Quality assurance of selective culture media for bacteria, moulds and yeasts: an attempt at standardization at the international level

To facilitate monitoring of culture media, a simple quantitative streaking technique, implying ever-decreasing numbers of colony-forming units per surface area, as in spiral plating, was developed. The procedure evaluates, in quantitative terms, the ability of media (1) to support the formation of colonies by organisms that it was designed to grow and (2) to resist colonization by organisms that it is expected to suppress. The procedure was therefore termed ecometric evaluation. The ecometric results appeared to agree well with observations made on productivity and selectivity of the media studied during routine examination of specimens. These encouraging results prompted further, rigorous standardization of ecometry. A template was developed to standardize inoculation and the depth of the agar was controlled to within +/- 10%. Finally the attributes of the inocula used were accurately defined. The standardized ecometric technique has been found useful for the following purposes: (1) to assess the practical significance of the inhibitory effect of gentamicin observed in some moulds and yeasts (this was solved by replacing poorer basal media by one particular richer modification, viz, yeast morphology agar); (2) the development of a blood-free selective enumeration medium for Campylobacter jejuni, i.e. sulphide iron motility agar plus the combination of antibiotics suggested by Skirrow (1977); and (3) verification of the absence of antimicrobial activity of enzyme preparations, e.g. catalase, used in culture media to remedy sublethal damage in certain groups of bacteria. Ecometric evaluation can now be recommended for (1) routine monitoring of consignments of dehydrated or ready-to-use, purchased media; and (2) in-house checking of the functioning of medium preparation departments. Only occasionally is it necessary to use conventional counting techniques to confirm the results.     

Partial hypoxanthine-guanine phosphoribosyl transferase deficiency with full expression of the Lesch-Nyhan syndrome

Summary A patient with the full clinical expression of the classical Lesch-Nyhan syndrome is presented with a residual hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity of 5–10% in erythrocyte lysate and about 30% in fibroblast lysate. The activities of other erythrocyte enzymes of purine metabolism were typical for a classical Lesch-Nyhan patient. The effects of allopurinol therapy on the excretion of urinary purine metabolites were studied by a newly developed isotachophoretic technique.The unusually high residual activity of HGPRT in erythrodytes and fibroblasts of the patient enabled the enzymologic characterization of the mutant enzyme: in fibroblasts the affinities for the substrates hypoxanthine and guanine were normal. However, there was an increased apparent K _m for phosphoribosylpyrophosphate (PRPP), a complete absence of product inhibition by IMP and GMP, and a decreased heat stability. Addition of PRPP did not stabilize the mutant enzyme. In addition to the altered properties of the fibroblast enzyme, the K _m of the erythrocyte enzyme for hypoxanthine was also increased.Immunoprecipitation experiments revealed the presence of an approximately normal amount of material cross-reacting with anti-human HGPRT antiserum. However, it appeared that this cross-reacting material had a decreased stability. When intact erythrocytes were incubated with radiolabeled purine bases, no formation of IMP or GMP could be detected, despite the relatively high residual activity of HGPRT in the hemolysate. The results fit the following hypothesis: as a consequence of a structural mutation affecting the PRPP-site of the enzyme and a decreased heat stability, the activity of the mutant enzyme under in vivo conditions is virtually zero.In the erythrocytes of the patient's mother a normal HGPRT-activity was found. However, the activity in her fibroblasts was lower than normal, while a decreased heat stability and an intermediate behavior towards IMP could be shown.Hair root analysis of several members of the patient's family confirmed the heterozygosity of the mother, whereas no other heterozygotes could be detected. The family anamnesis did not show other cases of Lesch-Nyhan syndrome. These findings were taken as evidence that the patient described in this paper might represent a mutation orginating from the gametes in either of the maternal grandparents.     

Artificial reporter gene providing MRI contrast based on proton exchange

Existing magnetic resonance reporter genes all rely on the presence of (super)paramagnetic substances and employ water relaxation to gain contrast. We designed a nonmetallic, biodegradable, lysine rich-protein (LRP) reporter, the prototype of a potential family of genetically engineered reporters expressing artificial proteins with frequency-selective contrast. This endogenous contrast, based on transfer of radiofrequency labeling from the reporter's amide protons to water protons, can be switched on and off.     

Patterns of peroxidative ethane emission from submerged rice seedlings indicate that damage from reactive oxygen species takes place during submergence and is not necessarily a post-anoxic phenomenon

Using ethane as a marker for peroxidative damage to membranes by reactive oxygen species (ROS) we examined the injury of rice seedlings during submergence in the dark. It is often expressed that membrane injury from ROS is a post-submergence phenomenon occurring when oxygen is re-introduced after submergence-induced anoxia. We found that ethane production, from rice seedlings submerged for 24–72 h, was stimulated to 4–37 nl gFW⁻¹, indicating underwater membrane peroxidation. When examined a week later the seedlings were damaged or had died. On de-submergence in air, ethane production rates rose sharply, but fell back to less than 0.1 nl gFW⁻¹ h⁻¹ after 2 h. We compared submergence-susceptible and submergence-tolerant cultivars, submergence starting in the morning (more damage) and in the afternoon (less damage) and investigated different submergence durations. The seedlings showed extensive fatality whenever total ethane emission exceeded about 15 nl gFW⁻¹. Smaller amounts of ethane emission were linked to less extensive injury to leaves. Partial oxygen shortage (O₂ levels <1%) imposed for 2 h in gas phase mixtures also stimulated ethane production. In contrast, seedlings under anaerobic gas phase conditions produced no ethane until re-aerated: then a small peak was observed followed by a low, steady ethane production. We conclude that damage during submergence is not associated with extensive anoxia. Instead, injury is linked to membrane peroxidation in seedlings that are partially oxygen deficient while submerged. On return to air, further peroxidation is suppressed within about 2 h indicating effective control of ROS production not evident during submergence itself.     

