Rapid Diagnosis of Mycobacterium marinum Infection by Next-Generation Sequencing: A Case Report.

We present the first report of histology- and culture-proven Mycobacterium marinum infection diagnosed by next-generation sequencing (NGS). It took <2 days to make a microbiological diagnosis using the Oxford Nanopore Technologies' MinION device, compared to 20 days for the mycobacterium to be isolated from the tissue biopsy. NGS is particularly useful for culture-negative and slow-growing microorganism infections, such as mycobacterial, fungal and partially treated pyogenic bacterial infections. Due to its low equipment cost, short turn-around-time and portable size, the Oxford Nanopore Technologies' MinION device is a useful platform for NGS in routine clinical microbiology laboratories.

Serum nitrite and nitrate: A potential biomarker for post-covid-19 complications?

Nitric oxide (NO) plays an important role in cardiovascular and immune systems. Quantification of blood nitrite and nitrate, two relatively stable metabolites of NO (generally as NOx), has been acknowledged, in part, representing NO bioactivity. Dysregulation of NOx had been reported in SARS-CoV-2 infected populations, but whether patients recovered from COVID-19 disease present with restored NOx is unknown. In this study, serum NO2- and NO3- were quantified and analyzed among 109 recovered adults in comparison to a control group of 166 uninfected adults. Nitrite or nitrate levels were not significantly different among mild-, common-, severe- and critical-type patients. However, these recovered patients had dramatically lower NO2- and NO2-/NO3- than the uninfected group (p < 0.0001), with significantly higher NO3- levels (p = 0.0023) than the uninfected group. Nitrate and nitrite/nitrate were positively and negatively correlated with patient age, respectively, with age 65 being a turning point among recovered patients. These results indicate that low NO2-, low NO2-/NO3- and high NO3- may be potential biomarkers of long-term poor or irreversible outcomes after SARS-CoV-2 infection. It suggests that NO metabolites might serve as a predictor to track the health status of recovered COVID-19 patients, highlighting the need to elucidate the role of NO after SARS-CoV-2 infection.

Urinary NOx, a novel potential biomarker for autism spectrum disorder.

Nitric oxide (NO) participates in many physiological and pathological processes in human. Urine tests tell a lot about health, which are convenient and harmless. Redox stress, including imbalance of reactive nitrogen species and its metabolites NOx, has been gaining increased attention in autism spectrum disorder (ASD) research. However, concentrations of urinary nitrite and nitrate among the ASD population stay unclear. In this study, nitrite and nitrate were precisely measured in urine specimens from 44 ASD children, 30 healthy children (the control group) and 28 healthy adults with an optimized and validated analytic method. For the first time, concentrations of urinary NOx in ASD and healthy children were reported. Nitrite in the ASD population is higher than in the control group, with concentrations of 0.8708 ±â€¯0.1121 µM (0.1556-3.0393 µM) and 0.5938 ±â€¯0.07276 µM (0.1134-2.1004 µM) (p = 0.0420), respectively. Nitrite in the adult groups is 0.5808 ±â€¯0.0985 µM (0.0808-1.9335 µM), which is similar to that in the control group. On the contrary, urinary nitrate concentration in ASD children is lower than that in the control group, which are 2.875 ±â€¯0.2716 mM (0.3264-7.1835 mM) and 4.558 ±â€¯0.5915 mM (1.1860-15.8555 mM) (p = 0.0133), respectively. Nitrate in adults is also significantly lower than that in the control, 2.799 ±â€¯0.3640 mM (0.2507-8.6978 mM) and 4.558 ±â€¯0.5915 mM (p = 0.0146), respectively. Nitrite/nitrate ratios for ASD and the control groups were 0.3496 ±â€¯0.04382 x 10-3 and 0.1604 ±â€¯0.01862 x 10-3 (p = 0.0002), which again indicated the probability of NOx as a novel biomarker. Furthermore, no correlation between NOx and gender, as well as sample collection timing was found. Taken together, the association between NOx and ASD was significant. Urinary nitrite, nitrate and NO2-/NO3-, might serve as a new biomarker for ASD diagnosis during pursuit of harmless, fast, and convenient diagnostic method. Further studies are needed for the metabolic pathways of NOx in ASD pathogenesis.

