BackgroundMore than 100 different pathogens can cause encephalitis. Testing of all the neurological pathogens by conventional methods can be difficult. Metagenomic next-generation sequencing (NGS) could identify the infectious agents in a target-independent manner. The role of this novel method in clinical diagnostic microbiology still needs to be evaluated. In present study, we used metagenomic NGS to search for an infectious etiology in a human immunodeficiency virus (HIV)-infected patient with lethally diffuse brain lesions. Sequences mapping to Toxoplasma gondii were unexpectedly detected.Case presentationA 31-year-old HIV-infected patient presented to hospital in a critical ill condition with a Glasgow coma scale score of 3. Brain magnetic resonance imaging showed diffuse brain abnormalities with contrast enhancement. Metagenomic NGS was performed on DNA extract from 300 μL patient’s cerebrospinal fluid (CSF) with the BGISEQ-50 platform. The sequencing detection identified 65,357 sequence reads uniquely aligned to the Toxoplasma gondii genome. Presence of Toxoplasma gondii genome in CSF was further verified by Toxoplasma gondii-specific polymerase chain reaction and Sanger sequencing. Altogether, those results confirmed the diagnosis of toxoplasmic encephalitis.ConclusionsThis study suggests that metagenomic NGS may be a useful diagnostic tool for toxoplasmic encephalitis. As metagenomic NGS is able to identify all pathogens in a single run, it may be a promising strategy to explore the clinical causative pathogens in central nervous system infections with atypical features.

Chronic-relapsing varicella zoster meningitis -Successfully treated with varicella vaccineDear Editor , We note with interest recent reports on the problems of diagnosing central nervous system (CNS) infections, 1 particularly the study comparing the presence of varicella zoster virus (VZV) DNA in blood and cerebrospinal fluid (CSF) in patients with VZV-related neurological symptoms. 2 Here we report a case of chronic-relapsing VZV meningitis, where the episodes occurred with frequent and debilitating regularity.A 62-year-old man, who worked as a General Practitioner at an asylum centre, presented with a 2-year history of headache, fatigue, diffuse myalgia, a low-grade fever, night sweats and mood disturbances. His symptoms waxed and waned every 1-3 weeks, without ever fully resolving, but never included seizures or focal neurological symptoms. He had lost 9 kg during this period. His work routinely exposed him to patients with various exotic and chronic infections, including a varicella outbreak 2 years previously.General physical examination was unremarkable, apart from some limb myalgia. Specifically, neither neck stiffness nor any other focal neurological signs were detected. Other investigations (chest X-ray, echocardiography, abdominal ultrasound) were all normal. An MRI-brain showed some non-specific lesions in the deep white matter, compatible with Virchow-Robin spaces.Full blood counts with differentiation, f erritin, lipid profile, thyroid stimulating hormone, CA19-9, liver function, serum glucose, urea and electrolytes and erythrocyte sedimentation rate were all normal, apart from a mild non-specific anaemia. Serological testing for HIV, cytomegalovirus (CMV), Epstein-Barr virus, herpes simplex virus (HSV), Brucella, Borrelia, Yersinia, Toxoplasma , and Mycoplasma pneumoniae were either negative or just indicated past infection.Cerebrospinal fluid examination revealed a slightly raised total protein (0.66 g/L) and a low glucose (1.9 mmol/L) with a glucose ratio of 0.44. No erythrocytes were seen, but 95 × 10 6 /L leucocytes were observed (primarily lymphocytes and monocytes). Polymerase chain reaction testing for CSF HSV and VZV was negative. Further CSF investigations using immuno-blotting showed intense IgG bands in the CSF, with fewer, weaker bands in a contemporaneous serum sample ( Fig. 1 ): CSF intrathecal VZV IgG titre was 2500, with a VZV-IgG index of 98.52, indicating intrathecal VZV-IgG synthesis.A diagnosis of chronic-relapsing VZV meningitis was made, and a 2-week course of intravenous acyclovir (10 0 0 mg TD) was given. The patient improved on treatment, but the meningitis symptoms recurred every 2 weeks once treatment had stopped.Further, repeated CSF examinations, during symptomatic and asymptomatic periods, showed similar profiles to the above. Con-

