology or dosage ofexercise for adults in the context of primary and secondary prevention of cardiovasculardiseases. Succinctly, the objective of this viewpoint to discuss and delimit thetherapeutic range of the aerobic exercise dose for the heart and make suggestions abouthow this dose can be individualized according to certain criteria and objectives.Free animals have been and will always be active, with confinement or sedentarystyles considered unnatural. Humankind, as it is known today, was able to set offfrom central regions and, through its own means of locomotion (exercise) spreadacross far areas of the planet. Seeking food and water, running from predators, fromchildhood to old age, have always been linked to exercise. Indeed, a sedentarylifestyle is very recent in the history of humankind.2 Among several terms, it is worth highlighting the differencebetween physical activity and exercise. While both involve movement and/or musclecontraction with energy expenditure, in exercise there is movement intention in a structured and repetitive manner, with the objective ofmaintenance or optimization of physical conditioning and/or health and/or bodyaesthetics.1Before approaching the theme of exercise and the heart, it is important toconceptualize some terms .1,2 AmonRegarding the pharmacological dose, it can generally be defined by a therapeuticrange with objective minimum and maximum limits. For a given individual, it is alsopossible to characterize an optimal dose with the best benefit/harm ratio. In therelation between aerobic exercise and the heart, it is also possible that there issomething similar; however, to better understand this matter, we will use a fewextreme examples.3analysed the cardiovascular effects of extreme inactivity. Five healthy youngindividuals were bedridden for 21 days, and at the end of this period, a reductionof 30% was observed in VO2max (maximum oxygen consumption), and it tookthem 60 days of training to recover pre-admission conditions. This 30% loss wasgreater then the "physiologic" reduction of aging observed in a re-evaluation done30 years later.4In 1968, the Dallas Bed Rest Studyhttp://www.petesfeetaa.com/).5 In England, Ben Smith burned over 2.5 million calories tocomplete 401 marathons (42.195 km) in 401 days  .6 These two integrate the growing contingent ofultra-runners - > 60,000 Americans in 2013 (https://www.ultrarunning.com/featured/which-state-has-the-most-ultrarunning-finishers-as-percentage-of-population/)7 - that is, those who participate inrunning competitions (most of which are off-road) over distances longer thanmarathons and, in general, more than 50 miles (80 km). These ultra-runners composean advanced and differentiated group when compared to the more than half a millionAmericans who complete a marathon every year.In October of 2016, two fantastic aerobic feats were accomplished. In the UnitedStates, Pete Kostelnick ran from San Francisco to New York , withan average of 110 km/day at different altitudes  and running several hours on consecutive days forlong periods of time, without even one day of rest. It objectively shows howexercise dosage is, in fact, very wide and more elastic than any cardiovasculardrug, and it exemplifies the cardiac tolerance of certain individuals to enormousdoses of aerobic exercise.2max) are associated tobetter health and longevity, while with a sedentary lifestyle, the oppositeoccurs.8 Indeed, theVO2max obtained  in an incremental and truly maximum exercise test is an excellenthealth indicator9 and has recentlybeen suggested as a vital sign.10There is a lot of epidemiological evidence that regular aerobic exercise and highfunctional capacity and/or aerobic condition  can be up to fivetimes, that is, respectively, something like 1% vs 5% per year. Furthermore, in anrecent example from Finland, Laukkanen et al.12 verified that, in healthy 50-year-old men, who increasedtheir aerobic condition (direct measure) in 1 MET after 11 years of follow-up, therisk of mortality decreased by five times, when compared to those who kept theiraerobic condition, and in up to 20 times, when compared to those withVO2max reduced by 4 or 5 METs in the same period. Thus, having aVO2max equal to or above the predicted (age/gender) should become agoal in the primary and secondary prevention of cardiovascular diseases.Even more importantly, VOIn the characterization of the exercise dose, it is appropriate to have a unit ofmeasurement. While there are several units proposed in literature, a very convenientone is METs-h/week, which consists of multiplying the number of exercise hours, in agiven week, by the mean intensity in METs. Thus, considering that walking fast (6km/h) and slow running (8 km/h) demand, respectively, 4 and 8 METs, we have that: ifthe individual completes the dose recommended in most guidelines and walks fast for30 minutes 5x/week  or runs slowly for 25minutes 3x/week , the exercise dose will bethe same in both examples - 2.5x4 or 1.25x8 - that is, ± 10 METs-h/week.13 identified that a maximum reduction in mortality was obtainedwith a dose of aerobic exercise corresponding to 3 to 5x the minimum doserecommended in the guidelines, that is, something between 20 and 50 METs-h/week(running 2 to 5 hours per week at 10 km/h) and that even a very high dose of 75-100METs-h/week (8 to 12 hours of running per