Cryptococcus neoformans can infect virtually any human organ, including the bone marrow, which is a rare presentation of cryptococcosis.Cryptococcosis is a common opportunistic infection in patients infected by Human Immunodeficiency Virus (HIV) and is the second leading cause of mortality in Acquired Immunodeficiency Syndrome (AIDS) patients worldwide. The most frequent presentation of cryptococcal infection is subacute meningitis, especially in patients with a CD4+ T Lymphocytes count below 100 cells/μL. However, in severely immunosuppressed individuals Cryptococcus sp. The bone marrow biopsy revealed a hypocellularity for age and absence of fibrosis. It was observed presence of loosely formed granuloma composed of multinucleated giant cells encompassing rounded yeast like organisms stained with mucicarmine, compatible with Cryptococcus sp. Then, the patient underwent a lumbar puncture to investigate meningitis, although he had no neurological symptoms and neurological examination was normal. The cerebrospinal fluid culture yielded Cryptococcus sp. The species and genotype identification step showed the infection was caused by Cryptococcus neoformans var. grubii (genotype VNI). The patient was initially treated with amphotericin B deoxycholate plus fluconazole for disseminated cryptococcosis, according to guideline recommendations. However, the patient developed acute kidney injury and the treatment was switched for fluconazole monotherapy. The symptoms disappeared completely with recovery of white blood cells and platelets counts. Cerebrospinal fluid cultures for fungi at one and two-weeks of treatment were negative.A 45-year-old HIV-infected male patient with a CD4+ T lymphocyte count of 26 cells/μL who presented to the emergency department with fever and pancytopenia. Throughout the diagnostic evaluation, the bone marrow aspirate culture yielded encapsulated yeasts in budding, identified as Cryptococcus neoformans is a rare presentation of cryptococcosis. The cryptococcal infection should be included for differential diagnosis in HIV-infected patients with fever and cytopenias, especially when CD4+ T lymphocytes count is below 100 cells/μL.Bone marrow infection caused by  Cryptococcosis remains the second most frequent cause of death in AIDS patients worldwide, only behind tuberculosis . Its mosCryptococcus neoformans can infect virtually any human organ system, including the bone marrow, which is a rare presentation of cryptococcosis.In turn, HIV-infected patients with severe immunodeficiency commonly present fever accompanied with cytopenias. The hematological abnormalities can be due to HIV infection itself or non-infectious and infectious causes. The most frequent opportunistic infections that cause pancytopenia are histoplasmosis and mycobacteriosis . In seveHere, we describe an uncommon presentation of disseminated cryptococcosis with invasive disease of bone marrow and asymptomatic meningitis.10). He was also using a trimethoprim-sulfamethoxazole double-strength tablet for Pneumocystis jirovecii prophylaxis. The patient had pulmonary tuberculosis 8 years ago, his AIDS-defining illness. In addition, he had several previous hospitalizations for chronic diarrhea. He used to drink distilled beverages thrice a week and denied tobacco use. The patient raised a dog and a parakeet as pets. He was born in Angicos City, rural area of Rio Grande do Norte, Brazil, an endemic region for visceral leishmaniasis and Chagas disease.A 45-year-old HIV-infected black man, mechanic, has sought emergency department referring progressive asthenia for two weeks, with difficulty on performing basic daily life activities, anorexia, fever and profuse sweating. During this time, he reported a weight loss of 3Kg. In the last 24 h, he had vomiting episodes preceded by nausea, in small volume, without relation to feeding. The patient denied other symptoms like headache, cough, abdominal pain and diarrhea. The HIV infection was diagnosed 8 years ago and his current antiretroviral therapy consisted of tenofovir disoproxil fumarate – lamivudine – efavirenz in a once-daily single-pill. His medical history was remarkable for poor adherence to antiretroviral therapy. His recent CD4+ and CD8+ T lymphocytes counts were 26 (1.92%) and 509 (37.9%) cells/μL respectively and the viral load was 252,624 copies/mL (5.402 Log2) and a mild hepatomegaly. He was lucid and oriented. There were no signs of meningeal irritation. The neurological examination was normal. Laboratory tests showed pancytopenia: hemoglobin of 100 g/L; white blood cell counts of 2.7 × 109/L (74% neutrophils and 22% lymphocytes) and platelets count of 123 × 109/L. The erythrocyte sedimentation rate was 68 mm [Reference Interval (RI): 0 – 15 mm] and serum C - reactive protein level was 28.5 nmol/L (RI: < 57.1 nmol/L). The serum creatinine was 0.058 mmol/L (RI: 0.044–0.106 mmol/L) and serum urea was 3.18 mmol/L (RI: 1.66–8.32 mmol/L). The alanine and aspartate aminotransferases levels were 43 and 23 IU/L . The alkaline phosphatase and gamma-glutamyl transferase levels were 201 IU/L and 49 IU/L . The serum albumin was 39.4 g/L (RI: 35 – 55 g/L), total bilirubin level was 8.55 μmol/L (RI: 3.42–20.52 μmol/L) and prothrombin time and activity were 13.5 s and 100% (RI: 9.5–13.5 s and ≥ 70%). The serum lactate dehydrogenase level was 477 IU/L (RI: 225 - 450 IU/L). Chest X-ray was normal. An abdominal ultrasonography showed hepatomegaly and cholelithiasis.On admission, vital signs were: axillary temperature 38.0 °C, blood pressure 100/80 mmHg, pulse rate of 110 bpm and respiration rate of 24 bpm. His physical examination was remarkable for cachexia  had a clear appearance, an opening pressure of 160mmH2O, a cell count of 2 leukocytes, protein of 44 mg/dL and glucose of 55 mg/dL, and numerous encapsulated yeasts in budding were seen on India ink stain. The CSF culture yielded Cryptococcus sp. Bone marrow aspirate culture was negative for bacteria, mycobacteria and Leishmania sp.He has been treated for febrile neutropenia with cefepime for 7 days, without changes in clinical symptoms. Blood cultures were negative for bacteria and fungi. The serologic tests were negative for hepatitis B and C, visceral leishmaniasis and Chagas disease. Tuberculin Test was non-reactive. Venereal disease research laboratory (VDRL) testing was negative too. Cryptococcal antigen testing in serum was not performed, because it is not available at our service. Cryptococcus sp.  were rarely observed. The myeloid series presented a preserved maturation scale with predominance of mature forms and the erythroid lineage was arranged in small islands without particularities. There were typical plasma cells scattered in hematopoietic tissue. Silver staining revealed a delicate network of reticulin fibers without fibrosis. The Masson’s trichrome staining showed no collagen fibers deposition. Giemsa, Fite-Faraco and Periodic Acid-Schiff staining were negative. In the mucicarmine staining, it was observed presence of loosely formed granuloma composed of multinucleated giant cells encompassing rounded yeast like organisms compatible with sp. Fig. .Fig. 1PhURA5 gene was amplified by Polymerase Chain Reaction (PCR) according to the method suggested by Meyer et al. [C. neoformans var. grubii (genotype VNI)  was extracted by initial mechanical rupture of yeast cell in liquid nitrogen using a pestle and mortar following the extraction protocol of McCullough et al. . The URANI) Fig. .Fig. 2GeThe patient was treated with amphotericin B deoxycholate at 1 mg/Kg daily, combined with fluconazole at 800 mg daily given intravenously. After a week of treatment, the patient developed acute kidney injury stage 3, according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria [9/L (65% neutrophils and 30% lymphocytes) and platelets count of 166 × 109/L. He was discharged from hospital with recommendations to keep oral fluconazole at 450 mg daily (maintenance phase) and outpatient follow-up at a regional HIV/AIDS care service.The patient remained hospitalized throughout the induction and consolidation phases of the treatment due to socioeconomic reasons. He could not afford the costs related to the hospitalization neither his medicines, that was why the patient went to our hospital, a public health facility, besides he did not have an appropriate family support to help him recover and manage his condition. Cranial computed tomography (CT) showed cerebellar atrophy with compensatory fourth ventricle ectasia and cerebral atrophy with non-hypertensive dilation of the supratentorial ventricles and enlargement of grooves, fissures and cisterns. Magnetic resonance imaging (MRI) confirmed a non-habitual volumetric reduction of encephalic parenchyma for age with compensatory ectasia of ventricular system; the brain parenchyma signal-intensity was preserved. The patient had no fever nor vomiting within the first week of treatment and recovered from asthenia after four weeks of treatment. After induction and consolidation phases of treatment, the hematological parameters have improved: hemoglobin level of 110 g/L; white blood