Oligoasthenoteratospermia and sperm tail bending in PPP4C-deficient mice.

Protein phosphatase 4 (PPP4) is a protein phosphatase that, although highly expressed in the testis, currently has an unclear physiological role in this tissue. Here, we show that deletion of PPP4 catalytic subunit gene Ppp4c in the mouse causes male-specific infertility. Loss of PPP4C, when assessed by light microscopy, did not obviously affect many aspects of the morphology of spermatogenesis, including acrosome formation, nuclear condensation and elongation, mitochondrial sheaths arrangement and '9 + 2' flagellar structure assembly. However, the PPP4C mutant had sperm tail bending defects (head-bent-back), low sperm count, poor sperm motility and had cytoplasmic remnants attached to the middle piece of the tail. The cytoplasmic remnants were further investigated by transmission electron microscopy to reveal that a defect in cytoplasm removal appeared to play a significant role in the observed spermiogenesis failure and resulting male infertility. A lack of PPP4 during spermatogenesis causes defects that are reminiscent of oligoasthenoteratospermia (OAT), which is a common cause of male infertility in humans. Like the lack of functional PPP4 in the mouse model, OAT is characterized by abnormal sperm morphology, low sperm count and poor sperm motility. Although the causes of OAT are probably heterogeneous, including mutation of various genes and environmentally induced defects, the detailed molecular mechanism(s) has remained unclear. Our discovery that the PPP4C-deficient mouse model shares features with human OAT might offer a useful model for further studies of this currently poorly understood disorder.

[The 472nd case: dyspnea, pulmonary shadows, abnormalities of whole blood cells].

A 54-year-old man was admitted to respiratory department with chief complaints of recurrent cough and dyspnea. Chest imaging showed multiple patchy shadows and interstitial changes. Evidence of infectious diseases was not definite, and antibiotic treatments were not effective. In the meantime, myelodysplasia syndrome was diagnosed with pancytopenia. The pathologic findings of transbronchoscopic lung biopsyshowed chronic inflammatory interstitial changes, suggesting a clinical diagnosis of organizing pneumonia. After glucocorticoids treatment, his condition aggravated. The second percutaneous lung biopsy showed the infiltration of a large number of neutrophils. Therefore, the final diagnosis of myelodysplasia syndrome with Sweet syndrome was made. Then glucocorticoids and supportive treatment were given This case may improve physicians' understanding of myelodysplasia syndrome complicated with Sweet syndrome.

[Relationship between sleep status and laryngopharyngeal reflux disease in adult patients in Otolaryngology clinic].

Objective: To explore the correlation between sleep and laryngopharyngeal reflux disease by epidemiological approaches. Methods: From May 1, 2017 to April 30, 2018, data of age, gender, height, weight, smoking, alcohol consumption, constipation and high fat diet in patients in Otorhinolaryngology specialist clinic, the Eighth Medical Center, General Hospital of the Chinese PLA were retrospectively analyzed. Reflux Symptom Index (RSI), Pittsburgh Sleep Quality Index (PSQI) and Hospital Anxiety and Depression Scale (HADS)were filled. According to RSI scores, patients were divided into case group and control group. The differences of the above indicators between the two groups were compared by Stata 12.0 software, and the risk factors of LPRD were analyzed by multivariate Logistic regression. Results: A total of 908 patients were enrolled, including 166 in the case group and 742 in the control group. There was no significant difference in BMI, smoking, drinking, constipation and high fat diet between the two groups (all P>0.05). The PSQI, anxiety and depression score of the case group were higher than those of the control group. The anxiety and depression scores of the patients with sleep disorders in the case group were significantly higher than those of the normal sleepers (all P<0.05). RSI of the patients with sleep disorders was higher than that of the patients with normal sleep(9.5[4.0,16.0]vs. 5.0[1.0,10.0], Z=-6.07, P<0.001). Multivariate analysis showed that sleep disorder was the risk factors of LPRD (OR=2.59, 95%CI 1.75-3.84). Conclusions: Sleep disorder is related to the occurrence of LPRD. The association between LPRD and sleep disturbances is bidirectional. Sleep disorder may also be related to the anxiety and depression in LPRD patients. Handling sleep disorder timely may benefit LPRD patients.

Targeting the vulnerability to NAD+ depletion in B-cell acute lymphoblastic leukemia.