week) maintained the benefits of mortalityreduction. Yet Gebel et al.14showed that, for a given weekly duration of exercise, benefits were greater when, inat least 30% of the duration, aerobic exercise was of high intensity. In fact, thesetwo important facts can be conflated towards the definition of an optimum dose, andalso characterize what could be denominated as extreme exerciser; that is, the onethat exceeds 100 METs-h/week for long periods of time, which is still scarcely foundin literature. We can then initially propose that the therapeutic range of aerobicexercise dose is between approximately 7.5 to 100 METs-h/week.Two articles from 2015 brought relevant contributions regarding the relation betweenall-cause mortality and exercise dose, considering, separately, relative volume andintensity of the exercise. Arem et al.2 max,regardless of the current level, is by incrementing the dose of aerobic exercise.Therefore, if there is a clinical objective of increasing VO2max toreduce future mortality rate, or for the individual's sport goal, we can define theoptimum dose as the one capable of offering the desired gains within apre-established period. On the other hand, if the objective is only to maintain anexcellent VO2max (> 120% of the predicted for age and gender), theoptimum aerobic exercise dose may be one that is able to ensure the maintenance ofthis privileged aerobic condition, according to cyclical adjustments recommended bythe science of physical training. 2maxobtained through assessment and based on the current exercise pattern.In our opinion, the issue of aerobic exercise dose should be individualized accordingto objectives to be reached. The only known way to increase VO15did a thorough medical evaluation of Boston Marathon runners before the competitionand one hour after completion (first place time of completion = 2h54m). Theyconcluded that participation in the marathon did not seem to cause significantdamage to the cardiovascular system of those young and healthy individuals. Aftermore than 110 years of this rich description, there is a lot of positive evidence ofathletes' and exercisers' cardiac health, such as the observation that athletes whoparticipate in longer sporting competitions16 and physically active adults tend to live longer,17 and there is also the fact thatdeaths are extremely rare in half-marathons and marathons, with a rate under1/100,000 participants.18 In thiscontext, it is worth mentioning the case of Alexandre Ribeiro, six-time Ultramanchampion, who, at 47 years of age, had accumulated 50,000 hours of aerobic training,in a typical dose of 250 METs-h/week. After extensive cardiologic, functional andimage evaluation, the only finding worth commenting on was a discreet increase inthe left atrium in the MRI.19In 1899, Williams & Arnold20 of having coronary arterycalcification, and presenting late enhancement or other abnormalities in cardiacimaging exams.21 However, to ourbest current knowledge, the clinical significance of these "abnormal" findings isstill unknown.18 In this context,further studies involving large samples of ultra-runners and/or individuals who havebeen exercisers for over 40 years may contribute to a better understanding of thereal cause-effect relation between exercise dose and benefit or harm and/orunfavorable cardiac outcomes.21The fact is that it is currently known that very low doses of aerobic exercise bringbenefits, while the maximum limit of the therapeutic dose of aerobic exercise isstill unknown. That is, from a medical perspective, there is no evidence that foundsa hypothesis of exercise exaggeration.22On the other hand, there are data from observational studies suggesting that thereseems to be an increased risk of developing atrial fibrillation,23 In Brazil,government data indicate that tens of millions of Brazilians - almost half the adultpopulation - do not exercise enough. At another extreme, it is estimated that,including athletes and exercisers, much less than 10,000 individuals do intenseaerobic training regularly and for periods longer than 10 hours per week or 100METs-h/week. That is, for every one "extreme" exerciser, there are approximately5,000 to 10,000 sedentary individuals. It is then clear that the question of a rareand unlikely theoretical excess of exercise for the heart is not a priority in termsof public healthcare.Recent global data suggest that around 5 million deaths per year  are caused by sedentary lifestyles. Moreover, it is estimatedthat annual worldwide costs due to low levels of physical activity/exercise surpass67 billion dollars.24Therefore, a priority for cardiologists must be the reduction or elimination of thesedentary lifestyle and stimulation of an increase in the exercise dose of thepopulation in general, thus obtaining an improvement in the aerobic condition, and,consequently, a higher life expectancy with the additional bonus of a wide range ofbeneficial physiological effects. On the other hand, in the case of those rare"extreme" exercisers, we can suggest that they be monitored by qualifiedprofessionals when dealing with health and training issues pertinent to thisperformance profile, while new evidence is gathered on the results of this "extreme"dose of aerobic exercise and its effect on the heart through the future analysis ofa cohort made up of 1,200 ultra-runners.


