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Abstract Background Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. Methods Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. Results Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. Conclusions Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

Cancer immunotherapy has transformed the landscape of adult cancer treatment and holds a great promise to treat paediatric malignancies. However, in vitro test coculture systems to evaluate the efficacy of immunotherapies on representative paediatric tumour models are lacking. Here, we describe a detailed procedure for the establishment of an ex vivo test coculture system of paediatric tumour organoids and immune cells that enables assessment of different immunotherapy approaches in paediatric tumour organoids. We provide a step-by-step protocol for an efficient generation of patient-derived diffuse intrinsic pontine glioma (DIPG) and neuroblastoma organoids stably expressing eGFP-ffLuc transgenes using defined serum-free medium. In contrast to the chromium-release assay, the new platform allows for visualization, monitoring and robust quantification of tumour organoid cell cytotoxicity using a non-radioactive assay in real-time. To evaluate the utility of this system for drug testing in the paediatric immuno-oncology field, we tested our in vitro assay using a clinically used immunotherapy strategy for children with high-risk neuroblastoma, dinutuximab (anti-GD2 monoclonal antibody), on GD2 proficient and deficient patient-derived neuroblastoma organoids. We demonstrated the feasibility and sensitivity of our ex vivo coculture system using human immune cells and paediatric tumour organoids as ex vivo tumour models. Our study provides a novel platform for personalized testing of potential anticancer immunotherapies for aggressive paediatric cancers such as neuroblastoma and DIPG.

Escherichia coli uses allantoin as the sole nitrogen source during anaerobic growth. In the final step of allantoin degradation, oxamic transcarbamylase (OXTCase) converts oxalurate to carbamoyl phosphate (CP) and oxamate. The activity of this enzyme was first measured in Streptococcus allantoicus in the 1960s, but no OXTCase enzyme or the encoding gene(s) have been found in any strain. This study discovered that allFGH ( fdrA , ylbE , and ylbF ) are the genes that encode the global orphan enzyme OXTCase. The three genes form an operon together with allK ( ybcF ), encoding catabolic carbamate kinase. The allFGHK operon is located directly downstream of the allECD operon that encodes enzymes for the preceding steps of OXTCase. The OXTCase kinetic parameters were analyzed using the purified protein composed of AllF-AllG-AllH (FdrA-YlbE-YlbF); for the substrate CP, K M and V max were 1.3 mM and 15.4 U/mg OXTCase, respectively, and for the substrate oxamate, they were 36.9 mM and 27.0 U/mg OXTCase. In addition, the OXTCase encoded by the three genes is a novel transcarbamylase that shows no similarity with known enzymes of the transcarbamylase family such as aspartate transcarbamylase, ornithine transcarbamylase, and YgeW transcarbamylase. The present study elucidated the anaerobic allantoin degradation pathway of E. coli . Therefore, we suggest that the genes fdrA , ylbE , and ylbF are renamed allF , allG , and allH , respectively.IMPORTANCEThe anaerobic allantoin degradation pathway of Escherichia coli includes a global orphan enzyme, oxamic transcarbamylase (OXTCase), which converts oxalurate to carbamoyl phosphate and oxamate. This study found that the allFGH ( fdrA , ylbE , and ylbF ) genes encode OXTCase. The OXTCase activity and kinetics were successfully determined with purified recombinant AllF-AllG-AllH (FdrA-YlbE-YlbF). This OXTCase is a novel transcarbamylase that shows no similarity with known enzymes of the transcarbamylase family such as aspartate transcarbamylase (ATCase), ornithine transcarbamylase (OTCase), and YgeW transcarbamylase (YTCase). In addition, OXTCase activity requires three genes, whereas ATCase is encoded by two genes, and OTCase and YTCase are encoded by a single gene. The current study discovered OXTCase, the last unknown step in allantoin degradation, and this enzyme is a new member of the transcarbamylase group that was previously unknown., Competing Interests: The authors declare no conflict of interest.

Anaerobic bacteria often use antiporters DcuB (malate/succinate antiport) or DcuA (l-aspartate/succinate antiport) for the excretion of succinate during fumarate respiration. The rumen bacterium Actinobacillus succinogenes is able to produce large amounts of succinate by fumarate respiration, using the DcuB-type transporter DcuE for l-malate/succinate antiport. Asuc_0142 was annotated as a second DcuB-type transporter. Deletion of Asuc_0142 decreased the uptake rate for l-[ 14 C]aspartate into A. succinogenes cells. Properties of transport by heterologously expressed Asuc_0142 were investigated in an Escherichia coli mutant deficient of anaerobic C4DC transporters. Expression of Asuc_0142 resulted in high uptake activity for l-[ 14 C]fumarate or l-[ 14 C]aspartate, but the former showed a strong competitive inhibition by l-aspartate. In E. coli loaded with l-[ 14 C]aspartate, [ 14 C]succinate or [ 14 C]fumarate, extracellular C4DCs initiated excretion of the intracellular substrates, with a preference for l-aspartate ex /succinate in or l-aspartate ex /fumarate in antiport. These findings indicate that Asuc_0142 represents a DcuA-type transporter for l-aspartate uptake and l-aspartate ex /C4DC in antiport, differentiating it from the DcuB-type transporter DcuE for l-malate ex /succinate in antiport. Sequence analysis and predicted structural characteristics confirm structural similarity of Asuc_0142 to DcuA, and Asuc_0142 was thus re-named as DcuA As . The bovine rumen fluid contains l-aspartate (99.6 µM), whereas fumarate and l-malate are absent. Therefore, bovine rumen colonisers depend on l-aspartate as an exogenous substrate for fumarate respiration. A. succinogenes encodes HemG (protoporphyrinogen oxidase) and PyrD (dihydroorotate dehydrogenase) for haem and pyrimidine biosynthesis. The enzymes require fumarate as an electron acceptor, suggesting an essential role for l-aspartate, DcuA As , and fumarate respiration for A. succinogenes growing in the bovine rumen.

Escherichia coli can use allantoin as its sole nitrogen source under anaerobic conditions. The ureidoglycolate produced by double release of ammonia from allantoin can flow into either the glyoxylate shunt or further catabolic transcarbamoylation. Although the former pathway is well studied, the genes of the latter (catabolic) pathway are not known. In the catabolic pathway, ureidoglycolate is finally converted to carbamoyl phosphate (CP) and oxamate, and then CP is dephosphorylated to carbamate by a catabolic carbamate kinase (CK), whereby ATP is formed. We identified the ybcF gene in a gene cluster containing fdrA - ylbE - ylbF - ybcF that is located downstream of the allDCE -operon. Reverse transcription PCR of total mRNA confirmed that the genes fdrA , ylbE , ylbF , and ybcF are co-transcribed. Deletion of ybcF caused only a slight increase in metabolic flow into the glyoxylate pathway, probably because CP was used to de novo synthesize pyrimidine and arginine. The activity of the catabolic CK was analyzed using purified YbcF protein. The V max is 1.82 U/mg YbcF for CP and 1.94 U/mg YbcF for ADP, and the K M value is 0.47 mM for CP and 0.43 mM for ADP. With these results, it was experimentally revealed that the ybcF gene of E. coli encodes catabolic CK, which completes anaerobic allantoin degradation through substrate-level phosphorylation. Therefore, we suggest renaming the ybcF gene as allK .