Although substantial progress has been made in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), the prognosis of patients with either refractory or relapsed B-ALL remains dismal. Novel therapeutic strategies are needed to improve the outcome of these patients. KPT-9274 is a novel dual inhibitor of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT). PAK4 is a serine/threonine kinase that regulates a variety of fundamental cellular processes. NAMPT is a rate-limiting enzyme in the salvage biosynthesis pathway of nicotinamide adenine dinucleotide (NAD) that plays a vital role in energy metabolism. Here, we show that KPT-9274 strongly inhibits B-ALL cell growth regardless of cytogenetic abnormalities. We also demonstrate the potent in vivo efficacy and tolerability of KPT-9274 in a patient-derived xenograft murine model of B-ALL. Interestingly, although KPT-9274 is a dual PAK4/NAMPT inhibitor, B-ALL cell growth inhibition by KPT-9274 was largely abolished with nicotinic acid supplementation, indicating that the inhibitory effects on B-ALL cells are mainly exerted by NAD+ depletion through NAMPT inhibition. Moreover, we have found that the extreme susceptibility of B-ALL cells to NAMPT inhibition is related to the reduced cellular NAD+ reserve. NAD+ depletion may be a promising alternative approach to treating patients with B-ALL.

Long non-coding RNA ROR is a novel prognosis factor associated with non-small-cell lung cancer progression.

OBJECTIVE: The aim of the present study was to determine the expression levels of long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) in non-small-cell lung cancer (NSCLC) patients and to further explore the prognostic value of this lncRNA. PATIENTS AND METHODS: In our investigation, we determined the expression of linc-ROR in human NSCLC tissues and matched normal lung tissues by quantitative Real-time-PCR analysis. Also, correlations between linc-ROR expression and the clinicopathological features were evaluated. Survival curves were plotted using the Kaplan-Meier method and differences in survival rates were analyzed using the log-rank test. Cox regression analyses were performed to explore the effect of linc-ROR as an independent predictor of survival. RESULTS: We found that linc-ROR had high expression in NSCLC specimens than that in matched adjacent normal lung tissues (p < 0.01). In addition, higher linc-ROR expression levels were positively correlated with advanced TNM stage (p = 0.007), positive distant metastasis (p = 0.001) and LN metastasis (p = 0.011). Furthermore, significantly shorter 5-year overall survival (OS) and disease-free survival (DFS) were observed in patients with higher expression of linc-ROR (both p < 0.001). In a multivariate Cox model, it was found that linc-ROR expression was an independent prognostic factor for both 5-years OS (p = 0.001) and 5-year DFS (p = 0.001) in NSCLC. CONCLUSIONS: Our findings indicate that linc-ROR plays an oncogenic role in NSCLC development and may function as a prognostic and predictive biomarker for NSCLC.

[The prevalence of metabolic syndrome among adults in rural areas of Ningxia Hui autonomous region].

Objective: To investigate the prevalence of metabolic syndrome (MS) among adults in rural areas of Ningxia Hui autonomous region. Methods: A cross-sectional study was conducted in 10 639 adults enrolled with a multistage method from Jingyuan County. The MS was identified according to Chinese type 2 diabetes prevention guide (2013). Results: Among all the subjects, 17.4% of them met the MS definition with the standardized prevalence of 14.7% after adjustment of sex and age. The prevalence and standardized rate of MS in men were 19.9% and 17.3%, and in women were 15.3% and 13.5%.The prevalence of MS in men was higher than that in women(P<0.001) and increased with aging in both genders. The prevalence and standardized rate of abdominal obesity, hyperglycemia, hypertension, high triglycerides, and low HDL-C were 19.5% and 16.7%, 15.0% and 12.9%, 42.0% and 37.1%, 25.8% and 23.1%, 28.5% and 27.7%, respectively. The rate of abdominal obesity was higher in women than in men (20.5% vs 18.2%, P=0.004), whereas the rate of hypertension, high triglycerides, and low HDL-C were higher in men than in women (all P<0.01). The prevalence of having one parameter of the MS was 68.4%. Conclusion: The prevalence of MS is higher in rural areas of Ningxia Hui autonomous region, suggesting that a series of comprehensive prevention measures should be carried out to prevent and control the MS so as to improve the public health conditions in rural areas.

Ordering of mutations in acute myeloid leukemia with partial tandem duplication of MLL (MLL-PTD).

Partial tandem duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated, whereas NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS), typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.

Effect of alpha-lipoic acid on relieving ammonia stress and hepatic proteomic analyses of broilers.