The Hokkaido Study on Environment and Children’s Health is an ongoing study consisting of two birth cohorts of different population sizes: the Sapporo cohort and the Hokkaido cohort. Our primary study goals are (1) to examine the effects of low-level environmental chemical exposures on birth outcomes, including birth defects and growth retardation; (2) to follow the development of allergies, infectious diseases, and neurobehavioral developmental disorders and perform a longitudinal observation of child development; (3) to identify high-risk groups based on genetic susceptibility to environmental chemicals; and (4) to identify the additive effects of various chemicals, including tobacco smoking. The purpose of this report is to update the progress of the Hokkaido Study, to summarize the recent results, and to suggest future directions. In particular, this report provides the basic characteristics of the cohort populations, discusses the population remaining in the cohorts and those who were lost to follow-up at birth, and introduces the newly added follow-up studies and case-cohort study design. In the Sapporo cohort of 514 enrolled pregnant women, various specimens, including maternal and cord blood, maternal hair, and breast milk, were collected for the assessment of exposures to dioxins, polychlorinated biphenyls, organochlorine pesticides, perfluoroalkyl substances, phthalates, bisphenol A, and methylmercury. As follow-ups, face-to-face neurobehavioral developmental tests were conducted at several different ages. In the Hokkaido cohort of 20,926 enrolled pregnant women, the prevalence of complicated pregnancies and birth outcomes, such as miscarriage, stillbirth, low birth weight, preterm birth, and small for gestational age were examined. The levels of exposure to environmental chemicals were relatively low in these study populations compared to those reported previously. We also studied environmental chemical exposure in association with health outcomes, including birth size, neonatal hormone levels, neurobehavioral development, asthma, allergies, and infectious diseases. In addition, genetic and epigenetic analyses were conducted. The results of this study demonstrate the effects of environmental chemical exposures on genetically susceptible populations and on DNA methylation. Further study and continuous follow-up are necessary to elucidate the combined effects of chemical exposure on health outcomes. Concentrations of PFOS and PFOA, the conventional perfluoroalkyl substances (PFASs), have been decreasing; however, the concentrations of newly developed PFASs such as perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) have been increasing [In 1997, Colborn et al. warned of the dangers of environmental chemicals as endocrine disruptors, which could lead to impaired reproductive capacity –4. Althocreasing . AccumulThe Hokkaido Study on Environment and Children’s Health: Malformation, Development and Allergy is an ongoing birth cohort study launched in 2002. The Hokkaido Study is one of the oldest birth cohort studies not only in Japan but also in Asia . The HokOur primary study goals include (1) examining the possible negative effects of perinatal environmental chemical exposure on birth outcomes, including birth defects and growth retardation; (2) following the development of allergies, infectious diseases, and neurobehavioral developmental disorders and performing a longitudinal observation of child development; (3) identifying high-risk groups with genetic susceptibilities to environmental chemical exposures; and (4) identifying the additive effects of lifestyle including tobacco smoking and folic acid intake encountered in the daily environment.Our previous cohort profile update in 2013  introducOver the 14 years of follow-ups, new research questions have arisen and the study focus has expanded beyond its primary goals. Studies have reported an increasing prevalence of children with developmental disabilities and behavioral problems , 9. The Emerging evidence has indicated that epigenetic modifications including DNA methylation may link the intrauterine environment to lifelong health trajectories . The effThe purpose of this report is to update the progress of the Hokkaido Study, to summarize the recent results, and to suggest its future directions. In particular, this report focuses on (1) providing the basic characteristics of the whole cohort population as the fixed data are now available after the closing of recruitment in 2012, and presenting the comparisons of the population remaining in the cohort with those who were lost to follow-up at birth; (2) introducing the nested case-cohort study design, in which a sub-cohort is used as a comparison group for all cases that occur in the cohort; and (3) introducing the details of the follow-up studies, including child neurobehavioral problems at preschool ages. This design is effective when a cohort is followed for health outcomes by avoiding the costs of collecting and processing covariate information and exposure assessment in the selected sub-cohort population. This strategy is applied to the Hokkaido cohort, which contains more than 20,000 participants.The details of the Hokkaido Study have been described in previous reports , 13. BriWe also established a sub-cohort of 4869 participants, which corresponded to 