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here, the interaction of fumarase FumB, aspartase AspA, and L-tartrate dehydratase TtdAB with the C4-dicarboxylate (C4-DC) transporters DcuA, DcuB, DcuC, and the L-tartrate transporter TtdT of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography using mSPINE (membrane Streptavidin protein interaction experiment). From the general C4-DC transporters, DcuB interacted with FumB and AspA, DcuA with AspA, whereas DcuC interacted with neither FumB nor AspA. Moreover, TtdT did not interact with TtdAB. The fumB-dcuB, the dcuA-aspA, and the ttdAB-ttdT genes encoding the respective proteins colocalize on the genome and each pair of genes forms cotranscripts, whereas the dcuC gene lies alone. The data suggest the formation of DcuB/FumB and DcuB/AspA metabolons for the uptake of L-malate, or L-aspartate, and their conversion to fumarate for fumarate respiration and excretion of the product succinate. The DcuA/AspA metabolon catalyzes uptake and conversion of L-aspartate to fumarate coupled to succinate excretion. The DcuA/AspA metabolon provides ammonia at the same time for nitrogen assimilation (ammonia shuttle). On the other hand, TtdT and TtdAB are not organized in a metabolon. Reasons for the formation (DcuA/AspA, DcuB/FumB, and DcuB/AspA) or nonformation (DcuC, TtdT, and TtdAB) of metabolons are discussed based on their metabolic roles., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Wild-type Escherichia coli was adapted to syntrophic growth with Methanobacterium formicicum for glycerol fermentation over 44 weeks. Succinate production by E. coli started to increase in the early stages of syntrophic growth. Genetic analysis of the cultured E. coli population by pooled sequencing at eight time points suggests that (i) rapid evolution occurred through repeated emergence of mutators that introduced a large number of nucleotide variants and (ii) many mutators increased to high frequencies but remained polymorphic throughout the continuous cultivation. The evolved E. coli populations exhibited gains both in fitness and succinate production, but only for growth under glycerol fermentation with M. formicicum (the condition for this laboratory evolution) and not under other growth conditions. The mutant alleles of the 69 single nucleotide polymorphisms (SNPs) identified in the adapted E. coli populations were constructed individually in the ancestral wild-type E. coli. We analyzed the phenotypic changes caused by 84 variants, including 15 nonsense variants, and found that FdrA D296Y was the most significant variant leading to increased succinate production. Transcription of fdrA was induced under anaerobic allantoin degradation conditions, and FdrA was shown to play a crucial role in oxamate production. The FdrA D296Y variant increased glyoxylate conversion to malate by accelerating oxamate production, which promotes carbon flow through the C4 branch, leading to increased succinate production. IMPORTANCE Here, we demonstrate the ability of E. coli to perform glycerol fermentation in coculture with the methanogen M. formicicum to produce succinate. We found that the production of succinate by E. coli significantly increased during successive cocultivation. Genomic DNA sequencing, evaluation of relative fitness, and construction of SNPs were performed, from which FdrA D296Y was identified as the most significant variant to enable increased succinate production by E. coli. The function of FdrA is uncertain. In this study, experiments with gene expression assays and metabolic analysis showed for the first time that FdrA could be the "orphan enzyme" oxamate:carbamoyltransferase in anaerobic allantoin degradation. Furthermore, we demonstrate that the anaerobic allantoin degradation pathway is linked to succinate production via the glyoxylate pathway during glycerol fermentation.

Cyclo(Phe-Pro) (cFP), produced by the Vibrio species, plays the dual roles of being a signaling molecule and a virulence factor. Acting modes of this compound have recently been characterized at the molecular level. Nevertheless, the method by which this compound passes across biological membranes remains obscure. Using radiolabeled cFP, we examined the kinetics of transport for this compound across membranes using V. vulnificus, Escherichia coli, and sheep red blood cells. We observed that cFP was taken up by these cells in a concentration-dependent manner and was not affected by the addition of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), suggesting that cFP is taken up by passive transport. The kinetics of uptake of cFP by the above three types of cells revealed no significant differences, indicating that no specific protein is involved in this process. When the intracellular accumulation of cFP in the tested cells was measured, the concentrations did not exhibit significant differences between the 1-min and 10-min time points after cFP was added to the culture. In contrast, the intracellular concentration of fumarate, which is well known to be taken up by cells via active transport, was significantly higher at the 10-min than at the 1-min time point after addition. Taken together, this study shows that cFP is a diffusible molecule that does not require energy for transportation across biological membranes, and that cFP does not need membrane machinery in order to cross membranes and consequently act as a virulence factor or signal. KEY POINTS: • Kinetics of cFP uptake into cells of V. vulnificus, E. coli, or RBS was studied. • The uptake was not saturated and required no energy, indicating passive transport. • The lack of cell specificity in cFP uptake means no specific protein is needed. • Therefore, the cFP moves across the biological membrane by simple diffusion.

Acetate is the main by-product from microbial succinate production. In this study, we performed acetate removal by Methanosarcina barkeri 227 for succinate fermentation by Actinobacillus succinogenes 130Z. The acetoclastic methanogen M. barkeri requires similar environmental factors to A. succinogenes, and the conditions required for co-cultivation were optimized in this study: gas used for anaerobicization, strain adaptation, medium composition, pH adjustment, and inoculation time points. M. barkeri 227 was adapted to acetate for 150 days, which accelerated the acetate consumption to 9-fold (from 190 to 1726 mmol gDW -1  day -1 ). In the acetate-adapted strain, there was a noticeable increase in transcription of genes required for acetoclastic pathway-satP (acetate transporter), ackA (acetate kinase), cdhA (carbon monoxide dehydrogenase/acetyl-CoA synthase complex), and mtrH (methyl-H 4 STP:CoM methyltransferase), which was not induced before the adaptation process. The activities of two energy-consuming steps in the pathway-acetate uptake and acetate kinase-increased about 3-fold. This acetate-adapted M. barkeri could be successfully applied to succinate fermentation culture of A. succinogenes, but only after pH adjustment following completion of fermentation. This study suggests the utility of M. barkeri as an acetate scavenger during fermentation for further steps towards genetic and process engineering.

We developed a formate-removing methanogenic inoculum (FRI) to facilitate succinate production from crude glycerol by Escherichia coli. FRI converted formate to methane, thereby enabling glycerol fermentation without additional electron acceptors under neutral pH. FRI was selectively enriched from sludge from the anaerobic digester of the Seonam sewage treatment plant (Seoul); this process was assessed via Illumina sequencing and scanning electron microscopy imaging. Methanobacterium congolense species occupied only 0.3% of the archaea community in the sludge and was enriched to 99.5% in complete FRI, wherein succinate-degrading bacteria were successfully eliminated. Co-culture with FRI improved glycerol fermentation and yielded 7.3 mM succinate from 28.7 mM crude glycerol, whereby FRI completely converted formate into methane. This study is the first to demonstrate methane production by M. congolense species, using formate. M. congolense-dominated FRI can serve as a renewable facilitator of waste feedstock fermentation and enable the production of commercially important compounds.

Actinobacillus succinogenes is a natural succinate producer, which is the result of fumarate respiration. Succinate production from anaerobic growth with C 4 -dicarboxylates requires transporters catalysing uptake and efflux of C 4 -dicarboxylates. Transporter Asuc_1999 (DcuE) found in A. succinogenes belongs to the Dcu family and was considered the main transporter for fumarate respiration. However, deletion of dcuE affected l-malate uptake of A. succinogenes rather than fumarate uptake. DcuE complemented anaerobic growth of Escherichia coli on l-malate or fumarate; thus, the transporter was characterized in E. coli heterologously. Time-dependent uptake and competitive inhibition assays demonstrated that l-malate is the most preferred substrate for uptake by DcuE. The V max of DcuE for l-malate was 20.04 μmol/gDW·min with K m of 57 μM. The V max for l-malate was comparable to that for fumarate, whereas the K m for l-malate was 8 times lower than that for fumarate. The catalytic efficiency of DcuE for l-malate was 7.3-fold higher than that for fumarate, showing high efficiency and high affinity for l-malate. Furthermore, DcuE catalysed the reversible exchange of three C 4 -dicarboxylates - l-malate, fumarate and succinate - but the preferred substrate for uptake was l-malate. Under physiological conditions, the C 4 -dicarboxylates were reduced to succinate. Therefore, DcuE is proposed as the l-malate/succinate antiporter in A. succinogenes., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Purpose: We compared how doses delivered via two-dimensional (2D) intracavitary brachytherapy (ICBT) and three-dimensional (3D) ICBT varied anatomically., Materials and Methods: A total of 50 patients who received 30 Gy of 3D ICBT after external radiotherapy (RT) were enrolled. We compared the doses of the actual 3D and 2D ICBT plans among patients grouped according to six anatomical variations: differences in a small-bowel V2Gy, small bowel circumference, the direction of bladder distension, bladder volume, sigmoid V3.5Gy, and sigmoid circumference. Seven dose parameters were measured in line with the EMBRACE recommendations., Results: In terms of bladder volume, the bladder and small-bowel D2cc values were lower in the 150-250 mL bladder volume subgroup; and the rectum, sigmoid, and bladder D2mL values were all lower in the >250 mL subgroup, for 3D vs. 2D ICBT. In the sigmoid V3.5Gy >2 mL subgroup, the sigmoid and bladder D2mL values were significantly lower for 3D than 2D ICBT. The bladder D2mL value was also significantly lower for 3D ICBT, as reflected by the sigmoid circumference. In patients with a small bowel V2.0Gy >10 mL or small bowel circumference >15%, most dose parameters were significantly lower for 3D than 2D ICBT. The bladder distension direction did not significantly affect the doses., Conclusion: Compared to 2D ICBT, a greater bladder volume can reduce the internal 3D ICBT organ dose without affecting the target dose.