Ammonia in poultry houses not only affects worker health but also induces a variety of poultry diseases. Alpha-lipoic acid (LA) is an effective antioxidant that protects cells against oxidative injury during various toxic and pathological processes. This study was designed to evaluate the mitigating effects of LA supplementation on ammonia stress and hepatic proteome changes in broilers. Male broilers (22 d old) were allocated to 3 groups: (1) a control group without ammonia stress (CTRL); (2) exposure to 70 ppm ammonia (AM); and (3) exposure to 70 ppm ammonia and dietary administration of 300 mg/kg LA (AM+LA). Ammonia exposure significantly decreased broiler growth performance and plasma glutathione peroxidase activity (P < 0.05), and increased plasma malondialdehyde content and glutamic-pyruvic transaminase activity (P < 0.05). These negative effects were eliminated by LA supplementation. Comparative proteomic analyses revealed 291 differentially expressed proteins in the AM group compared to the CTRL and AM+LA groups. A total of 30 proteins were differentially expressed between the AM/CTRL and (AM+LA)/AM groups. The addition of LA restored 24 of these proteins to control levels; these proteins were mainly related to transcription regulation, detoxification, protein translation and degradation, and immune and stress responses. The differentially expressed proteins included the high mobility group box (HMGB) and glutathione S-transferase (GST), which is closely related to immune response and oxidative stress, and collagens, which are implicated in liver injury. The addition of LA to broiler diet may reduce ammonia toxicity by maintaining the antioxidant system, xenobiotic metabolism, and metabolic pathways.

Down-regulated expression of Tim-3 promotes invasion and metastasis of colorectal cancer cells.

To explore how Tim-3 is expressed and how its expression influences invasion and metastasis of colorectal cancer (CRC) cells. A total of 188 CRC patients were prospectively collected for this study. Meanwhile, 135 normal controls were incorporated during the same period. Intestinal samples of the CRC radical cancerous tissues, paracancerous tissues ( 5.0 cm beyond the cancer tissue) were collected for the following experiment. Furthermore, peripheral venous blood samples (10 ml) were collected from each subject. Immunohistochemical analysis, quantitative real-time polymerase chain reaction (RT-qPCR) and western blot were performed for the detection of Tim-3 in different tissues. The immunohistochemical staining results showed that a positive Tim-3 signal was localized in the cytoplasm and nucleus, observed as yellow or brown granules. Tim-3 was largely expressed in colon carcinoma tissues and normal colon mucosa tissues but was rarely expressed in the cell membrane. RT-qPCR results indicated that Tim-3 mRNA levels were significantly lower in CRC tissues than in paracancerous tissues and normal colon mucosa tissues. A trend of decreased Tim-3 mRNA levels was also found in the paracancerous tissues compared with the normal colon mucosa tissues (all P < 0.05). Western blot results revealed reduced Tim-3 protein expression in CRC tissues compared with normal colon mucosa tissues and paracancerous tissues, and Tim-3 protein expression was much lower in the paracancerous tissues than in the normal colon mucosa tissues (all P < 0.05). Furthermore, obviously lower Tim-3 mRNA levels were found in the poorly differentiated CRC patients and in those with lymph node metastasis and distant metastasis (all P < 0.05). Collectively, Tim-3 expression was mainly located in the cytoplasm and nucleus, showing down-regulated expression in colon carcinoma tissues compared with normal and paracancerous tissues. Reduced Tim-3 expression may promote CRC invasion and metastasis providing a medical reference for the treatment of CRC.

Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

Rab3A, Rab27A, and Rab35 regulate different events during mouse oocyte meiotic maturation and activation.

Rab family members play important roles in membrane trafficking, cell growth, and differentiation. Almost all components of the cell endomembrane system, the nucleus, and the plasma membrane are closely related to RAB proteins. In this study, we investigated the distribution and functions of three members of the Rab family, Rab3A, Rab27A, and Rab35, in mouse oocyte meiotic maturation and activation. The three Rab family members showed different localization patterns in oocytes. Microinjection of siRNA, antibody injection, or inhibitor treatment showed that (1) Rab3A regulates peripheral spindle and cortical granule (CG) migration, polarity establishment, and asymmetric division; (2) Rab27A regulates CG exocytosis following MII-stage oocyte activation; and (3) Rab35 plays an important role in spindle organization and morphology maintenance, and thus meiotic nuclear maturation. These results show that Rab proteins play important roles in mouse oocyte meiotic maturation and activation and that different members exert different distinct functions.

Ammonia concentration and relative humidity in poultry houses affect the immune response of broilers.

