Background Accumulating evidence shows an over-abundance of Fusobacterium nucleatum in colorectal tumour tissues. Although stool DNA testing of Fusobacterium nucleatum might be a potential marker for ...the detection of colorectal tumours, the difficulty in detecting Fusobacterium nucleatum in stool by conventional methods prevented further explorations. Therefore, we developed a droplet digital polymerase chain reaction (PCR) assay for detecting Fusobacterium nucleatum in stool and investigated its clinical utility in the management of colorectal tumours in a Japanese population. Methods Feces were collected from 60 healthy subjects (control group) and from 11 patients with colorectal non-advanced adenomas (non-advanced adenoma group), 19 patients with colorectal advanced adenoma/carcinoma in situ (advanced adenoma/carcinoma in situ (CIS) group) and 158 patients with colorectal cancer of stages I to IV (colorectal cancer group). Absolute copy numbers of Fusobacterium nucleatum were measured by droplet digital PCR. Results The median copy number of Fusobacterium nucleatum was 17.5 in the control group, 311 in the non-advanced adenoma group, 122 in the advanced adenoma/CIS group, and 317 in the colorectal cancer group. In comparison with that in the control group, the Fusobacterium nucleatum level was significantly higher in the non-advanced adenoma group, the advanced adenoma/CIS group and the colorectal cancer group. Conclusions This study illustrates the potential of stool DNA testing of Fusobacterium nucleatum by droplet digital PCR to detect individuals with colorectal tumours in a Japanese population.
We monitored Chlamydia trachomatis growth in HeLa cells cultured with either DMEM or RPMI medium containing 10% FCS under 2% or 21% O2 conditions for 2 days. Bacterial numbers, host cell numbers, and ...fibrosis-related gene expression in the host cells were estimated by an inclusion forming unit assay, a cell counting assay, and a PCR array, respectively. In contrast to RPMI, bacterial growth under low oxygen conditions in DMEM rapidly decreased with increasing host cell density. The addition of supplements (glucose, glutamine, vitamin B12, D-biotin, non-essential amino acids, glutathione) to the media had no effect. The growth of host cells in DMEM under low oxygen conditions rapidly decreased, although the cells remained healthy morphologically. Furthermore, the downregulation of 17 genes was observed under low oxygen in DMEM. Whereas no effect on bacterial growth was observed when culturing in RPMI medium at low oxygen, and the downregulation of three genes (CTGF, SERPINE1, JUN) was observed following bacterial infection compared with the uninfected control cells. Thus, our findings indicate the need for carefully selected culture conditions when performing experiments with C. trachomatis under low-oxygen environments, and RPMI (rather than DMEM) is recommended when a low host cell density is to be used, proposing the major modification of cell culturing method of C. trachomatis in a low-oxygen environment.
•C. trachomatis growth is dampened in DMEM medium compared with RPMI medium, in 2% oxygen environment.•Subtle changes to host cell density under low-oxygen conditions is detrimental to the growth in 2% oxygen environment.•When performing experiments with C. trachomatis under low-oxygen environments, RPMI (rather than DMEM) is recommended.
Rac1, one of the Rho family small guanosine triphosphatases, has been shown to work as a "molecular switch" in various signal transduction pathways. To assess the function of Rac1 in the ...differentiation process of CD4 single-positive (CD4-SP) T cells from CD4CD8 double-positive (DP) cells, we used a DP cell line DPK, which can differentiate into CD4-SP cells upon TCR stimulation in vitro. DPK expressing dominant-negative (dn)Rac1 underwent massive apoptosis upon TCR stimulation and resulted in defective differentiation of CD4-SP cells. Conversely, overexpression of dnRac2 did not affect differentiation. TCR-dependent actin polymerization was inhibited, whereas early ERK activation was unaltered in dnRac1-expressing DPK. We found that TCR-dependent induction of Bcl-2 was suppressed greatly in dnRac1-expressing DPK, and this suppression was independent of actin rearrangement. Furthermore, introduction of exogenous Bcl-2 inhibited TCR-dependent induction of apoptosis and restored CD4-SP generation in dnRac1-expressing DPK without restoring TCR-induced actin polymerization. Collectively, these data indicate that Rac1 is critical in differentiation of CD4-SP from the DP cell line by preventing TCR-induced apoptosis via Bcl-2 up-regulation.
Background
We previously reported overexpression of heat-shock protein (HSP) 70 in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) using proteomic profiling and immunohistochemical ...staining (IHS). This suggested that HSP70 could be a molecular target for treatment of HCC.