A global transcriptome analysis of the natural succinate producer Actinobacillus succinogenes revealed that 353 genes were differentially expressed when grown on various carbon and energy sources, which were categorized into six functional groups. We then analyzed the expression pattern of 37 potential C 4 -dicarboxylate transporters in detail. A total of six transporters were considered potential fumarate transporters: three transporters, Asuc_1999 (Dcu), Asuc_0304 (DASS), and Asuc_0270-0273 (TRAP), were constitutively expressed, whereas three others, Asuc_1568 (DASS), Asuc_1482 (DASS), and Asuc_0142 (Dcu), were differentially expressed during growth on fumarate. Transport assays under anaerobic conditions with [ 14 C]fumarate and [ 14 C]succinate were performed to experimentally verify that A. succinogenes possesses multiple C 4 -dicarboxlayte transport systems with different substrate affinities. Upon uptake of 5 mmol/L fumarate, the systems had substrate specificity for fumarate, oxaloacetate, and malate, but not for succinate. Uptake was optimal at pH 7, and was dependent on both proton and sodium gradients. Asuc_1999 was suspected to be a major C 4 -dicarboxylate transporter because of its noticeably high and constitutive expression. An Asuc_1999 deletion (∆1999) decreased fumarate uptake significantly at approximately 5 mmol/L fumarate, which was complemented by the introduction of Asuc_1999. Asuc_1999 expressed in Escherichia coli catalyzed fumarate uptake at a level of 21.6 μmol·gDW -1 ·min -1 . These results suggest that C 4 -dicarboxylate transport in A. succinogenes is mediated by multiple transporters, which transport various types and concentrations of C 4 -dicarboxylates., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Escherichia coli can hardly grow anaerobically on glycerol without exogenous electron acceptor. The formate-consuming methanogen Methanobacterium formicicum plays a role as a living electron acceptor in glycerol fermentation of E. coli. Wild-type and mutant E. coli strains were screened for succinate production using glycerol in a co-culture with M. formicicum. Subsequently, E. coli was adapted to glycerol fermentation over 39 rounds (273 days) by successive co-culture with M. formicicum. The adapted E. coli (19.9 mM) produced twice as much succinate as non-adapted E. coli (9.7 mM) and 62% more methane. This study demonstrated improved succinate production from waste glycerol using an adapted wild-type strain of E. coli with wild-type M. formicicum, which is more useful than genetically modified strains. Crude glycerol, an economical feedstock, was used for the cultivation. Furthermore, the increase in methane production by M. formicicum during co-culture with adapted E. coli illustrated the possibility of energy-saving effects for the fermentation process.

Purpose: The purpose of this study was to evaluate the prognostic value of the lymph node ratio (LNR), which was defined as the proportion of involved nodes of all dissected nodes, in pN1 breast cancer., Materials and Methods: We retrospectively analyzed the clinical data of patients with pN1 breast cancer (N = 144) treated at Keimyung University Dongsan Medical Center, Daegu, Korea between 2001 and 2010. The median age was 46 years (range, 27 to 66 years). The LNR was 0.01-0.15 (low LNR) in 130 patients and >0.15 (high LNR) in 14 patients. Sixty-five patients (45.1%) had T1 tumors, 74 (51.4%) had T2 tumors, and 5 (3.5%) had T3 tumors. Eighty-eight patients (61.1%) underwent total mastectomy and 56 (38.9%) underwent partial mastectomy. Fifty-nine patients (41.0%) underwent radiotherapy and 12 (8.3%) underwent regional radiotherapy. The median follow-up period was 65 months., Results: The 5- and 10-year disease-free survival (DFS) rates were 92.7% and 82.4%, respectively. Univariate analyses revealed that high LNR (p = 0.004), total mastectomy (p = 0.006), no local radiotherapy (p = 0.036), and stage T2 or T3 (p = 0.010) were associated with worse DFS. In multivariable analysis, only high LNR (p = 0.015) was associated with worse DFS., Conclusion: High LNR is an independent prognostic factor in pN1 breast cancer and could be an indication for adjuvant radiotherapy in these patients.

AcrR, the toxic-compounds-response regulator, regulates motility in microorganisms, presumably to escape from toxic environments. In this study, the genome-wide target genes of AcrR were investigated in a Δ acrR mutant strain by microarray analysis. In the absence of AcrR, the transcription of most flagella/motility genes was highly increased. In addition, flagella formation was increased in this mutant strain. Motility assays revealed that AcrR modulates swimming motility, but not swarming.

C4-dicarboxylates and the C4-dicarboxylic amino acid l-aspartate support aerobic and anaerobic growth of Escherichia coli and related bacteria. In aerobic growth, succinate, fumarate, D- and L-malate, L-aspartate, and L-tartrate are metabolized by the citric acid cycle and associated reactions. Because of the interruption of the citric acid cycle under anaerobic conditions, anaerobic metabolism of C4-dicarboxylates depends on fumarate reduction to succinate (fumarate respiration). In some related bacteria (e.g., Klebsiella), utilization of C4-dicarboxylates, such as tartrate, is independent of fumarate respiration and uses a Na+-dependent membrane-bound oxaloacetate decarboxylase. Uptake of the C4-dicarboxylates into the bacteria (and anaerobic export of succinate) is achieved under aerobic and anaerobic conditions by different sets of secondary transporters. Expression of the genes for C4-dicarboxylate metabolism is induced in the presence of external C4-dicarboxylates by the membrane-bound DcuS-DcuR two-component system. Noncommon C4-dicarboxylates like l-tartrate or D-malate are perceived by cytoplasmic one-component sensors/transcriptional regulators. This article describes the pathways of aerobic and anaerobic C4-dicarboxylate metabolism and their regulation. The citric acid cycle, fumarate respiration, and fumarate reductase are covered in other articles and discussed here only in the context of C4-dicarboxylate metabolism. Recent aspects of C4-dicarboxylate metabolism like transport, sensing, and regulation will be treated in more detail. This article is an updated version of an article published in 2004 in EcoSal Plus. The update includes new literature, but, in particular, the sections on the metabolism of noncommon C4-dicarboxylates and their regulation, on the DcuS-DcuR regulatory system, and on succinate production by engineered E. coli are largely revised or new.