To investigate the effect of ammonia (NH3) and humidity on the immune response of broilers, broilers were exposed to 30 or 70 mg/kg atmospheric NH3 for 21 days. Additionally, birds were exposed to 35, 60, and 85% relative humidity (RH). The relative weights of lymphoid organs, serum total protein, serum globulin, serum albumin, serum lysozyme, proliferation index of peripheral blood lymphocytes, and splenic cytokine gene expression were determined. Exposure to 70 mg/kg NH3 decreased the relative weight of the spleen during the experimental period, serum lysozyme concentration in the first and second weeks, and serum globulin concentration in the third week. The proliferation of peripheral blood lymphocytes was reduced. High levels of NH3 caused increase in IL-1ß gene expression in the experimental period and IL-4 gene expression in the first week. Birds exposed to 85% RH had lower thymus and bursa of Fabricius weights in the third week and serum lysozyme concentration in the first week; IL-1ß and IL-4 expressions were higher in the second and third weeks and first and second weeks, respectively, than in birds exposed to 60% RH. IL-4 expression was lower during the first week, and IL-1ß expression was higher during the second week with 35% RH than with 60% RH. In conclusion, high NH3 level in the poultry house suppressed the immune response of broiler chickens. Neither high nor low RH benefited the immune response of broilers. Furthermore, there was an interactive effect between NH3 and RH on the immune response of broilers.

Germ cell-specific Atg7 knockout results in primary ovarian insufficiency in female mice.

Primary ovarian insufficiency (POI) is a common cause of infertility in around 1-2% of women aged <40 years. However, the mechanisms that cause POI are still poorly understood. Here we showed that germ cell-specific knockout of an essential autophagy induction gene Atg7 led to subfertility in female mice. The subfertility of Atg7 deletion females was caused by severe ovarian follicle loss, which is very similar to human POI patients. Further investigation revealed that germ cell-specific Atg7 knockout resulted in germ cell over-loss at the neonatal transition period. In addition, our in vitro studies also demonstrated that autophagy could protect oocytes from over-loss by apoptosis in neonatal ovaries under the starvation condition. Taken together, our results uncover a new role for autophagy in the regulation of ovarian primordial follicle reservation and hint that autophagy-related genes might be potential pathogenic genes to POI of women.

LNK (SH2B3): paradoxical effects in ovarian cancer.

LNK (SH2B3) is an adaptor protein studied extensively in normal and malignant hematopoietic cells. In these cells, it downregulates activated tyrosine kinases at the cell surface resulting in an antiproliferative effect. To date, no studies have examined activities of LNK in solid tumors. In this study, we found by in silico analysis and staining tissue arrays that the levels of LNK expression were elevated in high-grade ovarian cancer. To test the functional importance of this observation, LNK was either overexpressed or silenced in several ovarian cancer cell lines. Remarkably, overexpression of LNK rendered the cells resistant to death induced by either serum starvation or nutrient deprivation, and generated larger tumors using a murine xenograft model. In contrast, silencing of LNK decreased ovarian cancer cell growth in vitro and in vivo. Western blot studies indicated that overexpression of LNK upregulated and extended the transduction of the mitogenic signal, whereas silencing of LNK produced the opposite effects. Furthermore, forced expression of LNK reduced cell size, inhibited cell migration and markedly enhanced cell adhesion. Liquid chromatography-mass spectroscopy identified 14-3-3 as one of the LNK-binding partners. Our results suggest that in contrast to the findings in hematologic malignancies, the adaptor protein LNK acts as a positive signal transduction modulator in ovarian cancers.

Expression and significance of plasma 3-NT and ox-LDL in patients with Alzheimer's disease.

To examine the expression and clinical significance of plasma 3-nitrotyrosine (3-NT) and oxidized low-density lipoprotein (ox-LDL) levels in patients with Alzheimer's disease (AD), we examined 48 AD patients and 37 healthy control subjects. The Mini-Mental State Examination, Activities of Daily Living Scale, and Hachinski Ischemic Scale were examined in all subjects. AD patients were classified using the Global Deterioration Scale. The concentrations of plasma 3-NT and ox-LDL were detected using an enzyme-linked immunosorbent assay. We found that the plasma 3-NT concentration in the AD group (119.46 ± 21.82 nM) was significantly higher than that in the control group (55.09 ± 9.63 nM) (P < 0.05). Spearman analysis showed that plasma 3-NT level was negatively associated with the Mini-Mental State Examination results of AD patients. Plasma ox-LDL level in the AD group (112.25 ± 17.81 mg/L) was significantly higher than that in the control group (47.46 ± 10.04 mg/L) (P < 0.05). Spearman analysis showed that plasma ox-LDL level was positively correlated with AD severity in AD patients. However, plasma 3-NT level in the AD group was not associated with plasma ox-LDL level. Therefore, plasma 3-NT and ox-LDL levels in AD patients were significantly increased, which may be related to the degree of AD severity in AD patients.