cell counts of 5.9 × 10Cryptococcus neoformans in bone marrow aspirate culture, although the patient did not demonstrate clinical evidences of central nervous system (CNS) involvement. The finding of encapsulated yeasts in CSF was incidental, characterizing a subclinical or latent form of cryptococcal meningitis (CM).The most frequent clinical manifestation of cryptococcosis in HIV-infected patients is subacute meningitis. Due to the neurotropism, the isolation of Cryptococcus sp. in extra-neural sites should prompt to a lumbar puncture to rule out concomitant meningitis . In the Cryptococcus neoformans. He was submitted to a lumbar puncture, with numerous encapsulated yeasts observed in the CSF, subsequently identified as Cryptococcus neoformans.Subclinical CM has previously been reported. Liss and Rimland  describeCryptococcus neoformans in culture of extra-neural sites specimen: kidney, blood, lung, and a submandibular nodule. All patients had no symptoms of meningeal involvement, but one patient has been undergoing psychiatric treatment for personality change of several years duration. Smilack et al. [Cryptococcus neoformans was also isolated in CSF culture.In a series of 40 cases of CM, Butler and colleagues  found fok et al.  reportedThe studies of the prevalence of cryptococcal antigenemia in HIV-infected patients with CD4 + T lymphocyte count below 100 cells/μL demonstrated the CM may be common in clinical practice. In a recently published meta-analysis, Temfack et al.  found a With pre-emptive fluconazole therapy, the incidence of CM dropped from 21.4 to 5.7% among CrAg-positive patients and the all-cause mortality reduced from 39.7 to 17.4%, although it still remained significantly higher among CrAg-positive than CrAg-negative patients (around two-fold risk) . BesidesUnfortunately, the CrAg testing is not available at our service. However, it is worth pointing out that our patient was at high risk for cryptococcosis (CD4+ T lymphocytes count of 26 cells/μL) and would probably benefit from CrAg testing in blood for early detection of the cryptococcal infection, and once being positive, the CSF testing would be advantageous for early and timely diagnosis of meningeal involvement.As the course of CM is usually indolent over weeks and symptoms and signs may be subtle and non-specific, a high degree of clinical suspicion is required, especially in patients with advanced HIV infection. Vomiting and difficulty on performing basic daily activities, being non-specific, might be interpreted as neurological symptoms for this patient. In CM, vomiting is often a clinical manifestation of elevated intracranial pressure (ICP). In addition, hydrocephalus, a possible complication of CM and a cause of elevated ICP, may provoke dementia symptoms with impairment of daily activities. However, the opening pressure of CSF on lumbar puncture was normal and neuroimaging exhibited a normal brain parenchyma sign and excluded cerebral oedema or hydrocephalus as causes of elevated intracranial pressure.Cryptococcus neoformans in bone marrow aspirate prompted us to investigate the CNS before the symptoms appeared. Perhaps the atrophy of the brain parenchyma observed in the patient’s neuroimaging studies may have contributed to the absence of symptoms slowing the process of increasing ICP. The patient’s opening pressure of CSF was 160mmH2O. Despite this, the pathophysiology related to the symptoms and signs of CM is not fully elucidated.A possible explanation for the asymptomatic meningitis in our patient would be the early diagnosis of CNS involvement. The finding of 2O) as well as 79% of patients with normal ICP (≤190mmH2O) reported headache. In cryptococcal meningitis, elevated ICP is caused by impaired CSF reabsorption at the arachnoid granulations secondary to an inflammatory arachnoiditis or direct cryptococcal infiltration [In the study by Graybill et al. , clinicamal ICP ≤0mmH2O reltration  raised tCryptococcus neoformans has been reported to cause infection in any organ of the human body, including the bone marrow. In HIV-positive patients with disseminated cryptococcosis, the bone marrow involvement is observed from 13 to 42% of cases [In addition to CNS infections, HIV-infected patients also have more frequently disseminated cryptococcosis, with the finding of yeasts in extra-neural sites, associated to poorer prognosis , 9, 11. of cases , with a Cryptococcus neoformans can be absolutely asymptomatic in HIV-infected patients, and has been identified through screening cultures [Bone marrow infection by cultures . It can cultures . In extrcultures .Cryptococcus neoformans from tissue culture adds to diagnosis.The direct examination of bone marrow aspirate can show extra- and intracellular encapsulated yeast like organisms in budding or not, offering the advantage of a rapid turn-around time, while pending culture and biopsy results –24. In bCryptococcus neoformans. The culture of bone marrow aspirate for fungi requires a volume of 1 to 5 mL. Collecting this volume leads to an unavoidable dilution of bone marrow aspirate with peripheral blood [Cryptococcus sp. from this culture may result from: the true presence of yeast in bone marrow tissue; the presence of yeasts in peripheral blood (as part of cryptococcaemia) or both. In this case, the peripheral blood culture for fungi, incubated for 8 weeks, as part of diagnostic work-up of disseminated histoplasmosis, was negative. Moreover, the presentation of granulomatous reaction with encapsulated yeasts inside multinucleated giant cells is an unequivocal evidence of bone marrow infection by Cryptococcus sp.Here in Rio Grande do Norte, a state of Northeast of Brazil, there are other more frequent opportunistic causes of pancytopenia in patients with AIDS as visceral leishmaniasis, disseminated histoplasmosis and tuberculosis. Thus, even though pancytopenia may result from cryptococcosis, the diagnosis was completely unexpected. Our patient has fulfilled the criteria for invasive fungal infection of bone marrow by al blood . The yieIn immunocompromised patients, the cryptococcal disease may result from either primary infection or reactivation of a latent infection. There are epidemiological and biological evidence of the role of quiescence on pathogenesis of cryptococcosis , 28. In Cryptococcus neoformans has been isolated from the droppings of a variety of avian species [Cryptococcus sp. from exposure to aerosolized birds excrements to immunocompromised patients, suggesting a progressive primary infection [Furthermore, this patient had a remarkable epidemiological record of parakeet exposure.  species . Althougnfection , 32. TakIn disseminated cryptococcosis, the recommended treatment consists of an induction phase with an amphotericin B formulation given intravenously combined with oral flucytosine for, at least, two weeks. This combination is associated with more rapid sterilization of CSF and better survival . UnfortuLiposomal amphotericin B is preferable to conventional formulation, since it shows similar efficacy, but less toxicity . Our patCryptococcus neoformans is uncommon. This etiology should be included for differential diagnosis in HIV-infected patients with fever and cytopenias, especially when CD4 T lymphocytes count is below 100 cells/μL. This case illustrates an atypical presentation of disseminated cryptococcosis with bone marrow involvement and asymptomatic meningitis, highlighting the role of bone marrow aspirate and biopsy study in HIV-infected patients with fever and pancytopenia for early diagnosis and adequate treatment of opportunistic infections.In conclusion, bone marrow infection caused by


The prevalence of diabetes and potentially related complications, including stroke, is rapidly increasing in China. The long-term effects of lifestyle may affect glucose metabolism in the general population. Although some studies have shown an association between smoking and the risk of type 2 diabetes mellitus (T2DM), the relationship remains unclear. Furthermore, the relationship between smoking and stroke in patients with T2DM has not been fully elucidated.  We investigated the influence of cigarette smoking on T2DM and stroke in China. Detailed questionnaires about smoking status and anthropometric measurement were completed by participants, and oral glucose tolerance testing (OGTT), hemoglobin A1c (HbA1c), homeostasis model assessment of IR (HOMA-IR), and blood lipids were measured. 2 or waist circumference (WC) < 90 cm) compared with nonsmokers after adjusting for age, alcohol intake, physical activity, educational level, family history of diabetes, SBP, DBP, TG, TC, HDL-C, and LDL-C. However, no significant association was detected in male current smokers with overweight/obesity or female smokers. In addition, compared with nonsmokers without T2DM, current smokers with T2DM had a significantly higher risk of stroke  after adjusting for confounders.  