Purpose: To identify possible predictors of pathologic complete response (pCR) of rectal cancer after preoperative concurrent chemoradiotherapy (CCRT)., Materials and Methods: We conducted a retrospective review of 53 patients with rectal cancer who underwent preoperative CCRT followed by radical surgery at a single center between January 2007 and December 2012. The median radiotherapy dose to the pelvis was 54.0 Gy (range, 45.0 to 63.0 Gy). Five-fluorouracil-based chemotherapy was administered via continuous infusion with leucovorin., Results: The pCR rate was 20.8%. The downstaging rate was 66%. In univariate analyses, poor and undifferentiated tumors (p = 0.020) and an interval of ≥7 weeks from finishing CCRT to surgery (p = 0.040) were significantly associated with pCR, while female gender (p = 0.070), initial carcinoembryonic antigen concentration of <5.0 ng/dL (p = 0.100), and clinical stage T2 (p = 0.100) were marginally significant factors. In multivariate analysis, an interval of ≥7 weeks from finishing CCRT to surgery (odds ratio, 0.139; 95% confidence interval, 0.022 to 0.877; p = 0.036) was significantly associated with pCR, while stage T2 (odds ratio, 5.363; 95% confidence interval, 0.963 to 29.877; p = 0.055) was a marginally significant risk factor., Conclusion: We suggest that the interval from finishing CCRT to surgery is a predictor of pCR after preoperative CCRT in patients with rectal cancer. Stage T2 cancer may also be an important predictive factor. We hope to perform a robust study by collecting data during treatment to obtain more advanced results.

Background/objectives: Feeding in infancy is the most significant determinant of the intestinal microbiota in early life. The aim of this study was to determine the gut microbiota of Korean infants and compare the microbiota obtained between breast-fed and formula-fed Korean infants., Subjects/methods: We analyzed the microbial communities in fecal samples collected from twenty 4-week old Korean (ten samples in each breast-fed or formula-fed) infants using pyrosequencing., Results: The fecal microbiota of the 4-week-old Korean infants consisted of the three phyla Actinobacteria, Firmicutes, and Proteobacteria. In addition, five species, including Bifidocbacterium longum, Streptococcus salivarius, Strepotococcus lactarius, Streptococcus pseudopneumoniae, and Lactobacillus gasseri were common commensal intestinal microbiota in all infants. The predominant intestinal microbiota in the breast-fed infants (BFI) included the phylum Actinobacteria (average 70.55%), family Bifidobacteriacea (70.12%), genus Bifidobacterium (70.03%) and species Bifidobacterium longum (69.96%). In the microbiota from the formula-fed infants (FFI), the proportion of the phylum Actinobacteria (40.68%) was less, whereas the proportions of Firmicutes (45.38%) and Proteobacteria (13.85%) as well as the diversity of each taxonomic level were greater, compared to those of the BFI. The probiotic species found in the 4-week-old Korean infants were Bifidobacterium longum, Streptococcus salivarius, and Lactobacillus gasseri. These probiotic species accounted for 93.81% of the microbiota from the BFI, while only 63.80% of the microbiota from the FFI. In particular, B. longum was more abundant in BFI (69.96%) than in FFI (34.17%)., Conclusions: Breast milk supports the growth of B. longum and inhibits others. To the best of our knowledge, this study was the first attempt to analyze the gut microbiota of healthy Korean infants according to the feeding type using pyrosequencing. Our data can be used as a basis for further studies to investigate the development of intestinal microbiota with aging and disease status.

Acriflavine resistance regulator (AcrR), a local transcription factor, regulates the expression of the acrRAB genes associated with the AcrAB-TolC multidrug efflux pump. Screening of organic solvent tolerance (OST) with the overexpression of 13 genes in Escherichia coli revealed that the overexpression of acrR improved OST. Overexpression of AcrR in a background strain of wild-type E. coli and in the OST strain LMB015 (ΔfadR ΔmarR; acrR (+) and ΔfadR ΔmarR acrR (+) strain, respectively) significantly increased cell growth in the presence of n-hexane/cyclohexane, which attenuated the membrane reduction capacity of the wild-type strain below 50 % of the control level. This was recovered to control levels in the acrR (+) strain. Quantitative real-time PCR analysis of RNA from the wild-type, ΔacrR, and acrR (+) strains showed that AcrR represses the transcription of marRAB and soxRS, and its own gene cluster, acrRAB. Electrophoretic mobility shift assay demonstrated that AcrR binds directly to the promoter region of acrRAB, marAB, and soxRS, indicating that AcrR acts on global regulators to affect mar-sox-rob regulon. In the acrR (+) strain, soxS expression was significantly upregulated compared with the wild-type. The OST of the acrR (+) strain was completely lost in the ΔsoxS acrR (+) strain, indicating that SoxS mediated OST improvement in the acrR (+) strain. The observation that all genes associated with marRAB and soxRS are upregulated in the ΔacrR strain, and that there is only moderate induction of soxS (and marB) in the acrR (+) strain, provides insight into how acrR overexpression confers bacterial OST and the mar-sox-rob regulon control network.

Purpose: This study evaluated the efficacy of extended field irradiation (EFI) in patients with locally advanced cervical cancer without para-aortic nodal involvement., Materials and Methods: A total of 203 patients with locally advanced cervical cancer (International Federation of Gynecology and Obstetrics [FIGO] stage, IB2-IIIB) treated with radiotherapy at Keimyung University Dongsan Medical Center from 1996 to 2010 were retrospectively analyzed. The median patient age was 59 years (range, 29 to 83 years). None of the patients had para-aortic node metastases. Of the 203 patients, 88 underwent EFI and 115 underwent irradiation of the pelvis only. Concurrent chemoradiotherapy (CCRT) was administered to 133 patients. EFI field was used for treatment of 26 patients who received radiotherapy alone and 62 who received CCRT., Results: The median follow-up period was 60 months. The 2- and 5-year overall survival (OS) rates were 87.8% and 73.5%, respectively, and the 2- and 5-year disease-free survival rates were 81.7% and 75.0%, respectively, however, no survival differences were observed between the two treatment field groups. EFI tended to increase OS in the radiotherapy alone group, but not in the CCRT group., Conclusion: These findings suggest that EFI does not have a significant effect in patients with locally advanced cervical cancer, especially in patients receiving CCRT. Conduct of additional studies will be required in order to confirm these findings.

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Actinobacillus succinogenes, which is known to produce large amounts of succinate during fermentation of hexoses, was able to grow on C4-dicarboxylates such as fumarate under aerobic and anaerobic conditions. Anaerobic growth on fumarate was stimulated by glycerol and the major product was succinate, indicating the involvement of fumarate respiration similar to succinate production from glucose. The aerobic growth on C4-dicarboxylates and the transport proteins involved were studied. Fumarate was oxidized to acetate. The genome of A. succinogenes encodes six proteins with similarity to secondary C4-dicarboxylate transporters, including transporters of the Dcu (C4-dicarboxylate uptake), DcuC (C4-dicarboxylate uptake C), DASS (divalent anion : sodium symporter) and TDT (tellurite resistance dicarboxylate transporter) family. From the cloned genes, Asuc&#95;0304 of the DASS family protein was able to restore aerobic growth on C4-dicarboxylates in a C4-dicarboxylate-transport-negative Escherichia coli strain. The strain regained succinate or fumarate uptake, which was dependent on the electrochemical proton potential and the presence of Na(+). The transport had an optimum pH ~7, indicating transport of the dianionic C4-dicarboxylates. Transport competition experiments suggested substrate specificity for fumarate and succinate. The transport characteristics for C4-dicarboxylate uptake by cells of aerobically grown A. succinogenes were similar to those of Asuc&#95;0304 expressed in E. coli, suggesting that Asuc&#95;0304 has an important role in aerobic fumarate uptake in A. succinogenes. Asuc&#95;0304 has sequence similarity to bacterial Na(+)-dicarboxylate cotransporters and contains the carboxylate-binding signature. Asuc&#95;0304 was named SdcA (sodium-coupled C4-dicarboxylate transporter from A. succinogenes)., (© 2014 The Authors.)

