A comparison of the efficacy of trastuzumab deruxtecan in advanced HER2-positive breast cancer: active brain metastasis versus progressive extracranial disease alone.

BACKGROUND: Trastuzumab deruxtecan (T-DXd) has demonstrated efficacy in patients with brain metastasis (BM), a group historically with poor outcomes. The prevalence of BMs in patients commencing T-DXd is currently unknown. No direct comparisons have been made of the activity of T-DXd in patients with active BM versus those with extracranial progression alone. This real-world study explored the prevalence of BMs in patients commencing T-DXd, the efficacy of T-DXd in active BM versus extracranial progression alone and the safety of T-DXd. PATIENTS AND METHODS: Patients with human epidermal growth factor receptor 2-positive advanced breast cancer treated with T-DXd between June 2021 and February 2023 at our specialist cancer hospital were identified and notes reviewed. Clinicopathological information, prior treatment, the presence or absence of central nervous system (CNS) disease, outcomes and treatment-emergent adverse events (TEAEs) were recorded. RESULTS: Twenty-nine female patients, with a median age of 52 years (interquartile range 44-62 years), were identified; the prevalence of BM was 41%. Median number of lines of prior therapy was 2 (range 2-6). At a median follow-up of 13.8 months, median progression-free survival (PFS) for the overall population was 13.9 months [95% confidence interval (CI) 12.4 months-not estimable (NE)], 16.1 months (95% CI 15.1 months-NE) for active BMs and 12.4 months (95% CI 8.3 months-NE) for progressive extracranial disease alone. The 12-month overall survival (OS) rate was 74% (95% CI 59% to 95%) in the overall population, and 83% (95% CI 58% to 100%) and 66% (95% CI 45% to 96%) for active BMs and extracranial disease only, respectively. Most common TEAEs were fatigue, alopecia, and constipation. In nine patients (31%, including two deaths), pneumonitis occurred. CONCLUSION: In this real-world population, we demonstrate T-DXd to be effective in patients with active BMs and those with progressive extracranial disease alone. PFS and OS were numerically longer in those with active BMs. These data demonstrate that patients with active BM treated with T-DXd have at least comparable outcomes to those with extracranial disease alone. The high rate of pneumonitis warrants further consideration.

Molecular detection of Phytophthora pluvialis, the causal agent of red needle cast in Pinus radiata.

BACKGROUND: Phytophthora pluvialis was first described in 2013 and is the causal agent of red needle cast (RNC) in Pinus radiata as well as infection in Douglas fir (Pseudotsuga menziesii). A species-specific PCR is necessary for detection of this pathogen and diagnosis of RNC. OBJECTIVE: To design and validate a species-specific molecular assay for P. pluvialis using isolates from infected pine needles. METHODS: Species-specific PCR primers were generated from the ras-related GTP-binding protein 1 gene (ypt1) gene sequence, concentrating on DNA regions unique to P. pluvialis, and real-time and quantitative polymerase chain reaction (qPCR) were used to detect P. pluvialis from both artificially inoculated and naturally infected samples. RESULTS: The species-specific PCR assay was generated following P. pluvialis DNA sequence analysis. In vitro tests of the specificity of the probe-based, quantitative, polymerase chain reaction (qPCR) assay showed that no amplification was observed with other Phytophthora species including other closely-related clade 3 species, or with fungal species associated with pine or with pine DNA. The limit of detection of the qPCR assay was 2 pg/µl. When the qPCR assay was used to detect P. pluvialis in artificially-inoculated and naturally infected P. radiata needles, a PCR product was detected in all inoculated samples; the mean concentration ranges of P. pluvialis DNA in the inoculated and naturally infected samples tested were 5.9-124.5 pg/µl and 8.1-340.2 pg/µl, respectively. The assays described herein were used with serological diagnostic strips, providing the ability to identify to species level. CONCLUSIONS: The assay described herein detects P. pluvialis with high specificity and sensitivity from a range of DNA samples, including those extracted from infected plant material and serological diagnostic strips. The ability to detect and identify P. pluvialis, from infected tissues directly, provides value and practicality to diagnostics, biosecurity and research.

Functional dysbiosis within the gut microbiota of patients with constipated-irritable bowel syndrome.