In total, 8196 adults aged 40 years or older were included. We found a reduced risk of impaired glucose regulation (IGR) and T2DM in male smokers with normal weight (body mass index (BMI) < 25 kg/m Smoking was negatively associated with T2DM in Chinese men of normal body weight, but no significant association was found for men with overweight/obesity or women. In addition, smoking was positively associated with nonfatal stroke, especially in patients with T2DM. Further prospective studies are needed to examine the association between smoking, diabetes, and stroke in different ethnic groups. Smoking is a leading risk factor for noncommunicable diseases (NCDs), such as cardiovascular disease , 2, cancThe relationship between smoking and type 2 diabetes mellitus (T2DM) is unclear. Although most epidemiological evidence has demonstrated a positive association between smoking and T2DM –15, seveThe Risk Evaluation of Cancers in Chinese Diabetic Individuals: a community-based longitudinal (REACTION) study was a multicenter prospective observational study of adults aged 40 years or older. The primary purpose of the study was to assess the relationship between abnormal glucose metabolism and the risk of cancer in the Chinese population. Details of the study design have been published previously . The dat2 were excluded to eliminate the effect of morbidly lean patients on diabetes .All participants underwent an OGTT and provided blood samples which were tested for fasting plasma glucose (FPG), and 2-hour postprandial plasma glucose (2hPG), fasting insulin (FINS), hemoglobin A1c (HbA1c), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) according to China National Laboratory Accreditation . To estiAll participants were required to fast for at least 10 hours before undergoing a 75 g OGTT. Diagnosis of normal glucose tolerance (NGT), prediabetes, and T2DM was based on the 2017 T2DM diagnostic criteria of the Chinese Diabetes Society (CDS) . NGT wasχ2 test to draw an overall P value of comparison of three groups and then do the appropriate post hoc pairwise comparisons. The P values were corrected with the Bonferroni correction by multiple comparison between each status of glucose metabolism. Multinomial multivariable logistic regression was performed to assess the association between smoking status and glycemic regulation status  using the Enter method. Multivariable binary logistic regression was conducted to obtain the odds ratio (OR) with 95% CIs  for the association of smoking exposure and T2DM with stroke. A two-tailed test was used, and P values of <0.05 were regarded as statistically significant.All statistical analyses were conducted using SPSS version 23.0 . We first conducted analysis of variance (ANOVA), the Kruskal-Wallis test (as appropriate), or the β were significantly decreased. Subjects with T2DM had lower frequencies of physical activity and family histories of diabetes compared with the NGT group. The proportion of current smokers was 18.9%, 15.8%, and 16.3% in the NGT, prediabetes, and T2DM groups, respectively. The proportion of patients with nonfatal stroke was significantly higher in the T2DM group than in the NGT group. A comparison of baseline data between men and women is shown in Supplementary The clinical baseline characteristics of the total study population and the subgroups studied are summarized in After adjustment for age, BMI, gender, educational level, physical activity, alcohol consumption, family history of diabetes, SBP, DBP, TG, TC, HDL-C, and LDL-C, current smokers were associated with a 23%  and 20%  decreased risk of prediabetes and T2DM compared with nonsmokers, respectively.In addition, light smokers (1–19 cigarettes/day or 1–19 pack-years) exhibited a lower risk of having T2DM, while no significant association with risk of T2DM was found for heavy smokers and nonsmokers . The odd2, WC < 90 cm, and WHR < 1.0) showed that light and heavy smokers had a lower risk of prediabetes/T2DM than nonsmokers. Current smokers with overweight/obesity  also had no significant association with IGR or T2DM (P = 0.267) after adjustment for confounders .In men, stratification by normal weight  was significantly reduced in light smokers after adjustment for confounders. However, no significant association was found between smoking status and obesity in women . The association of stroke with an interaction term (between smoking status and T2DM) was significant (P = 0.023), after adjusting for confounders. Using nonsmokers without diabetes as a reference, current smokers with T2DM had a significantly higher risk of stroke  after adjusting for confounders  [2) had an adjusted relative risk (RR)  for diabetes of 0.54 (95% CI: 0.35–0.83) as compared with nonsmokers (mean BMI 28.2 kg/m2) in all adults [2) [Smoking is an established major risk factor for various chronic noncommunicable diseases –25, alth2) . Data fo2) . Similarl adults . In a foults [2) .2 compared with those who did not smoke. We also explored the relationship between smoking status and HOMA-IR and observed that an improvement of insulin resistance was only found in current smokers with BMI < 25 kg/m2. This association was not observed in male smokers with BMI ≥ 25 kg/m2, which supports data from the China National Diabetes and Metabolic Disorders Study in which no association was identified between smoking and newly diagnosed diabetes in males who were overweight/obese (BMI ≥ 25 kg/m2) [Similar to the studies described above, our findings show that cigarette smoking was associated with decreased odds of T2DM in men with BMI < 25 kg/m5 kg/m2) . These dGiven the different relationships identified between smoking and T2DM in different BMI/WC groups among men, we explored the relationship between smoking and obesity in the present. Logistic regression revealed significant negative associations between smoking and obesity in males, but no such association was found in women. Similarly, a previous study in Japan found that male current smokers had a lower OR for obesity compared with nonsmokers . HoweverP < 0.001).The inconsistent results between smoking and T2DM/obesity identified in our study were also found between men and women, as our data show that there was no evidence for an association between smoking and T2DM/obesity in women. The results of the Japanese study described above also showed that there were no differences in obesity between current smokers and nonsmokers . The MONDifferences between our findings and those reported in several previous studies might in part be attributable to the following factors. First, the BMI cutoff points to define overweight and obesity were different in different studies. In addition, some studies used measures other than BMI to define obesity, including WC, WHR, visceral fat thickness, fat percentage, and fat mass. These differences in the method used to estimate obesity may have led to differences in the results. Second, the participants enrolled in the present study were all Chinese, so we used the CDS diabetes diagnostic criteria. The FPG levels for IFG were higher in the 2017 CDS diagnostic criteria compared with the diagnostic criteria of the American Diabetes Association used in other studies. Third, some other studies used only self-reported data, while our study combined medical histories and standard OGTT results to diagnose T2DM. Finally, the inclusion of different patient populations in the various studies might have contributed to population bias, thus affecting the study conclusions to some extent. Further studies of the relationship between smoking and obesity in patients with diabetes and the possible mechanism responsible for such a relationship are therefore required.Diabetes is an established major risk factor for stroke . Of noteThis study had several limitations. First, although we enrolled 5422 women, there were only 87 current smokers in this group. The limited data might have affected the reliability of our conclusions for women. Further studies with a focus on women are thus required. Second, 27.1% of T2DM patients had received antidiabetic drugs, which might have affected the accuracy of the measurement of insulin levels and further affect HOMA-IR. Third, the diagnostic criteria for stroke were assessed based on patient self-reported data and medical history , which might have resulted in potential bias in the prevalence of stroke. Fourth, all subjects were of Chinese ethnicity, meaning that the results of the study might not be applicable to other populations. Finally, the use of a cross-sectional design means that it is not possible to infer a causal relationship between smoking status and prediabetes/T2DM. Prospective studies with larger sample sizes in specific populations are thus needed to clarify the relationship between smoking behavior and diabetes, related complications, and total morality.The results of this cross-sectional study indicate that smoking is negatively associated with IGR/T2DM in Chinese men with normal body weight, although no significant association was found for overweight/obese Chinese men or for women. In addition, smoking and T2DM had a combined positive correlation with stroke after adjusting for confounders (including BMI). Therefore, smoking cessation should be suggested to all current smokers, regardless of BMI.