Rationale: Interactions of biological molecules to form cluster species play a key role in biological processes and investigation of non-covalent complexes is one of the research fields using mass spectrometry. Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) is a useful method for the investigation of cluster formation of amino acids (AAs) by ion-molecule reactions., Methods: A mixture of 20 protein AAs was ionized by APCI and the product ions were analyzed. The ionization was performed in the positive and negative ion modes. Formation of the homo- and heterocluster ions of AAs was investigated. Mechanism for the formation of AA homo- and heterocluster ions was examined using hydrogen/deuterium (H/D)-exchange experiments., Results: In the positive ion mode, of the dimer species only the [2Pro+H](+) ion was detected. In the negative ion mode, the [2M - H](-) ions of His, Val, Ser, and Gln were observed. The deaminated dimers such as the [2Gln - H - NH3](-) and [His + Gln - H - NH3](-) ions were also observed. In the negative ion mass spectra of the His/Arg, His/Asn, and His/Lys binary mixture solutions, the [His + AA - H - NH3](-) ions of Asn, Arg, and Lys were also detected., Conclusions: The number and abundances of the negative product ions were much greater than those of the positive ones. Mechanism for the formation of [2Gln - H - NH3](-) and [His+AA - H - NH3](-) was examined by deuterium replacement of the amine and hydroxide groups to distinguish the deamination and dehydration reactions with a single quadrupole mass spectrometer. The [His + AA - H - NH3](-) ion is formed by ion-molecule reaction between the [His-H](-) ion and a neutral AA., (Copyright © 2014 John Wiley & Sons, Ltd.)

The aim of this study was assess the patient setup errors for various tumor sites based on clinical data from a sufficient number of treatments with volumetric-modulated arc therapy (VMAT) using daily pretreatment CBCT imaging guidance. In addition, we calculated and compared the planning target volume (PTV) margins for all disease sites based on an analysis of specific systematic and random errors in our institution. All patients underwent pretreatment kV-CBCT imaging. The various tumor sites were divided into four categories; 21 brain (438 fractions), 35 head-and-neck tumors (H&N, 933 fractions), 19 thorax and abdomen tumors (T&A, 313 fractions), and 17 prostate cancer tumors (546 fractions). Overall distributions of setup corrections in all directions, frequencies of 3D vector lengths, institution-specific setup error, and PTV margins were analyzed. The longitudinal distribution for the T&A site represented an asymmetric offset in the negative direction. Rotational distributions were comparable for all treatment sites, and the prostate site had the narrowest distribution of ≤ ± 2°. The cumulative frequencies of 3D vector length of ≥ 7 mm were rare for brain lesions and H&N, but more common for T&A and prostate lesions at 25.6% and 12.1%, respectively. The overall mean error for all treatment sites were within ± 1 mm and ± 0.1°, with the exception of the T&A site, which had overall mean error of 2 mm in the negative longitudinal direction. The largest magnitude of systematic error and random error for the brain lesions and H&N was 1.4 mm in the translational directions, and 3.3 mm for T&A and prostate lesions. The PTV margins required in this analysis are ≤ 4 mm for the brain lesions and H&N in all translational directions, but ranged from 4 to 10 mm for T&A and prostate lesions. Analysis of each institution's specific setup errors using daily CBCT is essential for determining PTV margins and reducing setup uncertainties, because setup errors vary according to each immobilization system and patient.

Considerable effort has been directed towards replacing lost teeth using tissue-engineering methods such as titanium implants. A number of studies have tried to modify bioinert titanium surfaces by coating them with functionally bioactive molecules for faster and stronger osseointegration than pure titanium surfaces. Recently, peptides have been recognized as valuable scientific tools in the field of tissue-engineering. The DLTIDDSYWYRI motif of the human laminin-2 α2 chain has been previously reported to promote the attachment of various cell types; however, the in vivo effects of the DLTIDDSYWYRI motif on new bone formation have not yet been studied. To examine whether a laminin-2-derived peptide can promote osseointegration by accelerating new bone formation in vivo, we applied titanium implants coated with the DLTIDDSYWYRI motif in a rabbit tibia model. The application of the DLTIDDSYWYRI motif-treated implant to tibia wounds enhanced collagen deposition and alkaline phosphatase expression. It significantly promoted implant osseointegration compared with treatment with scrambled peptide-treated implants by increasing the bone-to-implant contact ratio and bone area. These findings support the hypothesis that the DLTIDDSYWYRI motif acts as an effective osseointegration accelerator by enhancing new bone formation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Gö6976 is a nonglycosidic indolocarbazole compound widely used as a specific inhibitor of PKCα/β. In experiments probing for a role of PKCα in human laminin-2-integrin-mediated cell adhesion and spreading of PC12 cells, we observed unexpected enhancements of adhesion, spreading and stress fiber formation to 1 μM Gö6976 with concomitant increase in membrane translocation of PKCδ and autophosphorylation of focal adhesion kinase (FAK). Importantly, enhanced cellular behavior and membrane translocation of PKCδ induced by Gö6976 was retained in siRNA-transfected PC12 cells to knockdown PKCα expression. Gö6976 also induced laminin-dependent cell adhesion in NIH/3T3 and CV-1 fibroblasts, suggesting of a mechanism that may be common to multiple cell-types. A specific inhibitor of PKCδ, rottlerin, completely abrogated Gö6976-dependent increase in PC12 cell adhesion to laminin as well as the activation of small GTPases, Rac1 and Cdc42, that are downstream of PKCδ in adhesion receptor signaling. siRNA knockdown of Rac1 and Cdc42 expression inhibited cell spreading and lamellipodia formation in PC12 cells. Overall, these results suggest that Gö6976 may stimulate membrane recruitment of PKCδ through a mechanism that is independent of PKCα/β signaling. In addition, the activation of Rac1 and Cdc42 by human laminin-2-integrin-dependent activation of PKCδ/FAK signaling mediates cell spreading and lamellipodia formation in PC12 cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Rationale: Dissociation reactions of protonated amino acids (AAs) can be used as models for the fragmentation of protonated peptides. Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) provides a great deal of structural information in a short analysis time., Methods: In APCI-MS, the fragmentation patterns can be obtained by varying the cone voltage and some fragment ions are produced that can be used to identify the structure of an analyte. In general, the fragmentation of AAs has used liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, we studied the fragmentation of protonated AAs using a single quadrupole mass spectrometer., Results: The principal fragment ions were [M + H - H(2)O - CO](+), [M + H - H(2)O](+), and [M + H - NH(3)](+). AAs that only generated [M + H - H(2)O - CO](+) were alanine, glycine, histidine, isoleucine, leucine, proline, phenylalanine, and valine. AAs that generated [M + H - H(2)O](+) and [M + H - H(2)O - CO](+) were aspartic acid, glutamic acid, serine, and threonine, while AAs that generated [M + H - NH(3)](+) and [M + H - H(2)O - CO](+) were asparagine, cysteine, glutamine, methionine, tryptophan, and tyrosine. Arginine and lysine generated [M + H - H(2)O](+) and [M + H - NH(3)](+)., Conclusions: The relative abundances of the fragment ions increased with increase in the cone voltage. The experimental results were explained by the favorability of the intermediate structure and the stability of the fragment ion structure. The specific fragmentation patterns could be used for differentiating underivatized AAs., (Copyright © 2012 John Wiley & Sons, Ltd.)

Laminin-derived peptide coatings can enhance epithelial cell adhesion to implants, and the positive effect of these peptides on bone cell adhesion has been anticipated. The purpose of this study was to evaluate the improvement in bone cell attachment to and activity on titanium (Ti) scaffolds coated with a laminin-derived functional peptide, Ln2-P3 (the DLTIDDSYWYRI motif). Four Ti disc surfaces were prepared, and a human osteosarcoma (HOS) cell attachment test was performed to select two candidate surfaces for peptide coating. These two candidates were then coated with Ln2-P3 peptide, a scrambled peptide, or left uncoated to measure cell attachment to each surface, following which one surface was chosen to assess alkaline phosphatase (ALP) activity and osteogenic marker gene expression with quantitative real-time PCR. On the commercially pure Ti surface, the Ln2-P3 coating significantly increased cellular ALP activity and the expression levels of ALP and bone sialoprotein mRNA as compared with the scrambled peptide-coated and uncoated surfaces. In conclusion, although further in vivo studies are needed, the findings of this in vitro study indicate that the Ln2-P3-coated implant surface promotes bone cell adhesion, which has clinical implications for reducing the overall treatment time of dental implant therapy.