A Large Bladder Tumor Covered With a Thick "Shell" of Necrotic Material: Misdiagnosis of a Patient With Spina Bifida.

Bladder tumor arising in a spina bifida patient is rare and may be clinically latent. We report the case of a 61-year-old female patient with spina bifida, neurogenic bladder, and a history of recurrent urinary tract infections. A B-ultrasound and non-contrast computed tomography scan did not reveal any bladder mass, but an unexplained "well-filled" bladder was observed, which was confusing as the catheter was present and open. However, a subsequent cystoscopic evaluation revealed a large bladder mass measuring 9.5 × 9.0 × 6.5 cm³, which almost filled the entire bladder. The mass had coarse and flocculent surface and seemed to be free from each observed wall of the urinary bladder. It was diagnosed as an infectious necrotic mass based on its appearance. During transurethral resection of the mass, a bladder tumor was suspected as small blood vessels and bleeding appeared within the inner layer of the mass. Pathological examination revealed necrotic material, inflammatory cells, and urothelial carcinoma cells. Then, a radical cystectomy was performed, and the pathological results indicated stage pT3bN0M0 transitional cell carcinoma. In the gross specimen, the base of the tumor measured 3 × 3 cm² on the top of the back wall of the bladder. Bladder tumors may have atypical presentations in patients with spina bifida. Regular screening is helpful for earlier detection and improving outcomes of bladder tumors in such patients.

Fungus ball and emphysematous cystitis secondary to Candida tropicalis: A case report.

Fungus ball and fungal emphysematous cystitis are two rare complications of fungal urinary tract infection. A 53-year-old male patient presented with these complications caused by Candida tropicalis simultaneously. The predisposing factors were diabetes mellitus and usage of broad-spectrum antibiotics. The fungus ball, measuring 3.5 × 2.0 cm on the left wall of the urinary bladder, shrank significantly to 1.6 × 0.8 cm after 5 days of intermittent irrigation with saline before surgery. With transurethral removal of the fungus ball and antifungal treatment with fluconazole, the patient fully recovered. We conclude that a bladder fungus ball and fungal emphysematous cystitis should always be suspected in patients with diabetes mellitus with uncontrolled funguria and abnormal imaging. Treatment should include a systemic antifungal therapy and thorough surgical removal of the fungus ball. A systemic antifungal therapy combined with a local irrigation with saline or antifungal drugs might help decrease the dissemination of fungemia during an invasive manipulation.

Overexpression of a Novel Tumor Metastasis Suppressor Gene TMSG1/LASS2 Induces Apoptosis via a Caspase-dependent Mitochondrial Pathway.

The tumor metastasis suppressor gene 1 (TMSG1), also designated homo sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2), is a novel tumor metastatic suppressor gene. Although its effects on metastasis have been reported, its biological functions remain unclear. The purpose of this study was to investigate the effects of TMSG1/LASS2 protein on apoptosis and proliferation in human embryonic kidney cell lines HEK293 and 293 T and explore the potential mechanisms. Cell growth, morphology, expressions of apoptotic-related proteins and cell cycle distribution were evaluated in HEK293 and 293 T cells transfected with TMSG1/LASS2 expression plasmids or vector controls. MTT assays showed that overexpression of TMSG1/LASS2 inhibited cell proliferation; and morphological observations and flow cytometric assays with Annexin V/propidium iodide showed TMSG1/LASS2 overexpression increased apoptosis in these cells. Western blot analysis demonstrated that overexpression of TMSG1/LASS2 resulted in the downregulation of Bcl-2, release of cytochrome c from mitochondria, activation of procaspase-9 and procaspase-3, and the cleavage of PARP. Subsequent cell cycle analysis showed that TMSG1/LASS2 overexpression inhibited cell proliferation by mediating the induction of G0/G1 cell cycle arrest. Together, these results confirmed that TMSG1/LASS2 is a potential metastasis suppressor gene, and suggested that the mechanism involved the induction of apoptosis and inhibition of cell proliferation via a caspase-dependent mitochondrial pathway.