Tenebrio molitor (TM) meal inclusion on gut health of free-range chickens by intestinal microbiota, morphology and mucin composition characterization.Gut health in poultry depends on the balance between the host, intestinal microbiota, intestinal microscopic features and diet. The effects of insect meal  on chicken gut morphology have recently been reported, but no data about intestinal microbiota and mucin composition modulation are available. The present study evaluated the effects of dietary P < 0.05) and beta  diversity in birds fed TM diet than C. In comparison with C group, TM birds displayed significant increase and decrease, respectively, of the relative abundances of Firmicutes and Bacteroidetes phyla, with higher Firmicutes:Bacteroidetes ratios . The relative abundance of Clostridium, Oscillospira, Ruminococcus, Coprococcus and Sutterella genera was higher in TM chickens than C (FDR < 0.05). On the contrary, TM birds displayed significant decrease of the relative abundance of Bacteroides genus compared to the C group (FDR < 0.05). Gut morphology evaluation by morphometric analysis on small intestine revealed similar villus height, crypt depth and villus height to crypt depth ratio between C and TM birds. Characterization of gut mucin composition by periodic-acid Schiff, Alcian Blue pH 2.5 and high iron diamine staining on small and large intestine showed unaffected mucin staining intensity in TM chickens when compared to C group.One hundred forty female medium-growing hybrids were divided into 2 dietary treatments  and slaughtered at 97 days of age . The gut microbiota assessment on cecal content samples by 16S rRNA amplicon based sequencing showed higher alpha  contains supplementary material, which is available to authorized users. Gut health has been a focus of major research efforts in production animals, since it can be considered a synonymous to animal health and is of vital importance to animal performance . The latGut health depends on the maintenance of the delicate balance between the host, intestinal microbiota, intestinal barrier (in terms of microscopic structure) and dietary compounds . In partFeed may be considered the most important entity in the poultry industry in terms of animal health and producers revenue and profit. As a first aspect to consider, feed substances are capable of exposing the birds to potentially harmful organisms and/or components through the gastrointestinal tract . Feed isTenebrio molitor (TM) has recently been tested in diets for broiler [Insect meal utilization as alternative protein source for animal feeding has already been pointed out , 15, bec broiler –20 and f broiler  as prote broiler , 18, soy broiler , 20, or  broiler . In part broiler , 18–21.  broiler –21, but The present study aims to investigate the effects of TM meal utilization on intestinal microbiota, morphology and mucin composition of free-range chickens, also proposing a standardized multidisciplinary post mortem approach for the assessment of gut health in poultry when dietary modifications occur.A total of 530,550 raw reads (2x250bp) were obtained after sequencing. After joint and quality filtering, a total of 104,081 reads passed the filters applied through QIIME, with an average value of 10,408 reads/sample. In order to avoid biases due to different sequencing depths, all samples were rarefied at 3600 reads after raw read quality filtering.P < 0.05) in free-range chickens fed with TM compared to C diet, whereas Chao1 and PD Whole Tree indexes and observed species richness showed no significant differences (P > 0.05) between C and TM groups . These differences were demonstrated by Principal Component Analysis (PCA), which showed a clear separation of the microbiota as a function of the diet   of Bacteroidales order, Alistipes, Parabacteroides and Coprobacter were identified as predominant OTUs in both birds fed with C and TM diets . On the contrary, the relative abundance of Bacteroidetes was lower in TM chickens than C (FDR < 0.05). The birds fed with TM also showed higher Firmicutes:Bacteroidetes ratios compared to the C group (FDR < 0.05). At genus level . On the contrary, the birds fed with TM displayed significant increase of the relative abundance of Sutterella, Ruminococcus, Oscillospira, Clostridium and Coprococcus genera compared to the C group (FDR < 0.05).Compared to the C group Fig. , the birvel Fig. , the relP > 0.05, Fig. P < 0.01) and Cd  than the other gut segments and higher Vh/Cd ratio  than the ileum. The detailed results of the intestinal morphology of the chickens of the current trial are reported by Biasato et al. [Dietary TM meal inclusion did not affect the intestinal morphometric indices of the free-range chickens of the present study P > 0.0, Fig. 5.o et al. .Fig. 5GrP < 0.001). However, there was no significant influence of dietary TM meal inclusion (P > 0.05) on the histochemical findings (Table P < 0.001) than neutral and acidic sulfated. Furthermore, lower mucin staining intensity was found in the caecal crypts (P < 0.001) compared with the other gut segments and in the jejunal crypts (P < 0.001) compared with the duodenum and ileum, respectively. Crypt base also showed greater mucin staining intensity (P < 0.001) than the midsection and tip  on mucin staining intensity for the intestinal villi, whereas both gut segment and villus fragment significantly influenced  the histochemical findings (Table P < 0.001) compared with the other gut segments and in the jejunum (P < 0.001) compared with the duodenum, respectively  than the base  and diversity, has the longest residence time (12–20 h) of digesta in the gastrointestinal tract, and is an important site for recycling of urea, water regulation, and carbohydrate fermentations contributing to intestinal health and nutrition [Characterization of the cecal microbiota of free-range poultry has already been reported in previous studies about Chinese Dagu chickens , Italianutrition .Bacteroidetes, Firmicutes and Proteobacteria, as previously reported in normal chickens [Bacteroidetes over Firmicutes and Proteobacteria is in agreement with the previous findings about cecal microbiota of Dagu [Bacteroidetes, Firmicutes and Proteobacteria phyla was observed in Italian local free-range breeds, but a comparative reduction of Firmicutes and a concomitant increase of Bacteroidetes was still identified in these birds when compared to broilers [In the free-range chickens fed with basal diet of the present study, cecal microbiota was mainly colonized by the phyla chickens –28. In p of Dagu  and Berm of Dagu  free-ranbroilers .Clostridium, Ruminococcus, Lactobacillus and Bacteroides [Prevotella as the most abundant genus. In particular, Bacteroides genus, U.m. of Prevotellaceae family, U. m. of Ruminococcaceae family and U. m. of Bacteroidales order have been reported as the most predominant members of cecal microbiota of Bermuda free-range chickens [Bacteroides, U. m. of Bacteroidales order, Clostridium and Ruminococcus as the most predominant genera, can fit into the overall view of these previous studies.Differently from phyla, at genus level there is some controversy over the predominant taxa in chicken cecal microbiota. The majority of studies observed that the most predominant genera found in chicken cecum are teroides –32, whilteroides  also idechickens . The curWith regards to gut microbiota modulation by insect meal utilization, the cecal microbiota of the free-range chickens of the present study showed higher α- and β-diversity when fed with TM meal compared to C feed. This represents a quite relevant finding, since high levels of diversity have been reported to help maintain the stability of intestinal microbiota after environmental stress  and deteBacteroidetes, Firmicutes and Proteobacteria. Furthermore, the free-range chickens fed with TM showed Bacteroides, U. m. of Bacteroidales order, Clostridium and Ruminococcus as the most predominant genera of their cecal microbiota. These findings suggest that TM meal utilization does not alter the physiological cecal community at both phylum and genus levels.Similarly to what observed for C birds, the cecal microbiota of the free-range chickens fed with TM diet of the present study was mainly colonized by the phyla Firmicutes and Bacteroidetes phyla, respectively, along with higher Firmicutes:Bacteroidetes ratios. This represents a quite relevant finding, since bacteria within Firmicutes phylum have an important role in the digestion of feed and the host health [Firmicutes:Bacteroidetes ratios have been associated with bacterial profile with higher capacity of energy harvesting [Clostridium, Oscillospira, Ruminococcus, Coprococcus and Sutterella genera in their cecal community. As already pointed out, Clostridium and Ruminococcus are some of the most predominant genera found in chicken cecum [Clostridium genus, along with Oscillospira and Coprococcus, also encompasses bacteria capable of producing butyrate [Ruminococcus genus can also produce other short chain fatty acids  through glucose metabolism and digest cellulose in food [Sutterella genus, which has been reported to be a potential probiotic present in pigeon “milk” that can improve the rate of growth and feed conversion ratio in chickens [Investigating the compositional differences in the cecal microbiota between C and TM birds, the free-range chickens fed with insect meal showed increased and decreased abundances of t health  and grearvesting . At genuen cecum –30. On tbutyrate , 41. Butbutyrate . Further in food . It is w in food . Anotherchickens . The incBacteroides genus within