Purpose: To compare failure patterns and evaluate prognostic factors related to survival rates after concurrent chemoradiotherapy (CCRT) or radiotherapy (RT) alone in cervical cancer., Materials and Methods: From January 1996 to December 2006, 218 patients with cervical cancer (FIGO Stage IB2 - III) treated with CCRT or RT alone as primary treatments were included, retrospectively. One-hundred eight patients were treated with CCRT and 110 with RT alone., Results: There was no significant difference in failure patterns between the treatment groups, but distant metastasis was the predominant pattern in both groups. The frequent metastatic sites were supraclavicular lymph node, lung, and brain. Treatment group, diabetes, and FIGO Stage were found to be significant for overall survival (OS) and disease-free survival (DFS), and initial hemoglobin level for DFS., Conclusion: Distant metastasis is the predominant failure pattern and diabetes is one of the independent prognostic factors to survival rates in this study.

The improvement of bacterial tolerance to organic solvents is a main prerequisite for the microbial production of biofuels which are toxic to cells. For targeted genetic engineering of Escherichia coli to increase organic solvent tolerances (OSTs), we selected and investigated a total of 12 genes that participate in relevant mechanisms to tolerance. In a spot assay of 12 knockout mutants with n-hexane and cyclohexane, the genes fadR and marR were finally selected as the two key genes for engineering. Fatty acid degradation regulon (FadR) regulates the biosynthesis and degradation of fatty acids coordinately, and the multiple antibiotic resistance repressor (MarR) is the repressor of the global regulator MarA for multidrug resistance. In the competitive growth assay, the ΔmarR mutant became dominant when the pooled culture of 11 knockout mutants was cultivated successively in the presence of organic solvent. The increased OSTs in the ΔmarR and ΔfadR mutants were confirmed by a growth experiment and a viability test. The even more highly enhanced OSTs in the ΔfadR ΔmarR double mutant were shown compared with the two single mutants. Cellular fatty acid analysis showed that the high ratio of saturated fatty acids to unsaturated fatty acids plays a crucial role in OSTs. Furthermore, the intracellular accumulation of OST strains was significantly decreased compared with the wild-type strain.

Purpose: To investigate the prognostic factors and effectiveness of postoperative radiotherapy alone for endometrial carcinoma., Materials and Methods: Sixty four patients with stage I-III endometrial cancer (EC) treated with postoperative radiotherapy alone between January 1989 and December 2008 at the Keimyung University Dongsan Medical Center were chosen for the present study. Typically, total hysterectomy, salpingo-oophorectomy and lymphadenectomy were performed on the patient's pelvis. Total dose from 50.4 Gy to 63 Gy was irradiated at pelvis or extended field. Thirteen patients were treated with Co-60 or Ir-192 intracavitary radiotherapy. Follow-up periods were from 7 to 270 months, with a median of 56 months., Results: Five year overall survival (OS) rate was 58.7%, respectively. Five year disease-free survival (DFS) rate was 59.2%, respectively. In univariate analysis for OS and DFS, stage, menopausal age, type of operation, serosal invasion, and lymph node involvement were found to be statistically significant. Histologic type was marginally significant. In multivariate analysis for OS and DFS, stage, types of operation, histologic type were also found to be statistically significant. Treatment failure occurred in 14 patients. The main pattern of failure was found to be distant metastasis. Time to distant metastasis was from 3 to 86 months (median, 12 months). There were no grade 3 or 4 complications., Conclusion: Stage, types of operation, and histologic type could be the predictive prognostic factors in patients. We contemplated postoperative radiation as effective and safe treatment method for EC. Additional treatment would be needed to reduce distant metastasis.

Laminin α2 chain plays an important role in basement membrane assembly and peripheral myelinogenesis; however, the integrin binding motif within human laminin α2 chain and the signaling pathways downstream of this ligand-receptor interaction are poorly understood. We identified a motif, RNIPPFEGCIWN (Ln2-LG3-P2), within LG3 domain of human laminin α2 chain as a major site for both α3β1 integrin and cellular activities such as cell adhesion, spreading, and migration. Binding of α3β1 integrin with Ln2-LG3-P2 induced the membrane recruitment of protein kinase Cδ (PKCδ) and stimulated its tyrosine phosphorylation. The cellular activities induced by Ln2-LG3-P2 and the phosphorylation of focal adhesion kinase (FAK) were inhibited by rottlerin, a PKCδ inhibitor, but not by Gö6976, a PKCα/β inhibitor. These results indicate that RNIPPFEGCIWN motif within human laminin α2 chain is a major ligand for α3β1 integrin, and that binding of α3β1 integrin mediates cellular activities through membrane recruitment and tyrosine phosphorylation of PKCδ and FAK phosphorylation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Glucose uptake by the heterofermentative lactic acid bacterium Oenococcus oeni B1 was studied at the physiological and gene expression levels. Glucose- or fructose-grown bacteria catalyzed uptake of [(14)C]glucose over a pH range from pH 4 to 9, with maxima at pHs 5.5 and 7. Uptake occurred in two-step kinetics in a high- and low-affinity reaction. The high-affinity uptake followed Michaelis-Menten kinetics and required energization. It accumulated the radioactivity of glucose by a factor of 55 within the bacteria. A large portion (about 80%) of the uptake of glucose was inhibited by protonophores and ionophores. Uptake of the glucose at neutral pH was not sensitive to degradation of the proton potential, Δp. Expression of the genes OEOE&#95;0819 and OEOE&#95;1574 (here referred to as 0819 and 1574), coding for secondary transporters, was induced by glucose as identified by quantitative real-time (RT)-PCR. The genes 1574 and 0819 were able to complement growth of a Bacillus subtilis hexose transport-deficient mutant on glucose but not on fructose. The genes 1574 and 0819 therefore encode secondary transporters for glucose, and the transports are presumably Δp dependent. O. oeni codes, in addition, for a phosphotransferase transport system (PTS) (gene OEOE&#95;0464 [0464] for the permease) with similarity to the fructose- and mannose-specific PTS of lactic acid bacteria. Quantitative RT-PCR showed induction of the gene 0464 by glucose and by fructose. The data suggest that the PTS is responsible for Δp-independent hexose transport at neutral pH and for the residual Δp-independent transport of hexoses at acidic pH.

Although previous studies indicate that skin-derived precursors (SKPs) are multipotent dermal precursors that share similarities with neural crest stem cells (NCSCs), a shared ability for multilineage differentiation toward neural crest lineages between SKPs and NCSCs has not been fully demonstrated. Here, we report the derivation of SKPs from adult mouse skin and their directed multilineage differentiation toward neural crest lineages. Under controlled in vitro conditions, mouse SKPs were propagated and directed toward peripheral nervous system lineages such as peripheral neurons and Schwann cells, and mesenchymal lineages, such as osteogenic, chondrogenic, adipogenic, and smooth muscle cells. To ask if SKPs could generate these same lineages in vivo, a mixture of SKP-derived mesenchymal stem cells and hydroxyapatite/tricalcium phosphate was transplanted into the rat calvarial defects. Over the ensuing 4 weeks, we observed formation of osteogenic structure in the calvarial defect without any evidence of teratomas. These findings demonstrate the multipotency of adult mouse SKPs to differentiate into neural crest lineages. In addition, SKP-derived mesenchymal stem cells represent an accessible, potentially autologous source of precursor cells for tissue-engineered bone repair.