Methods
Twelve patients with HCV-related HCC were enrolled in a phase 1 clinical trial. Dendritic cells (DCs) transfected with HSP70 mRNA (HSP70-DCs) induced by electroporation were injected intradermally. Patients were treated three times every 3 weeks. The number of HSP70-DCs injected was 1 × 10
7
as the lowest dose, then 2 × 10
7
as the medium dose, and then 3 × 10
7
as the highest dose. Immunological analyses were performed.
Findings
No adverse effects of grade III/IV, except one grade III liver abscess at the 3 × 10
7
dose, were observed. Thus, we added three more patients to confirm whether 3 × 10
7
is an appropriate dose. Eventually, we chose 3 × 10
7
as the recommended dose of DCs. Complete response (CR) without any recurrence occurred in two patients, stable disease in five, and progression of disease in five. The two patients with CR have had no recurrence for 44 and 33 months, respectively. IHS in one patient who underwent partial hepatectomy showed infiltration of CD8+ T cells and granzyme B in tumors, indicating that the dominant immune effector cells were cytotoxic T lymphocytes with tumor-killing activity.
Interpretation
This study demonstrated that HSP70-DCs therapy is both safe and feasible in patients with HCV-related HCC. Further clinical trials should be considered.
Phage tail fibres are elongated protein assemblies capable of specific recognition of bacterial surfaces during the first step of viral infection
. The folding of these complex trimeric structures ...often requires a phage-encoded tail fibre assembly (Tfa) protein
. Despite the wide occurrence of Tfa proteins, their functional mechanism has not been elucidated. Here, we investigate the tail fibre and Tfa of Escherichia coli phage Mu. We demonstrate that Tfa forms a stable complex with the tail fibre, and present a 2.1 Å resolution X-ray crystal structure of this complex. We find that Tfa proteins are comprised of two domains: a non-conserved N-terminal domain that binds to the C-terminal region of the fibre and a conserved C-terminal domain that probably mediates fibre oligomerization and assembly. Tfa forms rapidly exchanging multimers on its own, but not a stable trimer, implying that Tfa does not specify the trimeric state of the fibre. We propose that the key conserved role of Tfa is to ensure that fibre assembly and multimerization initiates at the C terminus, ensuring that the intertwined and repetitive structural elements of fibres come together in the correct sequence. The universal importance of correctly aligning the C termini of phage fibres is highlighted by our work.
Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive ...immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85α regulatory subunit) are impaired in nitric oxide (NO) production upon lipopolysaccharide and interferon-γ stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.
Tumor necrosis factor receptor superfamily member 19 (
) is involved in the Wnt/β-catenin signaling pathway and interacts with leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5), ...which is a well-known biomarker of cancer stem cells and a prognostic marker of colorectal cancer (CRC). Because there have been no studies to evaluate the prognostic significance of
, we performed the present study to determine whether
can be a prognostic biomarker in CRC patients. We evaluated
expression levels in 100 CRC tissues by quantitative real-time PCR and investigated the association of
expression levels with clinicopathologic features. Cancer stage and
expression level were found to be independent prognostic factors of disease-free survival. Moreover,
overexpression was the sole independent prognostic factor of disease-free survival in patients with stage II and III CRC. These results suggest that analysis of
might help predict clinical outcome in patients with CRC. To support our findings, confirmatory studies using independent data sets are needed.
We studied the comprehensive
DNA
methylation status in the naturally derived gastric adenocarcinoma cell line
SNU
‐719, which was infected with the
E
pstein–
B
arr virus (
EBV
) by methylated
C
p
G
...island recovery on chip assay. To identify genes specifically methylated in
EBV
‐associated gastric carcinomas (
EBV
a
GC
), we focused on seven genes,
TP73
,
BLU
,
FSD1
,
BCL7A
,
MARK1
,
SCRN1
, and
NKX3.1
, based on the results of methylated
C
p
G
island recovery on chip assay. We confirmed
DNA
methylation of the genes by methylation‐specific
PCR
and bisulfite sequencing in
SNU
‐719. The expression of the genes, except for
BCL7A
, was upregulated by a combination of 5‐
A
za‐2′‐deoxycytidine and trichostatin A treatment in
SNU
‐719. After the treatment, unmethylated
DNA
became detectable in all seven genes by methylation‐specific
PCR
. We verified
DNA
methylation of the genes in 75 primary gastric cancer tissues from 25 patients with
EBV
a
GC
and 50
EBV
‐negative patients who were controls. The methylation frequencies of
TP73
,
BLU
,
FSD1
,
BCL7A
,
MARK1
,
SCRN1
, and
NKX3.1
were significantly higher in
EBV
a
GC
than in
EBV
‐negative gastric carcinoma. We identified seven genes with promoter regions that were specifically methylated in
EBV
a
GC
. Inactivation of these genes may suppress their function as tumor suppressor genes or tumor‐associated antigens and help to develop and maintain
EBV
a
GC
.
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