the cecal community of the free-range chickens fed with TM meal of the present study may be considered a potential negative finding. Indeed, apart from being one of the most predominant genera found in chicken cecum [Clostridium perfringens sporulation by its fermentation products [Bacteroides genus could be problematic for chickens, since it may predispose the animal gut to Clostridium perfringens infection and gastroenteritis [Bacteroides genus still outnumbered the other taxa in TM group of the present study. The birds fed with insects also showed similar growth performance to those fed with C diet [Despite this overall positive modulation, the reduction of en cecum , 31, 33,en cecum  and the products . As a conteritis . Howeverh C diet , thus fuDietary TM meal inclusion in free-range chickens of the current trial did not affect the gut morphometric indices, as already reported in details by Biasato et al. . FeatureIndependently of TM meal utilization, the duodenum of the free-range chickens of the current trial showed greater development in relation to the other gut segments. It is well known that the intestine possesses an inherent ability to create and maintain regional differences with regard to mucosal structure , thus deThe present study is the first one to investigate the effects of dietary TM meal inclusion on gut mucin composition in poultry. Furthermore, the characterization of the three types of mucins  in the two intestinal mucosal compartments  of four defined segments  represents a unique histochemical approach.Dietary TM meal inclusion in free-range chickens of the current trial did not influence the mucin staining intensity either in the intestinal crypts or villi. This finding could reflect an innate feeding habit, since chickens naturally consume insects when reared in free-range systems .Independently of TM meal utilization, the intestinal crypts of the birds of the current trial showed greater acidic sialylated mucins staining intensity. This represents an interesting finding, since sialic acid groups have protective properties  and incrThe intestinal crypts of both chickens fed with control and TM diets of the present research showed lower mucin staining intensity in the cecum compared with the other gut segments. This finding is in agreement with what observed by Tsirtsikos et al. , even ifIndependently of dietary TM meal inclusion, the intestinal villi of the free-range chickens of the present study showed greater mucin staining intensity in the ileum compared with the other gut segments. This is consistent with previous findings in chickens , 59, whiThe intestinal crypts of both birds fed with control and TM diets of the current trial showed greater mucin staining intensity in the base compared with the other crypt fragments. This is in agreement with previous studies, in which the decreased stain in the crypt tip has been suggested to be related to the process of proliferation and maturation of goblet cells , 56. SimIn conclusion, dietary TM meal inclusion may positively modulate the gut microbiota of the free-range chickens without influencing the intestinal morphology and mucin composition. The identification of physiological cecal community, gut morphological development and mucin dynamics also suggests that insect meal utilization does not negatively affect the gut health of the birds. Furthermore, since the rapid growth of chickens directly depends on morphological and functional integrity of the digestive tract (as herein confirmed), the gut health assessment by a post mortem multidisciplinary approach appears to be fundamental.The present study is a part of ongoing research work that aims to investigate the effects of TM meal inclusion on growth performance, haematochemical profile, carcass traits, histological features and gut health of free-range chickens . In ordeA total of 140 43-days-old female Label Hubbard hybrid chickens , a medium-growing genotype, were randomly allotted to two dietary treatments. Each of them consisted of five replicate floor pens, with 14 chicks per pen. The experimental unit was the pen. Two diets were formulated: a control corn-soybean-gluten meal-based diet (C), normally used by the breeder, and an experimental diet (TM7.5), in which full-fat TM larvae meal  was included at 75 g/kg in complete substitution of corn gluten meal. Details of the diets are shown in Additional file A total of ten chickens per treatment (two birds per pen) were randomly selected and slaughtered in a commercial abattoir at 97 days of age. Animals were euthanized by electrical stunning and bleeding. The remaining birds were slaughtered using the same euthanasia procedures and carcasses were submitted to proper disposal methods. Cecal content was collected into sterile plastic tubes that were promptly refrigerated (for a maximum of 2 h) and frozen at − 80 °C until DNA extraction. Intestinal segment samples (approximately 5 cm in length) of duodenum, jejunum, ileum and cecum were excised and flushed with 0.9% saline to remove all the content. The collected segments of intestine were the loop of the duodenum (duodenum), the tract before Meckel’s diverticulum (jejunum), the tract before the ileocolic junction (ileum) and the apex of the caeca (cecum). Gut segments were fixed in Carnoy’s and 10% buffered formalin solutions for morphometric analysis and histochemical staining, respectively. Tissues were routinely embedded in paraffin wax blocks, sectioned at 5 μm thickness and mounted on glass slides.v/v) of PhiX.A pool of the cecal content from two chickens per pen (five pools per feeding group) was submitted to DNA extraction and sequencing. DNA was extracted with a commercial kit  according to the manufacturer’s instructions. One μl of RNase  was added to digest RNA in the DNA samples, with an incubation of 1 h at 37 °C. DNA was quantified using the NanoDrop and standardized at 5 ng/μl. Microbial diversity was studied by sequencing the amplified V3-V4 region of the 16S rRNA gene by using primers and PCR conditions previously reported . SamplesCarnoy-fixed and paraffin-embedded intestinal sections of ten chickens per feeding group (two birds per pen) were submitted to Haematoxylin & Eosin staining and one slide per each intestinal segment was examined by light microscopy. Each slide was captured with a Nikon DS-Fi1 digital camera coupled to a Zeiss Axiophot microscope using a 2.5× objective lens. NIS-Elements F software was used for image capturing. Morphometric analysis was performed by Image®-Pro Plus software. The evaluated morphometric indices were villus height , crypt depth  and the villus height to crypt depth ratio (Vh/Cd) . MorphomFormalin-fixed and paraffin-embedded intestinal sections of ten chickens per feeding group (two birds per pen) were submitted to three different histochemical staining, in order to characterize the three types of mucins .Neutral mucins were identified by periodic-acid Schiff staining. Sections were brought to water, immersed in 0.5% periodic acid solution for 20 min, washed in running tap water for 5 min and immersed in Schiff’s reagent for a further 30 min. Sections were successively rinsed in running tap water for 10 min, dehydrated and mounted. Neutral mucins stained magenta .Acidic sialylated mucins were identified by Alcian Blue pH 2.5 staining. Sections were brought to water, immersed in 8 G X alcian blue in 3% acetic acid solution for 30 min, washed in running tap water for further 5 min and successively dehydrated and mounted. Acidic sialylated mucins stained blue .Acidic sulfated mucins were identified by high iron diamine staining. Sections were brought to water, oxidized in 1% periodic acid solution for 10 min and washed in running tap water for further 5 min. Sections were successively immersed in the high iron diamine solution  for 18 h, rinsed very rapidly in tap water, dehydrated and mounted. Acidic sulfated mucins stained purple-black .One slide per histochemical staining for each intestinal segment was examined by light microscopy. A total of 10 crypts and 10 villi for each slide were evaluated. Crypts and villi were divided into three fragments , according to Tsirtsikos et al. , 10. MucThe experimental unit was the pen for 16S rRNA sequences and bird for morphometric and histochemical data.diversity function of the vegan package [https://www.r-project.org). OTU table showed the higher taxonomy resolution reached by the 16S data. When the taxonomy assignment was not able to reach the genus level, the phylum, class, order or family were displayed. Pairwise Kruskal-Wallis tests were used to find significant differences in microbial taxa abundance according to the dietary treatment. The experimental unit was the pen. P-values were adjusted for multiple testing and a false discovery rate (FDR) < 0.05 considered as significant.Paired-end reads were first merged using FLASH software  with def package . A pairwt test for independent samples. Morphometric data were also analyzed by means of One-way ANOVA (post hoc test: Duncan’s multiple range test) to evaluate the influence of intestinal segment within each dietary treatment. Results were expressed as mean and pooled standard error of the mean (SEM). P values < 0.05 were considered statistically significant [The statistical analysis of morphometric and histochemical data was performed using IBM SPSS Statistics V20.0.0 software. The influence of diet on intestinal morphometric measurements was tested using Student’s nificant . Histochnificant , 10. TheAdditional file 1:Tenebrio molitor meal (TM7.5) diets. Description column indicates the 5 