Bacteria are able to grow at the expense of both common (succinate, L-malate, fumarate and aspartate) and uncommon (L-tartrate and D-malate) C(4)-dicarboxylates, which are components of central metabolism. Two types of sensors/regulators responding to the C(4)-dicarboxylates function in Escherichia coli, Bacillus, Lactobacillus and related bacteria. The first type represents membrane-integral two-component systems, while the second includes cytoplasmic LysR-type transcriptional regulators. The difference in location and substrate specificity allows the exogenous induction of metabolic genes by common C(4)-dicarboxylates, and endogenous induction by uncommon C(4)-dicarboxylates. The two-component sensors, DcuS and CitA, are composed of an extracellular Per-Arnt-Sim (PAS) domain, two transmembrane helices, a cytoplasmic PAS and the kinase domain. The structures of the extracellular PAS domains of DcuS and CitA have been determined in the ligand-bound and the apo form. Binding of the ligand results in closing and compaction of the binding site, and the structural change gives rise to piston-type movement of the adjacent membrane-spanning helix-2, and signal transmission to the cytoplasmic side. For DcuS, a membrane-embedded construct has been developed that suggests (by experimentation and modeling) that plasticity of the cytoplasmic PAS domain is central to signal transduction from the membrane to the kinase. Sensor kinase DcuS of E. coli requires the C(4)-dicarboxylate transporters DctA or DcuB as co-sensors for function under aerobic and anaerobic conditions, respectively. DcuB contains a regulatory site that controls the function of DcuS and is independent from the transport region. Therefore, DcuS senses C(4)-dicarboxylates in two independent modes, responding to the effector concentration and the metabolic flux of extracellular C(4)-dicarboxylates.

Although >60% of oral cancer cases are not related to human papillomavirus (HPV) infection, most studies of oral carcinogenesis in human cells in vitro are carried out with human oral keratinocytes immortalized by HPV DNA. To explore whether human oral keratinocytes can spontaneously transform without HPV infection, we attempted to establish spontaneously immortalized and tumorigenic-transformed human oral keratinocytes by serial subculture to the post-mitotic stage. Here we report two spontaneously transformed human oral keratinocyte lines from adult human gingival samples. These lines were obviously immortal (>140 passages) and transformed phenotypes in vitro. One of the lines, Spi-HOK1, remained non-tumorigenic in nude mice, whereas the other line, Spt-HOK80, showed tumorigenicity. These lines showed epithelial origi-nality, but did not contain high-risk types of HPV DNAs. On karyotyping, Spi-HOK1 was aneuploid with a unique stable marker chromosome. Both cell lines revealed a mutation in the p53 gene, loss of p21WAF1/Cip1 and overexpression of p-Rb-Ser807/811. These cell lines were resistant to cisplatin-induced apoptosis by suppressing induction of apoptotic proteins. These results clearly demonstrate that spontaneous immortalization and spontaneous tumorigenic transformation of primary human oral keratinocytes can occur in vitro without HPV infection and are associated with chromosomal alterations, p53 mutation and impaired apoptosis. To our knowledge, this is the first report demonstrating that the Spi-HOK1 and Spt-HOK80 lines are novel cell lines that are spontaneously transformed from primary human oral keratinocytes.

Escherichia coli K-12 is able to grow under aerobic conditions on D-malate using DctA for D-malate uptake and the D-malate dehydrogenase DmlA (formerly YeaU) for converting D-malate to pyruvate. Induction of dmlA encoding DmlA required an intact dmlR (formerly yeaT) gene, which encodes DmlR, a LysR-type transcriptional regulator. Induction of dmlA by DmlR required the presence of D-malate or L- or meso-tartrate, but only D-malate supported aerobic growth. The regulator of general C(4)-dicarboxylate metabolism (DcuS-DcuR two-component system) had some effect on dmlA expression. The anaerobic L-tartrate regulator TtdR or the oxygen sensors ArcB-ArcA and FNR did not have a major effect on dmlA expression. DmlR has a high level of sequence identity (49%) with TtdR, the L- and meso-tartrate-specific regulator of L-tartrate fermentation in E. coli. dmlA was also expressed at high levels under anaerobic conditions, and the bacteria had D-malate dehydrogenase activity. These bacteria, however, were not able to grow on D-malate since the anaerobic pathway for D-malate degradation has a predicted yield of < or = 0 ATP/mol D-malate. Slow anaerobic growth on D-malate was observed when glycerol was also provided as an electron donor, and D-malate was used in fumarate respiration. The expression of dmlR is subject to negative autoregulation. The network for regulation and coordination of the central and peripheral pathways for C(4)-dicarboxylate metabolism by the regulators DcuS-DcuR, DmlR, and TtdR is discussed.

Escherichia coli catabolizes L-tartrate under anaerobic conditions to oxaloacetate by the use of L-tartrate/succinate antiporter TtdT and L-tartrate dehydratase TtdAB. Subsequently, L-malate is channelled into fumarate respiration and degraded to succinate by the use of fumarase FumB and fumarate reductase FrdABCD. The genes encoding the latter pathway (dcuB, fumB and frdABCD) are transcriptionally activated by the DcuS-DcuR two-component system. Expression of the L-tartrate-specific ttdABT operon encoding TtdAB and TtdT was stimulated by the LysR-type gene regulator TtdR in the presence of L- and meso-tartrate, and repressed by O(2) and nitrate. Anaerobic expression required a functional fnr gene, and nitrate repression depended on NarL and NarP. Expression of ttdR, encoding TtdR, was repressed by O(2), nitrate and glucose, and positively regulated by TtdR and DcuS. Purified TtdR specifically bound to the ttdR-ttdA promoter region. TtdR was also required for full expression of the DcuS-DcuR-dependent dcuB gene in the presence of tartrate. Overall, expression of the ttdABT genes is subject to L-/meso-tartrate-dependent induction, and to aerobic and nitrate repression. The control is exerted directly at ttdA and in addition indirectly by regulating TtdR levels. TtdR recognizes a subgroup (L- and meso-tartrate) of the stimuli perceived by the sensor DcuS, which responds to all C(4)-dicarboxylates; both systems apparently communicate by mutual regulation of the regulatory genes.

Escherichia coli is able to grow under anaerobic conditions on D: -tartrate when glycerol is supplied as an electron donor (D-tartrate fermentation). D-Tartrate was converted to succinate. Growth was lost in strains deficient for DcuB, the fumarate/succinate antiporter of fumarate respiration. The L-tartrate/succinate antiporter TtdT of L-tartrate fermentation, or the C4-dicarboxylate carriers DcuA and DcuC, were not able to support D-tartrate transport and fermentation. Deletion of fumB demonstrated, that fumarase B is required for growth on D-tartrate. The mutant lost most (about 79%) of D-tartrate dehydratase activity. L-Tartrate dehydratase (TtdAB), and fumarase A or C, showed no or only a small contribution to D-tartrate dehydratase activity. Therefore D-tartrate is metabolised by a sequence of reactions analogous to that from L-tartrate fermentation, including dehydration to oxaloacetate, which is then converted to malate, fumarate and succinate. The stereoisomer specific carrier TtdT and dehydratase TtdAB of L-tartrate fermentation are substituted by enzymes from general anaerobic fumarate metabolism, the antiporter DcuB and fumarase B, which have a broader substrate specificity. No D-tartrate specific carriers and enzymes are involved in the pathway.

Escherichia coli ferments L-tartrate under anaerobic conditions in the presence of an additional electron donor to succinate. The carrier for L-tartrate uptake and succinate export and its relation to the general C(4)-dicarboxylate carriers DcuA, DcuB, and DcuC were studied. The secondary carrier TtdT, encoded by the ttdT (previously called ygjE) gene, is required for the uptake of L-tartrate. The ttdT gene is located downstream of the ttdA and ttdB genes, encoding the L-tartrate dehydratase TtdAB. Analysis of mRNA by reverse transcription-PCR showed that ttdA, ttdB, and ttdT are cotranscribed. Deletion of ttdT abolished growth by L-tartrate and degradation of L-tartrate completely. Bacteria containing TtdT catalyze L-tartrate or succinate uptake and specific heterologous L-tartrate/succinate antiporting. D-Tartrate is not a substrate for TtdT. TtdT operates preferentially in the direction of tartrate uptake and succinate excretion. The Dcu carriers do not support anaerobic growth on L-tartrate or L-tartrate transport. TtdT is related in sequence and function to CitT, which catalyzes heterologous citrate/succinate antiporting in citrate fermentation.