A microscopy study of hyphal growth of Penicillium rubens on gypsum under dynamic humidity conditions

To remediate indoor fungal growth, understanding the moisture relations of common indoor fungi is crucial. Indoor moisture conditions are commonly quantified by the relative humidity (RH). RH is a major determinant of the availability of water in porous indoor surfaces that fungi grow on. The influence of steady‐state RH on growth is well understood. Typically, however, the indoor RH constantly changes so that fungi have to endure frequent periods of alternating low and high RH. Knowledge of how common indoor fungi survive and are affected by the low‐RH periods is limited. In particular, the specific effects of a drop in RH on the growth of the mycelium remain unclear. In this work, video microscopy was used to monitor hyphal growth of Penicillium rubens on gypsum substrates under controlled dynamic humidity conditions. The effect of a single period of low RH (RH = 50–90%) interrupting favourable conditions (RH = 97%) was tested. It was found that hyphal tips ceased to extend when exposed to any tested decrease in RH. However, new hyphal growth always emerges, seemingly from the old mycelium, suggesting that this indoor fungus does not rely only on conidia to survive the humidity patterns considered. These findings are a fundamental step in unravelling the effect of RH on indoor fungal growth.     

Comparison of coenzyme F420 from Methanobacterium bryantii with 7- and 8-hydroxy-10-methyl-5-deazaisoalloxazine

Syntheses of 7-hydroxy-10-methyl-5-deazaisoalloxazine (7-HMDI) and 8-hydroxy-10-methyl-5-deazaisoalloxazine (8-HMDI) are described. The physicochemical and biological properties of 8-HMDI, in contrast to those of 7-HMDI, are very analogous to those of F₄₂₀, a coenzyme found in methanogenic bacteria.     

Modulation of hypoxia in murine liver metastases of colon carcinoma by nicotinamide and carbogen

There is increasing evidence that modulation of tumor hypoxia may improve therapy outcome. However, most preclinical data are derived from subcutaneous rather than orthotopic tumor models. We investigated the effect of the hypoxia-modulating agents nicotinamide and carbogen on tumor hypoxia, tumor blood perfusion, and proliferative activity in liver metastases of the murine colon carcinoma line C26a. In untreated C26a liver metastases, we observed a considerable amount of hypoxia, similar to the amount in liver metastases of patients with colorectal cancer. Compared to untreated mice, we observed a significantly smaller hypoxic fraction in the liver metastases of mice treated with nicotinamide and carbogen breathing as single treatments or in combination. In the group of mice that underwent carbogen breathing, perfusion was significantly lower than in the untreated group, but the decrease was only marginal. The proliferative activity was similar in all groups. In C26a subcutaneous tumors, a similar effect on hypoxia has been observed that was, however, combined with a decrease in proliferative activity. The different effects of nicotinamide and carbogen on parameters of the tumor microenvironment in liver metastases and subcutaneous tumors suggest that the host tissue influences the mechanism by which nicotinamide and carbogen exert their effects. Since tumor hypoxia may be a clinical problem in colorectal liver metastases, our results open possibilities for further research on the effect of hypoxia modifiers on colorectal liver metastases to improve therapy outcome.     

18F-FDG PET-CT after Neoadjuvant Chemoradiotherapy in Esophageal Cancer Patients to Optimize Surgical Decision Making

Background

Prognosis of esophageal cancer patients can be significantly improved by neoadjuvant chemoradiotherapy (nCRT). Given the aggressive nature of esophageal tumors, it is conceivable that in a significant portion of patients treated with nCRT, dissemination already becomes manifest during the period of nCRT. The aim of this retrospective study was to determine the value and diagnostic accuracy of PET-CT after neoadjuvant chemoradiotherapy to identify patients with metastases preoperatively in order to prevent non-curative surgery.

Methods

From January 2011 until February 2013 esophageal cancer patients deemed eligible for a curative approach with nCRT and surgical resection underwent a PET-CT after completion of nCRT. If abnormalities on PET-CT were suspected metastases, histological proof was acquired. A clinical decision model was designed to assess the cost-effectiveness of this diagnostic strategy.

Results

156 patients underwent a PET-CT after nCRT. In 31 patients (19.9%) PET-CT showed abnormalities suspicious for dissemination, resulting in 17 cases of proven metastases (10.9%). Of the patients without proven metastases 133 patients were operated. In 6 of these 133 cases distant metastases were detected intraoperatively, corresponding to 4.5% false-negative results. The standard introduction of a post-neoadjuvant therapy PET-CT led to a reduction of overall health care costs per patient compared to a scenario without restaging with PET-CT ($34,088 vs. $36,490).

Conclusion

In 10.9% of esophageal cancer patients distant metastases were detected by standard PET-CT after neoadjuvant chemoradiotherapy. To avoid non-curative resections we advocate post-neoadjuvant therapy PET-CT as a cost-effective step in the standard work-up of candidates for surgery.