Background UFM1 has been found to be involved in the regulation of tumor development. This study aims to clarify the role and potential molecular mechanisms of UFM1 in the invasion and metastasis of gastric cancer. Methods Expression of UFM1 in gastric tumor and paired adjacent noncancerous tissues from 437 patients was analyzed by Western blotting, immunohistochemistry, and realtime PCR. Its correlation with the clinicopathological characteristics and prognosis of gastric cancer patients was analyzed. The effects of UFM1 on the invasion and migration of gastric cancer cells were determined by the wound and trans-well assays, and the effect of UFM1 on subcutaneous tumor formation was verified in nude mice. The potential downstream targets of UFM1 and related molecular mechanisms were clarified by the human protein kinase assay and co-immunoprecipitation technique. Results Compared with the corresponding adjacent tissues, the transcription level and protein expression level of UFM1 in gastric cancer tissues were significantly downregulated (P < 0.05). The 5-year survival rate of gastric cancer patients with low UFM1 expression was significantly lower than the patients with high UFM1 expression (42.1% vs 63.0%, P < 0.05). The invasion and migration abilities of gastric cancer cells with stable UFM1 overexpression were significantly decreased, and the gastric cancer cells with UFM1 stable knockdown showed the opposite results; similar results were also obtained in the nude mouse model. Further studies have revealed that UFM1 could increase the ubiquitination level of PDK1 and decrease the expression of PDK1 at protein level, thereby inhibiting the phosphorylation level of AKT at Ser473. Additionally, the effect of UFM1 on gastric cancer cell function is dependent on the expression of PDK1. The expression level of UFM1 can improve the poor prognosis of PDK1 in patients with gastric cancer. Conclusion UFM1 suppresses the invasion and metastasis of gastric cancer by increasing the ubiquitination of PDK1 through negatively regulating PI3K/AKT signaling.

Background Acinetobacter baumannii is a gram-negative aerobic bacillus that is commonly causes of hospital-acquired infections. Community-acquired pneumonia caused by Acinetobacter baumannii (CAP-Ab) is rare but fatal if diagnosis and treatment are delayed. Conventional culture of clinical specimens is the main method for clinical diagnosis of A. baumannii infections which may suffer from limited positive rate and is time consuming. Timely and precise diagnosis of CAP-Ab remains challenging. Case presentation A 66-year-old man with 24 h history of acute fever and dyspnea was admitted to our hospital. He was diagnosed as severe community acquired pneumonia (CAP), septic shock, respiratory failure and acute kidney injury. Next-generation sequencing (NGS) was performed on the patient’s sputum and blood, which identified numerous A. baumannii nucleotide sequences in the sample of sputum and led to the rapid diagnosis and treatment of community acquired pneumonia caused by A. baumannii. This result was confirmed by subsequent sputum culture. Conclusions This case described that the successful application of the next generation sequencing assisting the speedy diagnosis of A. baumannii infection provides a new idea for the timely diagnosis of CAP-Ab and highlights that NGS is a promising tool in rapid etiological diagnosis of acute and severe infectious diseases.

Cyclin-dependent kinase 5 regulatory subunitassociated protein 3 (CDK5RAP3 or C53) is involved in the development of various types of tumor, and alternative splicing of C53 results in numerous transcription variants that encode different isoforms. The present study aimed to clone human C53 isoform d (IC53d) and explore its role in the proliferation of gastric cancer cells. Reverse transcription-quantitative polymerase chain reaction was used to detect the expression levels of IC53d in 80 primary gastric adenocarcinoma tissues and adjacent normal tissues. In addition, the association between IC53d and clinicopathological parameters was determined. Gastric cancer cell lines stably overexpressing IC53d were established to observe its effects on cell proliferation, invasion and migration, and on in vivo tumorigenicity, and the mechanism of action was explored. The results of the presen study demonstrated that IC53d was upregulated in gastric cancer tissues and was associated with tumor T-stage. Furthermore, overexpression of IC53d promoted the proliferation, colony formation and G 1 /S phase transition of gastric cancer cells, leading to enhancement of tumorigenesis in vitro and in vivo. Overexpression of IC53d also promoted phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3β (GSK3β), which increased the expression of cyclin D1. In addition, high cyclin D1 expression was associated with a significantly worse prognosis for patients compared with in patients with low cyclin D1 expression. These results indicated that IC53d may promote the phosphorylation of AKT and GSK3β, which in turn may increase cyclin D1 expression, enhancing G 1 /S phase transition, accelerating cell cycle progression, promoting the proliferation of gastric cancer cells, and inducing a poor prognosis in patients with gastric cancer.