23.3% of all participants in the Hokkaido Study. In this sub-cohort, we included 500 participants who were randomly selected from each enrollment year between 2003 and 2011, and all 369 participants from the enrollment year 2012.Details of the information collected at the time of enrollment and birth have been described previously , 13. A sThe details of the follow-up questionnaires and tests are shown in Table Observations were made to assess the prevalence of birth defects, asthma and allergies, and neurobehavioral developmental disorders . The defThe details of specimen collection are described in our previous cohort profile . In the In the Sapporo cohort, the levels of 29 dioxin and dioxin-like PCB (DL-PCB) congeners, 58 other PCB congeners, and 5 hydroxylated PCB (OH-PCB) congeners in maternal blood and breast milk were measured –18. The In the Hokkaido cohort, simultaneous analysis of 11 PFASs in maternal plasma collected during the third trimester of pregnancy was conducted . In addiAs outcomes, thyroid, steroid, and reproductive hormone levels were measured using cord blood samples. Adipokine levels were also measured in cord blood samples. In addition to hormone levels, IgE and IgA levels were measured.IGF2), H19, and long interspersed element 1 (LINE1) DNA methylation in cord blood were analyzed by pyrosequencing using a PyroMark Q24 system  and assessed using the Qiagen Pyro Q-CpG Software [Genotyping of single nucleotide polymorphisms (SNPs) was performed using allelic discrimination assays, with fluorogenic probes and 5′ nuclease (TaqMan)   or a nanSoftware . Cord blSoftware , 28.n = 514) and Hokkaido  cohorts are shown in Tables The fixed dataset as of the end of 2015 included 20,926 women who had initially registered. However, 121 of the participants requested that their data not be included in any analysis; thus, we included data from 20,805 participants. The basic characteristics of the Sapporo n = 51 and HokkThe prevalence of pregnancy complications was also examined. The most common complication was pregnancy hypertension, observed in 491 women 2.5%). Other complications included gestational diabetes mellitus , pregnancy with preexisting diabetes , oligoamnios , thyroid dysfunction , placenta previa , and placental abruption . Table %. Other p,p′-dichlorodiphenyldichloroethylene . The detailed concentrations of organochlorine pesticides can be found in our previous literature [The exposure levels of environmental chemicals in the Sapporo cohort are shown in Table terature . MEHP waIn the Hokkaido cohort, the concentrations of 11 PFASs in the third trimester maternal plasma were measured Table . PFOS anThe effects of PCDD/PCDF and DL-PCB, PFOS, and PFOA exposure on birth size were presented in our 2012 cohort profile , 34. SinMaternal and infant thyroid hormone levels were investigated in association with prenatal chemical exposure –41. The Steroid and reproductive hormone levels were also investigated in the Sapporo cohort Table . We examIn addition to the effects of PCDD/PCDF on the Bayley Scales of Infant Development second edition scores at 6 months reported in our previous update , 46, theThe prevalence rates of wheezing, eczema, food allergy, and otitis media at 1.5 years of age in the Sapporo cohort were 11.3, 11.3, 17.0, and 18.7%, respectively. In the Hokkaido cohort, the prevalence rates of wheezing, eczema, and allergic rhino-conjunctivitis symptoms at 2 years of age were 19.3, 17.8, and 4.4%, respectively, and at 4 years of age were 18.7, 19.0, and 5.4%, respectively. The prevalence rates observed in the Hokkaido Study are slightly higher than those reported in the Japanese population .Since our last cohort update , 53, 54,Maternal fatty acids (FAs) are essential for fetal growth. In our study, nine types of FAs and triglycerides (TGs) were investigated in association with PFAS and DEHP exposure. Our study found that PFOS but not PFOA was negatively associated with the levels of palmitic, palmitoleic, oleic, linoleic, α-linolenic, and arachidonic acids and TG. PFOS was associated with reduced essential FAs, including omega-3 and omega-6 FAs . We alsoAhR), cytochrome P450 1A1 (CYP1A1), glutathione S-transferase mu 1, nicotinamide adenine dinucleotide phosphate, cytochrome P450 2EI, 5,10-methylenetetrahydrofolate reductase, and X-ray cross-complementing gene 1.The effects of maternal smoking during pregnancy and maternal genetic polymorphisms on infant birth weight were examined in the Sapporo and Hokkaido cohorts , 59–63. AhR  and CYP1A1  genes were associated with maternal dioxin concentration [The associations between maternal exposure to dioxins during pregnancy and maternal genetic polymorphisms were also examined in the Sapporo cohort. Maternal polymorphisms in the ntration . Moreoventration  Fig. 1)AhR  was launched in 2012, and 24 cohorts from 11 countries are currently participating . EnvironThe results of the Hokkaido Study suggest that even relatively low levels of exposure to environmental chemicals may have adverse effects on child health. When implementing policies and guidelines, it is important to consider individual genetic susceptibility to these effects. Further studies on the combined effects of chemical exposure, contribution to infant health via effects on DNA methylation, and long-term follow-up of the participants are essential.