Substantial pain is experienced by a lot of patients with cancer, and undertreated pain significantly undermines their quality of life. Despite international and national guidelines on cancer pain management, the practical effectiveness of management is still problematic. We did a prospective cross-sectional survey on pain prevalence, pain intensity, its impact on daily activity, and adequacy of pain management in 823 patients treated by medical oncologists and radiologic oncologists. At the time of the survey, 29.7% of the patients had pain that was moderate or severe. Performance status and tumor extent were significant predictors of pain presence and its intensity. Pain severity and the proportion of the patients whose function is impaired are highly associated. Among the patients with pain, only 37% rated pain relief as satisfactory. Forty-one percent of the patients with pain received inadequate pain management. Physicians did not adjust the analgesic prescription in about 53% of the patients who reported severe pain. These results demonstrate the alarming degree of undertreatment of pain in patients with cancer in Korea, and indicate the need to improve the management of cancer-related pain. Future research should elucidate the factors that impede adequate pain management in order to overcome obstacles to adequate treatment., (Copyright 2003 S. Karger AG, Basel)

Quintuple mutants of Escherichia coli deficient in the C(4)-dicarboxylate carriers of aerobic and anaerobic metabolism (DctA, DcuA, DcuB, DcuC, and the DcuC homolog DcuD, or the citrate/succinate antiporter CitT) showed only poor growth on succinate (or other C(4)-dicarboxylates) under oxic conditions. At acidic pH (pH 6) the mutants regained aerobic growth on succinate, but not on fumarate. Succinate uptake by the mutants could not be saturated at physiological succinate concentrations (< or =5 mM), in contrast to the wild-type, which had a K(m) for succinate of 50 microM and a V(max) of 35 U/g dry weight at pH 6. At high substrate concentrations, the mutants showed transport activities (32 U/g dry weight) comparable to that of the wild-type. In the wild-type using DctA as the carrier, succinate uptake had a pH optimum of 6, whereas succinate uptake in the mutants was maximal at pH 5. In the mutants succinate uptake was inhibited competitively by monocarboxylic acids. Diffusion of succinate or fumarate across phospholipid membranes (liposomes) was orders of magnitude slower than the transport in the wild-type or the mutants. The data suggest that mutants deficient in DctA, DcuA, DcuB, DcuC, DcuD (or CitT) contain a carrier, possibly a monocarboxylate carrier, which is able to transport succinate, but not fumarate, at acidic pH, when succinate is present as a monoanion. Succinate uptake by this carrier was inhibited by addition of an uncoupler. Growth by fumarate respiration (requiring fumarate/succinate antiport) was also lost in the quintuple mutants, and growth was not restored at pH 6. In contrast, the efflux of succinate produced during glucose fermentation was not affected in the mutants, demonstrating that, for succinate efflux, a carrier different from, or in addition to, the known Dcu and CitT carriers is used.

Background: Significant antitumor activity has been reported with the combined use of 13-cis-retinoic acid (cRA) and interferon-alpha2a (IFN-alpha) in the treatment of advanced-stage cervical cancers and skin cancers. Since IFN-alpha has been shown to be a modest radiation enhancer for selected malignant tumor cells and the cytotoxic activity is more enhanced by combining cRA and IFN-alpha, we hypothesized that the exposure of selected human carcinoma cells to combined cRA and IFN-alpha would render the cells highly radiosensitive., Methods and Materials: Two human cervical carcinoma cell lines, ME-180 and HeLa-S3, were chosen for the present study because of the different characteristics of the retinoic acid receptor status of the cell lines. To demonstrate the effects of combined cRA and IFN-alpha treatment on radiation response, we exposed the cells to cRA, IFN-alpha, or a combination of the drugs for 72 h before radiation. Experiments were carried out at minimally cytotoxic concentrations of the drug for radiation studies. End points of the study were cell growth inhibition and clonogenic ability of the single-plated cells. Effects of cRA and IFN-alpha on radiation response were quantitatively analyzed by constructing the radiation cell survival curves of ME-180 and HeLa cells., Results: ME-180 cells exhibited varying degrees of cytotoxicity with cRA and IFN-alpha, while HeLa cells showed no toxic effects with the same treatment. Combined treatment of cRA and IFN-alpha produced an additive cytotoxic effect in ME-180 cells. Radiosensitization was minimal when ME-180 cells were treated with either cRA or IFN-alpha before radiation. When ME-180 cells were exposed to 10 microM cRA for 48 h and 1000 U/ml IFN-alpha for 24 h prior to radiation, there was a significant enhancement in radiation-induced cell killing; the dose modification factor was 2.1 +/- 0.9 at the 1% cell-survival level. On the other hand, HeLa-S3 cells exhibited no increased cytotoxicity or radiation enhancement under the same experimental conditions., Conclusion: The present data provide a radiobiological basis for using cRA and IFN-alpha as a combination radiosensitizer in selected human carcinoma cells.

Purpose: To demonstrate in a well-characterized tumor model that the radiosensitivity of tumor cells transduced with a herpes simplex virus thymidine kinase gene (HS-tk) would be selectively enhanced by antiviral agents., Methods and Materials: Rat 9L gliosarcoma cells transduced with a retroviral vector containing an HS-tk gene, 9L-tk cells were exposed to various doses of irradiation under either in vitro or in vivo conditions. The radiation sensitizing potential of two antiviral drugs, bromovinyl deoxyuridine (BVdU) and dihydroxymethyl ethyl methyl guanine (acyclovir), was evaluated in vitro. The radiosensitizing ability of BVdU was also evaluated with a 9L-tk tumor growing in the rat brain. Tumors growing in the right hemisphere of rat brains were irradiated stereotactically with single-dose irradiation., Results: The radiation response of 9L-tk cells was selectively enhanced by antiviral agents relative to nontransduced cells. In the cell culture, when a 24-h drug exposure (20 micrograms/ml) preceded radiation, the sensitizer enhancement ratio (SER) for BVdU and acyclovir was 1.4 +/- 0.1 and 1.3 +/- 0.1, respectively. Exposure of cells to 10 micrograms/ml acyclovir for two 24-h periods both pre- and postirradiation resulted in a SER of 1.6 +/- 0.1. In vivo, a significant increase in median survival time of rats with 9L-tk tumors was found when BVdU was administered prior to single-dose irradiation relative to the survival time of similar rats receiving radiation alone., Conclusion: An antiviral agent can enhance cell killing by radiation with selective action in cells transduced with the herpes simplex virus thymidine kinase gene. The results suggest that the three-pronged therapy of HS-tk gene transduction, systemically administered antiviral drug, and stereotactically targeted radiation therapy will improve the effectiveness of radiation therapy for the treatment of radioresistant tumors.

Survival and extracellular matrix gene expression were studied by viable cell count assay and Northern transfer analysis to compare the sensitivity of normal skin and keloid fibroblasts towards x-irradiation. As the dosage of radiation increased, the numbers of viable cells in irradiated groups were remarkably decreased exponentially, with no significant difference between normal and keloid cell lines. By Northern blot analysis, there was no change in size of the mRNAs for pro alpha 1(I) collagen, fibronectin and beta-actin. By slot-blot hybridization, pro alpha 1(I) collagen mRNA levels in x-irradiated fibroblasts were markedly decreased compared with non-irradiated controls. The amounts of fibronectin and beta-actin mRNAs were also decreased. This study suggests that both normal skin and keloid fibroblasts are sensitive to x-irradiation, and that extracellular matrix gene expression is also affected by such exposure.