No abstract

Background: To investigate the feasibility of a knowledge-based automated intensity-modulated radiation therapy (IMRT) planning technique for locally advanced nasopharyngeal carcinoma (NPC) radiotherapy. Methods: One hundred forty NPC patients treated with definitive radiation therapy with the step-and-shoot IMRT techniques were retrospectively selected and separated into a knowledge library (n = 115) and a test library (n = 25). For each patient in the knowledge library, the overlap volume histogram (OVH), target volume histogram (TVH) and dose objectives were extracted from the manually generated plan. 5-fold cross validation was performed to divide the patients in the knowledge library into 5 groups before validating one group by using the other 4 groups to train each neural network (NN) machine learning models. For patients in the test library, their OVH and TVH were then used by the trained models to predict a corresponding set of mean dose objectives, which were subsequently used to generate automated plans (APs) in Pinnacle planning system via an in-house developed automated scripting system. All APs were obtained after a single step of optimization. Manual plans (MPs) for the test patients were generated by an experienced medical physicist strictly following the established clinical protocols. The qualities of the APs and MPs were evaluated by an attending radiation oncologist. The dosimetric parameters for planning target volume (PTV) coverage and the organs-at-risk (OAR) sparing were also quantitatively measured and compared using Mann-Whitney U test and Bonferroni correction. Results: APs and MPs had the same rating for more than 80% of the patients (19 out of 25) in the test group. Both AP and MP achieved PTV coverage criteria for no less than 80% of the patients. For each OAR, the number of APs achieving its criterion was similar to that in the MPs. The AP approach improved planning efficiency by greatly reducing the planning duration to about 17% of the MP (9.85 ± 1.13 min vs. 57.10 ± 6.35 min).

BackgroundAngiogenesis plays critical roles in the progression and metastasis of malignant tumors. Gastric neuroendocrine carcinoma is an uncommon stomach cancer that is rich in blood vessels and exhibits highly malignant biological behavior with a poor prognosis. The role of CDK5RAP3 in GNEC has not been reported to date.MethodsImmunohistochemistry was used to assess the expression of CDK5RAP3 in GNEC tissues and adjacent non-tumor tissues. Cell lines with stable overexpression or knockdown of CDK5RAP3 were constructed using lentiviral transfection. Wound-healing assays, invasion and metastasis assays, tube formation assays, and tumor xenograft transplantation assays were performed to evaluate the effect of CDK5RAP3 on GNEC angiogenesis in vitro and in vivo. Real-time PCR, ELISA, western blot analysis, and confocal-immunofluorescence staining were used to explore the molecular mechanism of CDK5RAP3′s effect on angiogenesis.ResultsCompared with their respective adjacent non-tumor tissues, protein levels of CDK5RAP3 were significantly decreased in GNEC tissues. Furthermore, low expression of CDK5RAP3 was correlated with more advanced TNM stage, increased tumor microvessel density, and poor prognosis. Functionally, we found that GNEC cells with CDK5RAP3 knockdown promoted human umbilical vein endothelial cells migration and tube formation via activation of AKT/HIF-1α/VEGFA signaling, resulting in increased levels of VEGFA in GNEC cell supernatant. In addition, CDK5RAP3 overexpression in GNEC cells caused the opposing effect. Consistent with these results, nude mouse tumorigenicity assays showed that CDK5RAP3 expression downregulated angiogenesis in vivo. Lastly, patients with low CDK5RAP3 expression and high VEGFA expression exhibited the worst prognosis.ConclusionsThis study demonstrated that CDK5RAP3 inhibits angiogenesis by downregulating AKT/HIF-1α/VEGFA signaling in GNEC and improves patient prognosis, suggesting that CDK5RAP3 could be a potential therapeutic target for GNEC.