replicate pens of control  and 7.5% inclusion level of Tenebrio molitor meal  dietary treatments. (XLSX 9 kb)Good’s coverage and α-diversity measures of cecal microbiota of free-range chickens fed with control (C) and 7.5% inclusion level of Additional file 2:Tenebrio molitor meal (TM7.5) diets. (XLSX 10 kb)Relative abundance of the main bacterial phyla and genera of cecal microbiota of free-range chickens fed with control (C) and 7.5% inclusion level of Additional file 3:cis9, C16:1 cis9, C18:1 cis11, C20:0, C18:3 n6, C20:1 cis9, C20:1 cis11. TM, Tenebrio molitor; AME, apparent metabolizable energy; DM, dry matter; CP, crude protein; C = control; TM7.5 = 7.5% inclusion level of Tenebrio molitor. (XLSX 10 kb)Ingredients and chemical composition of the experimental diets. The mineral-vitamin premix  Italy) given values are supplied per kg: 650.000 IU of vitamin A; 65.000 IU of vitamin D3; 650 IU of vitamin E; 80 mg of vitamin K; 80 mg of vitamin B1; 150 mg of vitamin B2; 770 mg of vitamin B3; 80 mg of vitamin B6; 0.5 mg of vitamin B12; 240 mg of pantothenic acid; 4700 mg of betaine; 1750 mg of Iron (II) carbonate; 1835 mg of Magnesium oxide; 1612 mg of Zinc oxide; 178 mg of Copper (II) oxide; 18.3 mg of Potassium iodide; 6.6 mg of Sodium selenite; 4100 mg of DL-methionine; 5500 mg of L-lysine; 120 g Calcium carbonate; 450 g Calcium phosphate; 11.5 g of Sodium chloride. SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids. Other fatty acids : C12:0, C14:0, C14:1


Human dental pulp stem cell (DPSC)-mediated regenerative endodontics is a promising therapy for damaged teeth; however, the hypoxic environment in root canals can affect tissue regeneration. In this study, we investigate the characteristics and possible regulatory mechanisms of DPSC function under hypoxic conditions.2) and hypoxia (3% O2). DPSC proliferation and osteo/odontogenic differentiation potential were assessed by Cell Counting Kit-8 (CCK8) assay, carboxyfluorescein succinimidyl ester (CFSE) assay, alkaline phosphatase (ALP) activity, Alizarin red staining, real-time RT-PCR assays, and western blot analysis. Microarray and bioinformatic analyses were performed to investigate the differences in the mRNA, lncRNA, and miRNA expression profiles of DPSCs.Human DPSCs were cultured under normoxia , 47 lncRNAs (20 upregulated and 27 downregulated), and 14 miRNAs (7 upregulated and 7 downregulated) in DPSCs were differentially expressed in the hypoxia group compared with the normoxia group. Bioinformatic analysis identified that 7 mRNAs  and 5 lncRNAs ) may be associated with DPSCs during hypoxia according to CNC network analysis, while 28 mRNAs  and 13 miRNAs (including hsa-miR-3916 and hsa-miR-192-5p) may be involved according to miRNA target gene network analysis. The depletion of one candidate lncRNA, STL, inhibited the osteo/odontogenic differentiation potentials of DPSCs.Our results revealed that hypoxia could enhance the proliferation ability and impair the osteo/odontogenic differentiation potential of DPSCs in vitro. Furthermore, our results identified candidate coding and noncoding RNAs that could be potential targets for improving DPSC function in regenerative endodontics and lead to a better understanding of the mechanisms of hypoxia’s effects on DPSCs.The online version of this article (10.1186/s13287-019-1192-2) contains supplementary material, which is available to authorized users. Pulpitis and periapical periodontitis are the most prevalent oral diseases, and root canal treatment is the current primary treatment. Although the treatment has been shown to be highly effective, after treatment, teeth can become devitalized, brittle, and susceptible to fracture, leading to a higher incidence of extraction , 2. Duri2) in vitro, which is markedly different from their typical in vivo environment. Previous studies have indicated that after being cultured in normoxic conditions, dental MSCs recultured in hypoxic conditions possess altered proliferation capacities and differentiation potentials; however, the results are inconclusive due to different cell sources and experimental conditions. Most studies have shown that hypoxia can promote the proliferation of dental stem cells [2) or anoxia [The microenvironmental niche of MSCs, which maintains and regulates their function, is the key element for determining the characteristics of MSCs. Oxygen in the microenvironment is necessary for the normal growth and differentiation of cells. Oxygen tensions in human organs and tissues vary from 2 to 9% in physiological conditions. In pathologic conditions or in some relatively hypoxic areas, oxygen tensions can drop to 1% or, in some cases, lower . Oxygen em cells –24. Howeem cells –28. Someem cells , 29–31, em cells . In addir anoxia . MSC-medr anoxia . There i2 tension was used to mimic hypoxia, and the characteristics and gene expression profiles of DPSCs in hypoxia were investigated. In addition, bioinformatic analysis was conducted to predict the interactions of coding and noncoding RNA and identify core regulatory factors of DPSCs in hypoxia. Furthermore, loss-of-function assays were performed to detect the effect of one candidate noncoding RNA, six-twelve leukemia (STL), on DPSCs. These results may help reveal the molecular mechanisms of DPSCs in a hypoxic environment, which may contribute to the identification of the key regulators that are necessary for DPSC-mediated dental tissue regeneration.Here, 3% O2 and 5% CO2, with a balance of 92% N2 at 37 °C by using the hypoxic incubator  which can accurately adjust the O2 concentration of 1–21%.Human teeth were collected with informed patient consent and based on the protocol approved by Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, . Human dental pulps were obtained from healthy teeth extracted for impaction or other orthodontic reasons. The tissues from the crown and superior 2/3 root pulp were gently separated, and DPSCs were isolated, cultured, and characterized as previously described . The cul2), the culture medium was replaced with 100 μL MEM containing 10 μL CCK8, then incubated for 2 h at 37 °C. Absorbance (OD) at 450 nm was measured in a multiwell spectrophotometer.The proliferation of DPSCs was determined using the CCK8 kit, according to the manufacturer’s instructions . A volume of 100 μL of DPSC suspension (3000 cells/well) was dispensed into the wells of a 96-well plate. After preincubation for 2 or 3 days in a humidified incubator  with 635 nm excitation and emission filter appropriate for far red. The proliferation index was calculated by Modfit LT .Carboxyfluorescein succinimidyl ester (CFSE) assays were performed according to the CellTrace™ CFSE Cell Proliferation Kit protocol  for labeling cells in suspension as previously described . Briefly2PO4, and 10 nM/L dexamethasone (Sigma-Aldrich). Cells were induced for 5 days, and an ALP activity assay was performed as previously described [To detect mineralization, DPSCs were grown in mineralization-inducing medium consisting of α-MEM, 15% FBS, 2 mM/L glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μM/L ascorbic acid, 10 mM/L β-glycerophosphate , 1.8 mM/L of KHescribed . DPSCs wescribed .−△△CT calculation. Each reaction was run in triplicate, and the entire procedure was repeated three times. The primers were designed by applying the online D-LUX DesignerTM program (Invitrogen) and are shown in Additional file Real-time RT-PCR was used to detect the expression levels of osteo/odontogenic marker genes and to verify the changes in gene expression that were detected by microarray analysis. Total RNA was isolated from DPSCs with TRIzol reagent (Invitrogen). cDNA was synthesized using reverse transcriptase with random primers for mRNA and lncRNA and with a special stem-loop primer for miRNA. The QuantiTect SYBR Green PCR kit  was used in real-time PCR reactions, and the IcycleriQ multicolor real-time PCR detection system was used . The gene expression levels were evaluated by normalizing the PCR signal to that of GAPDH (mRNA and lncRNA) or U6 (miRNA) and applying the 2To detect proteins of osteo/odontogenic differentiation genes, DPSCs were cultured in mineralization-inducing medium for 2 weeks, and then western blot analysis was performed as previously described . Primaryp value < 0.05.To detect differential coding and noncoding transcript expression profiles, DPSCs from three different individuals were cultured in normoxia or hypoxia for 3 days. Total RNA was then extracted from DPSCs using TRIzol and the RNeasy mini kit (Qiagen). The quality and quantity of RNA were verified by multiImager and spectrophotometer  analysis. The RNA labeling, hybridization, and scanning of the chips  were performed as outlined in the Affymetrix technical manual by Cnkingbio Corp . The threshold set for up- or downregulated mRNA, lncRNA, or miRNA was a fold change > 1.5 and a The microarray raw data were normalized for follow-up analysis with Expression Console software  using the MAS 5.0 statistical algorithm. The bioinformatic analysis was performed after normalized data was compared and filtered. Hierarchical clusters were performed by EPCLUST. Gene ontology (GO) analysis was used to analyze the genes’ primary functions. Pathway analysis was used to determine the significantly changed pathways of the differential genes based on KEGG, Biocarta, and Reatome. A coding-noncoding gene coexpression (CNC) network and miRNA target gene network were constructed to identify the interactions among genes and to locate core regulatory factors that played an important role in these networks.STL shRNA (STLsh) and control shRNA (Consh) were purchased from Genepharma . The lentiviruses were transfected into DPSCs in the presence of polybrene  for 12 h. After 48 h, the transfected DPSCs were selected with 1 μg/mL puromycin for 3 days. The target sequences for the shRNA were STLsh, 5′-CGGCATGACTAAGAGATATCG-3′ and Consh, 5′-TTCTCCGAACGTGTCACGT-3′.To detect adipogenic differentiation, DPSCs were grown in adipogenic-inducing medium for 3 weeks under normoxic conditions, and then Oil Red O staining was performed as previously described . The OD t test or one-way ANOVA analysis. For all tests, p < 0.05 was considered statistically significant.The statistical analyses were performed by using SPSS 16.0 software . Significance was determined by the Student’s To investigate the effects of hypoxia on DPSCs, DPSCs were cultured in both normoxic and hypoxic conditions. CCK8 assays showed that the cell numbers of DPSCs were increased in hypoxic conditions compared with normoxic conditions at days 2 and 3 Fig. a. CFSE at test was applied to filter the genes that were differentially expressed, and the differentially expressed genes with 1.5-fold changes and p values < 0.05 were selected for subsequent analysis. From the microarray data, a total of 60 mRNAs with differential expression in DPSCs were identified between the hypoxia and normoxia groups, of which 25 were upregulated and 35 were downregulated in the hypoxia group compared with those in the normoxia group  were obtained, of which 28 were upregulated and 63 were downregulated in the hypoxia group. The negative logarithm of the p value (− LgP) was applied to show the correlation of gene expression levels and the relevant biological processes  were identified, among which 5 were upregulated and 12 were downregulated in the hypoxia group  were determined, and the reliability of the microarray data was confirmed by real-time RT-PCR. In total, 60 differentially expressed mRNAs, 47 differentially expressed lncRNAs, and 14 differentially expressed miRNAs were identified. These differentially expressed coding and noncoding RNAs may be candidates for key regulators controlling the proliferation and differentiation of DPSCs in hypoxia and warrant further bioinformatic analysis.By GO and pathway analysis, we screened out the significantly altered functions and pathways associated with DPSCs in hypoxia, including the Wnt and HIF-1 signaling pathways. The canonical Wnt/β-catenin signaling pathway has different functions in hard tissue development and regeneration, depending on cell type and stage of development. Previous reports found that the proliferation and differentiation potentials of DPSCs could be regulated via the Wnt/β-catenin signaling pathway , 36. HIF2 (5%) according to microarray analysis and further validation, which is consistent with our results [According to CNC network and miRNA target gene network analysis, some of the differentially expressed RNAs in the two groups were screened out as possible regulatory factors of DPSCs in hypoxia, including ERO1L, NQO1, STL, hsa-miR-192-5p, and several others. NQO1 can function as a direct superoxide anion radical scavenger or a major target of the Nrf2 pathway to maintain cellular redox homeostasis and attenuate cellular oxidative stress. In addition to oxidation-reduction process, NQO1 is related to obesity, hypertension, renal injury, bone metabolism, tumor growth, and other processes –41. It w results . NQO1 ma results . ERO1L b results . It was  results , 45. ERO results –51. As a results –54. Emer results –57. Cons results , 59. As  results –64. Some results , 65. It  results –68. AccoIndeed, we found that the expression of STL in DPSCs was enhanced along with osteo/odontogenic differentiation of DPSCs, indicating that STL may play a positive role in this process. We therefore conducted loss-of-function assays to investigate the functions of STL in DPSCs. Our results showed that the knockdown of STL inhibited the osteo/dentinogenic differentiation potential of DPSCs, indicating that STL was involved in the positive regulation of osteo/dentinogenic differentiation ability of DPSCs. Consistent with the CNC network, NQO1 was downregulated while ERO1L was upregulated when STL was knocked down in DPSCs. In addition, STL and NQO1 were downregulated and ERO1L was upregulated in hypoxia, indicating that decreased STL might be one factor for the impaired osteo/dentinogenic differentiation ability of DPSCs in hypoxia and that this process might be downregulated via NQO1 and upregulated via ERO1L. It is possible that STL works on DPSCs via interactions with these two genes, and therefore, STL may be considered as a possible therapeutic target for dental tissue regeneration. However, further studies are needed to verify this hypothesis and the underlying mechanism.In conclusion, our results revealed that 3% oxygen tension could enhance the proliferative ability and impair the osteo/odontogenic differentiation potential of DPSCs, indicating that hypoxia is an important factor affecting DPSC-mediated dental tissue regeneration. Furthermore, our results identified that candidate coding and noncoding RNA could be potential targets for improving the DPSC characteristics in hypoxia and lead to a better understanding of the mechanisms of hypoxia’s effects on DPSCs. In addition, our results highlighted the significant involvement of one lncRNA, STL, in the positive regulation of the osteo/odontogenic differentiation ability of DPSCs and indicated that STL could be a potential target in regenerative endodontics.Additional file 1:Table S1. Primer sequences used in real-time RT-PCR analysis. (DOCX 16 kb)Additional file 2:Table S2. The differentially expressed mRNAs of hDPSCs in hypoxic and normoxic conditions. (DOCX 18 kb)Additional file 3:Table S3. The differentially expressed lncRNAs of hDPSCs in hypoxic and normoxic conditions. (DOCX 20 kb)Additional file 4:Table S4. The differentially expressed miRNAs of hDPSCs in hypoxic and normoxic conditions. (DOCX 14 kb)Additional file 5:Figure S1. Significant GOs of upregulated genes of DPSCs in hypoxia. The x axis shows − LgP and the y axis shows GO category. A larger – LgP indicates a smaller p value for the difference. (TIFF 1845 kb)Additional file 6:Figure S2. Significant GOs of downregulated genes of DPSCs in hypoxia. The x axis shows − LgP and the y axis shows GO category. A larger − LgP indicates a smaller p value for the difference. (TIFF 2370 kb)Additional file 7:Figure S3. Significant pathways of upregulated genes of DPSCs in hypoxia. The x axis shows − LgP and the y axis shows pathways category. A larger – LgP indicates a smaller p value for the difference. (TIFF 512 kb)Additional file 8:Figure S4. Significant pathways of downregulated genes of DPSCs in hypoxia. The x axis shows − LgP and the y axis shows pathways category. A larger – LgP indicates a smaller p value for the difference. (TIFF 1140 kb)Additional file 9:Figure S5. Knockdown of STL inhibited the adipogenic differentiation of DPSCs in vitro.  Oil red staining (a) and a quantitation analysis (b) revealed a significant decrease in lipid deposits in STL-depleted DPSCs compared to the control groups 3 weeks after adipogenic induction. One-way ANOVA was performed to determine statistical significance. All error bars represent SD (n = 3). NS; no significant difference, **p < 0.01. Scale bar 200 μm. (TIFF 1495 kb)


In glomerular injury dendrin translocates from the slit diaphragm to the podocyte nucleus, inducing apoptosis. We analyzed dendrin expression in IgA glomerulonephritis and Henoch Schönlein purpura (IgAN/HSP) versus in podocytopathies minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), and compared it to pathohistological findings and renal function at the time of biopsy and the last follow-up.Twenty males and 13 females with median of age 35 years (min-max: 3–76) who underwent percutaneous renal biopsy and had diagnosis of glomerular disease (GD) were included in this retrospective study. Fifteen patients had IgAN/HSP and eighteen podocytopathy. Control group consisted of ten patients who underwent nephrectomy due to renal cancer. Dendrin expression pattern , number of dendrin positive nuclei and proportion of dendrin negative glomeruli were analyzed.P = 0.004 and P = 0.003, respectively). Number of dendrin positive nuclei was higher in podocytopathies than in IgAN/HSP, 3.90 versus 1.67 (P = 0.028). Proportion of dendrin negative glomeruli correlated to higher rates of interstitial fibrosis (P = 0.038), tubular atrophy (P = 0.011) and globally sclerotic glomeruli (P = 0.008). Dual and nuclear dendrin expression pattern were connected with lower rate of interstitial fibrosis and tubular atrophy than negative dendrin expression pattern . Proportion of dendrin negative glomeruli correlated with lower creatinine clearance (CC) at the time of biopsy and the last follow-up . Dendrin expression pattern correlated to CC at the last follow-up (P = 0.009), being lower in patients with negative than nuclear or dual dendrin expression .In GD and the control group significant differences in number of dendrin positive nuclei and proportion of dendrin negative glomeruli were found (In this pilot study the number of dendrin positive nuclei was higher in podocytopathies than in inflammatory GD. Negative dendrin expression pattern correlated to chronic tubulointerstitial changes and lower CC, which needs to be confirmed in a larger series. Dendrin is a proline - rich protein of still unclear function that was originally identified in telencephalic dendrites of sleep-deprived rats . Apart fcapillary loop stage and it is