A novel tumor metastasis suppressor gene LASS2/TMSG1 interacts with vacuolar ATPase through its homeodomain.

LASS2/TMSG1 was a novel tumor metastasis suppressor gene, which was first cloned by our laboratory from non-metastatic and metastatic cancer cell variants of human prostate carcinoma PC-3M using mRNA differential display in 1999. LASS2/TMSG1 could interact with the C subunit of vacuolar ATPase (V-ATPase, ATP6V0C) and regulate V-ATPase activity. In an attempt to provide molecular mechanism of the interaction between LASS2/TMSG1 and V-ATPase, we constructed four variant transfectants containing different functional domain of LASS2/TMSG1 and stably transfected the variants to human prostate cancer cell line PC-3M-1E8 cell with high metastatic potential. Results showed that there were no obvious differences of V-ATPase expression among different transfected cells and the control. However, V-ATPase activity and intracellular pH was significantly higher in the variant transfectants with Homeodomain of LASS2/TMSG1 than that in the control using the pH-dependent fluorescence probe BECEF/AM. Immunoprecipitation, immunofluorescence and immuno-electron microscope alone or in combination demonstrated the direct interaction of Homeodomain of LASS2/TMSG1 and ATP6V0C. Loss of Homeodomain markedly enhanced the proliferation ability but weakened the apoptotic effect of LASS2/TMSG1 in PC-3M-1E8 cells. These lines of results for the first time contribute to the conclusion that LASS2/TMSG1 could regulate V-ATPase activity and intracellular pH through the direct interaction of its Homeodomain and the C subunit of V-ATPase. Their interaction could play important roles in the apoptosis of tumor cells.

Phosphoprotein associated with glycosphingolipid microdomains 1 inhibits the proliferation and invasion of human prostate cancer cells in vitro through suppression of Ras activation.

Phosphoprotein associated with glycosphingolipid microdomains 1 (PAG) is an important negative regulator of immune signaling in T lymphocytes. However, newly emerging evidence has indicated that PAG may play important roles in tumor cells. Our previously reported cDNA microarray experiments identified PAG as a gene down-regulated in the high metastatic potential prostate cancer cell line PC-3M-1E8. In this study, we investigated the role of PAG in the proliferation, invasion and metastasis of prostate cancer cells and the underlying mechanisms. We confirmed that the expression of PAG at both the mRNA and protein levels was low in PC-3M-1E8 and DU145 cells compared to low metastatic potential prostate cancer cells PC-3M-2B4. In addition, we demonstrated that the reintroduction of PAG to PC-3M-1E8 and DU145 cells led to reduced proliferation through cell cycle arrest, decreased anchorage-independent growth and reduced invasion ability of tumor cells in vitro. This is the first report demonstrating that PAG inhibits the proliferation and invasion potential of prostate cancer cells via the interaction with RasGAP to recruit RasGAP to the cell membrane, where RasGAP hydrolyzes GTP to GDP, reduces the level of activated Ras, and ultimately suppresses the activation of ERK1/2, cyclin D1 and other effectors of the Ras signaling pathway. Morphologically, we observed that PAG could diminish the formation of pseudopodia on the cell surface and redistribute the intracellular F-actin in PC-3M-1E8 cells, which directly leads to the decreased invasion and metastasis potential of tumor cells. Taken together, these results suggest that PAG acts to inhibit the development and metastasis of prostate cancers and represents a novel therapeutic target for prostate cancer.

KLF6/Sp1 initiates transcription of the tmsg-1 gene in human prostate carcinoma cells: an exon involved mechanism.