never expressed in podocyte nuclei [During fetal development of human kidney, dendrin is first detected at the e nuclei , 3, 8. De nuclei . The expe nuclei , 7. Intee nuclei . AccordiThe aim of our pilot study was to evaluate possible difference in dendrin expression in inflammatory and non-inflammatory GD. We analyzed 11 IgAN, 4 HSP and 18 podocytopathies (11 primary FSGS and 7 MCD) and correlated with histological parameters and renal function at the time of the biopsy and the last follow-up.Thirty-three patients whose percutaneous renal biopsy was performed from 1996 to 2015 in University Hospital Centre Split, Split, Croatia were enrolled in this study. Paraffin blocks of their biopsies were collected from the Department of Pathology and clinical data at the time of biopsy and the last follow-up in 2017 were collected from the hospital records. All biopsies were analyzed by light, immunofluorescence and electron microscopy making the diagnoses of IgAN, HSP, MCD or primary FSGS. Inclusion criteria were enough material in the paraffin block for immunohistochemical analysis and complete clinical data. The patients without adequate laboratory findings or paraffin blocks were excluded. In control group we analyzed glomeruli from ten patents with normal renal function, who underwent nephrectomy due to clear cell renal carcinoma. Five micrometer thin slides were cut from the paraffin blocks. Indirect immunohistochemistry was performed in Benchmark . The primary antibody was rabbit polyclonal anti-dendrin  diluted at 1:200 and incubated for 1 h. Secondary antibody was Optiview Detection kit  and incubation lasted 30 min. In immunofluorescence we used rabbit anti-dendrin , diluted at 1:200 and goat anti-nephrin antibody , diluted at 1:300. They were applied simultaneously and left overnight in a humidified chamber at room temperature. The secondary antibodies were donkey anti-goat IgG , diluted at 1:300 and donkey polyclonal anti-rabbit IgG , diluted at 1:300. After washing in phosphate-buffered saline (PBS), DAPI  was applied and washed again. Stained sections were viewed and photographed, using a BX51 microscope  equipped with a DP71 digital camera (Olympus), and processed with CellA Imaging Software for Life Sciences Microscopy (Olympus). Dendrin translocation to nuclei was presented by co-localization of dendrin, nephrin and DAPI in the same cell on immunofluorescence  per slide. The same was done on immunofluorescence microscope. Since there was no statistical difference in number of dendrin positive nuclei between these two methods, only the results of the immunohistochemistry were presented in the further text. Number of dendrin positive nuclei was divided with the number of analyzed glomeruli and thus median (min-max) was calculated for each patient. Number of dendrin negative glomeruli was divided with number of analyzed glomeruli to get their proportion. Control slides were analyzed on one low magnification field (7 glomeruli in average) and analyzed on the same way. A pattern of dendrin expression in each glomerulus was assigned as membranous, dual, nuclear or negative. Only membranous staining was assigned as membranous, simultaneously positive membranous and nuclear staining as dual, only nuclear staining as nuclear and both negative membranous and nuclear staining with positive external control as negative Fig. .Fig. 2DeP < 0.05, and all confidence intervals (CI) were at the 95% level. Spearman’s correlation coefficient (Rho) was used to calculate degree of connection. Statistical significance of the difference in categorical characteristics of several independent variables was calculated by using χ2 test. Fisher’s exact test was used in the analysis of contingency tables when tested sample sizes were small. Analysis of statistical significance of differences in several numerical variables was performed with the Kruskal-Wallis test and of differences between two groups with the Mann-Whitney test. The study was performed according to the Helsinki Declaration. Informed signed consent form was obtained from each patient (or parents if a child).Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software . Statistical significance was set at In the study there were 20 61%) males, with median of age 38.5 (min-max: 3–76 years) and 13 (39%) females, with median of age 35 (min-max: 4–62 years). Eleven (33.33%) patients had IgAN, four (12.12%) HSP, seven (21.21%) MCD and eleven (33.33%) FSGS. The distribution according to the type of glomerular disease and clinical parameters is presented in Table % males, Patients with several GD were not significantly different in any clinical parameters. In further analysis types of GD and the control group were analyzed according to median of number of nuclear dendrin expression and proportion of dendrin negative glomeruli Table .Table 2NP = 0.028, Z = 2.2, r = 0.38, 95% CI 0.12–4.34). In further analysis MCD and FSGS were analyzed separately. Nuclear dendrin expression was different in IgAN/HSP, MCD, FSGS and control group . Median nuclear dendrin expression was higher in MCD than IgAN/HSP (P = 0.033) and in MCD than the control group (P = 0.003). No difference was found between IgAN/HSP and FSGS (P = 1.000). Median of nuclear dendrin expression was not significantly different in MCD and FSGS (P = 0.363). Proportion of dendrin negative glomeruli was statistically different between GD . In IgAN/HSP and FSGS proportions of dendrin negative glomeruli were higher than in the control group . There was no difference between IgAN/HSP and FSGS (P = 1.000). Difference was not found between MCD and control group (P = 1.000).In IgAN/HSP median number of dendrin positive nuclei was 1.67 (min - max: 0.00–5.60) and in podocytopathies 3.90 (min – max: 0.00–23.10), (P = 0.403). According to Spearman’s coefficient of correlation, CC at the time of biopsy and proportion of dendrin negative glomeruli showed negative correlation . CC at the last follow-up significantly correlated to proportion of dendrin negative glomeruli  and to median of dendrin positive nuclei . No difference was found in correlation between the level of proteinuria and median of dendrin positive nuclei , as well as proteinuria and proportion of dendrin negative glomeruli .CC at the time of biopsy did not significantly correlate to median of dendrin positive nuclei . Proportion of dendrin negative glomeruli in the first group was 0.50 (min-max: 0.00–1.00), and in the former 0.33 (0.00–1.00) (P = 0.668). According to International Study of Kidney Disease in Children (ISKDC) classification, three patients with HSP were grade 3, and one grade 6.In eight IgAN samples with crescents median of dentrin positive nuclei was 1.58 (min – max: 0.00–3.85) and in seven IgAN samples without crescents 2.30 (min – max: 0.00–5.60) (P = 0.299). Also there was no difference in dual/nuclear versus negative expression pattern (P = 0.418). Dendrin expression pattern in 33 patients was correlated to pathohistological findings and clinical parameters of renal function , dual in sixteen 48.48%) and negative in eight (24.24%) patiens. There was no difference between IgAN/HSP and podocytopathies in distribution of dual or nuclear dendrin expression pattern . Mann-Whitney test in post-hoc analysis did not find any difference between the patients with nuclear or dual expression . In patients with negative dendrin expression pattern, median of CC was significantly lower than in patients with nuclear expression pattern  and for 79.5 lower than in patients with dual expression pattern . CC at the time of biopsy and proteinuria were not significantly different between dendrin expression patterns .Dendrin expression pattern significantly correlated to median of CC at the last follow-up (χP = 0.018). Significant correlations between number of dendrin positive nuclei and interstitial fibrosis  or tubular atrophy  were not found. Statistically significant positive Spearman’s correlations were found in proportions of interstitial fibrosis , tubular atrophy  and globally sclerotic glomeruli  with proportion of dendrin negative glomeruli.According to Spearman’s coefficient of correlation, proportion of globally sclerotic glomeruli and number of dendrin positive nuclei showed negative correlation   Table .Table 5D2 = 9.60; P < 0.001). In MCD there was 3.9 times more glomeruli with nuclear dendrin expression pattern than in IgAN, but only one glomerulus with negative dendrin expression . IgAN had 3.1 times less glomeruli with nuclear dendrin expression than FSGS . Nuclear dendrin expression was found in MCD, in contrast to HSP . Dual pattern was 1.8 times more common in HSP than FSGS . In MCD one glomerulus had negative expression pattern and in FSGS 17% glomeruli .In IgAN nuclear pattern was found in 13% glomeruli and none in HSP . These changes could be explained by the differences in the pathophysiological mechanisms, the clinical course and dynamics of a particular disease.Relocation of dendrin from membrane to nucleus is a dynamic, gradual and multi-level process controlled and affected by numerous factors and signal pathways. We have found that dendrin translocation occurs in different types of glomerular diseases, including MCD, and dendrin expression presents in different patterns. The proportion of dendrin negative glomeruli could be a novel adverse prognostic factor of the kidney function. We propose that dendrin translocation and its correlation with kidney failure and pathological parameters should be interpreted in the context of dendrin expression pattern, not only as an absolute number of dendrin positive nuclei.