The tumor metastasis suppressor gene-1 (tmsg-1) was first cloned as a new tumor suppressor gene in our laboratory several years ago. Since then, however, despite the substantial progression that has been made in investigation of the biologic roles played by this gene, the manner in which it exerts its regulatory influence is still unknown. With transfection of various deletion or mutation constructs, we identified a potential enhancer and three potential silencers in the 5'-flanking region. However, it was particularly interesting to find that a region (+59 to +123 bp) of exon 1 exhibited a strong role in initiation of tmsg-1 gene transcription. Deletion of this region led to essentially complete loss of driving activity of exon-1 sequence on luciferase. Further analysis showed that transcription factors KLF6 and Sp1 are able to interact with each other and bind to their elements in this region. Co-transfection of pGL3-114/+123 with KLF6- and/or Sp1-expressing plasmids resulted in an elevation of luciferase activity and transcription level of tmsg-1, which was abolished by knockdown of KLF6 or Sp1. Analysis of metastatic capacity showed that cells with high metastatic capability exhibited a lower level of KLF6/TMSG-1 proteins with higher invasive capability and vice versa. Thus, we concluded that interaction of KLF6 and Sp1, together with their binding of elements in exon 1 are critical events in initiation of transcription of the tmsg-1 gene. These results reveal a hitherto unreported mechanism for initiation of transcription of the tmsg-1 gene.

[Transcriptional activation of TMSG-1 by complex of KLF6 and Sp1].

OBJECTIVE: To investigate the regulatory mechanism of the transcription of tumor metastasis suppressor gene TMSG-1. METHODS: Luciferase reporter assay and site-directed mutagenesis were used to analyze the regulatory region of TMSG-1. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were carried out to verify the interaction of KLF6 and Sp1 with the regulatory region of TMSG-1. Co-immunoprecipitation (CoIP) was performed to analyze the interaction between KLF6 and Sp1. TMSG-1 and wt-KLF6 mRNA expressions in cells with different metastatic capacities were quantitated by real-time PCR. Cell invasive capability was determined by Matrigel invasion assay. RESULTS: A 63 bp inducible regulatory region (+59 bp - +123 bp) in exon 1 was identified by luciferase assay using reporter plasmids with a series of TMSG-1 regulatory region deletions. Mutations in KLF6/Sp1 binding sites of this region resulted in a decrease of luciferase activity, while cotransfection with KLF6 or Sp1 expressing plasmids led to a remarkable increase of luciferase activity. EMSA and ChIP demonstrated that KLF6 as well as Sp1 interacted with this region. CoIP also indicated a possible interaction between KLF6 and Sp1 proteins. In the highly metastatic cell sublines, a low level of wild type KLF6 was associated synchronously with a low TMSG-1 level. Prostate carcinoma cells overexpressing KLF6 exhibited a higher TMSG-1 level and a lower invasive capability. CONCLUSIONS: Transcription factor complex of KLF6 and Sp1 may participate in the inducible transcriptional regulation of TMSG-1, and a decreased wild type KLF6 expression is likely associated with a low TMSG-1 level in the highly metastatic cell sublines.

[Identification of nucleolar localization signal sequence of tumor metastasis suppressor gene-1].

OBJECTIVE: To identify the putative specific localization signal sequence of tumor metastasis suppressor gene-1 (TMSG-1) and to explore the mechanism of subcellular localization of TMSG-1 protein. METHODS: Vectors expressing green fluorescence protein (GFP) tagged different TMSG-1 fragments were generated and transfected into human embryo kidney 293 (HEK293) cells. The expression of those fusion proteins was detected by Western blotting and their subcellular localizations were observed by laser confocal microscope. RESULTS: GFP was fused with the native TMSG-1(aa1-380) or different fragments including T1 (aa1-70), T2 (aa1-128), T3 (aa129-380), T4 (aa71-128), T5 (aa71-179) and T6 (aa71-380). Anti-GFP Western blotting showed that these fusion proteins were successfully expressed. Under laser confocal microscope, GFP fused with fragment T4 (aa71-128) localized mainly in the nucleolus; GFP fused with fragment T6 (aa71-380) localized diffusely in the nucleus; while other fusion proteins with TMSG-1 (aa1-380) or fragment T1 (aa1-70), T2 (aa1-128), T3 (aa129-380) and T5 (aa71-179) localized in the cytoplasm. Fragment T4(Δ119-128) was generated from T4 with deletion of 10 amino acid of the C terminal. GFP fused with fragment T4(Δ119-128) remained in the nucleus, but no longer in the nucleolus. CONCLUSIONS: There is a nucleolar localization signal (aa119-128 RRRRNQDRPS) within TMSG-1. This finding may have laid the foundation for further investigations into subcellular localization and function of TMSG-1.