BACKGROUND: The role of the gut microbiota in patho-physiology of irritable bowel syndrome (IBS) is suggested by several studies. However, standard cultural and molecular methods used to date have not revealed specific and consistent IBS-related groups of microbes. AIM: To explore the constipated-IBS (C-IBS) gut microbiota using a function-based approach. METHODS: The faecal microbiota from 14 C-IBS women and 12 sex-match healthy subjects were examined through a combined strictly anaerobic cultural evaluation of functional groups of microbes and fluorescent in situ hybridisation (16S rDNA gene targeting probes) to quantify main groups of bacteria. Starch fermentation by C-IBS and healthy faecal samples was evaluated in vitro. RESULTS: In C-IBS, the numbers of lactate-producing and lactate-utilising bacteria and the number of H(2) -consuming populations, methanogens and reductive acetogens, were at least 10-fold lower (P < 0.05) compared with control subjects. Concomitantly, the number of lactate- and H(2) -utilising sulphate-reducing population was 10 to 100 fold increased in C-IBS compared with healthy subjects. The butyrate-producing Roseburia - E. rectale group was in lower number (0.01 < P < 0.05) in C-IBS than in control. C-IBS faecal microbiota produced more sulphides and H(2) and less butyrate from starch fermentation than healthy ones. CONCLUSIONS: A major functional dysbiosis was observed in constipated-irritable bowel syndrome gut microbiota, reflecting altered intestinal fermentation. Sulphate-reducing population increased in the gut of C-IBS and were accompanied by alterations in other microbial groups. This could be responsible for changes in the metabolic output and enhancement in toxic sulphide production which could in turn influence gut physiology and contribute to IBS pathogenesis.

Human colonic microbiota associated with diet, obesity and weight loss.

BACKGROUND: It has been proposed that the development of obesity in humans is influenced by the relative proportions of the two major phyla of bacteria (Bacteroidetes and Firmicutes) present in the large intestine. OBJECTIVE: To examine the relationships between body mass index, weight loss and the major bacterial groups detected in fecal samples. DESIGN: Major groups of fecal bacteria were monitored using fluorescent in situ hybridization (FISH) in obese and non-obese subjects under conditions of weight maintenance, and in obese male volunteers undergoing weight loss on two different reduced carbohydrate weight-loss diets given successively for 4 weeks each. RESULTS: We detected no difference between obese and non-obese individuals in the proportion of Bacteroidetes measured in fecal samples, and no significant change in the percentage of Bacteroidetes in feces from obese subjects on weight loss diets. Significant diet-dependent reductions in a group of butyrate-producing Firmicutes were, however, detected in fecal samples from obese subjects on weight loss diets. CONCLUSIONS: Diets designed to achieve weight loss in obese subjects can significantly alter the species composition of the gut microbiota, but we find no evidence that the proportions of Bacteroidetes and Firmicutes among fecal bacteria have a function in human obesity.

Understanding the effects of diet on bacterial metabolism in the large intestine.

Recent analyses of ribosomal RNA sequence diversity have demonstrated the extent of bacterial diversity in the human colon, and have provided new tools for monitoring changes in the composition of the gut microbial community. There is now an excellent opportunity to correlate ecological niches and metabolic activities with particular phylogenetic groups among the microbiota of the human gut. Bacteria that associate closely with particulate material and surfaces in the gut include specialized primary degraders of insoluble substrates, including resistant starch, plant structural polysaccharides and mucin. Butyrate-producing bacteria found in human faeces belong mainly to the clostridial clusters IV and XIVa. In vitro and in vivo evidence indicates that a group related to Roseburia and Eubacterium rectale plays a major role in mediating the butyrogenic effect of fermentable dietary carbohydrates. Additional cluster XIVa species can convert lactate to butyrate, while some members of the clostridial cluster IX convert lactate to propionate. The metabolic outputs of the gut microbial community depend not only on available substrate, but also on the gut environment, with pH playing a major role. Better understanding of the colonic microbial ecosystem will help to explain and predict the effects of dietary additives, including nondigestible carbohydrates, probiotics and prebiotics.

Cultivable bacterial diversity from the human colon.

Knowledge of the composition of the colonic microbiota is important for our understanding of how the balance of these microbes is influenced by diet and the environment, and which bacterial groups are important in maintaining gut health or promoting disease. Molecular methodologies have advanced our understanding of the composition and diversity of the colonic microbiota. Importantly, however, it is the continued isolation of bacterial representatives of key groups that offers the best opportunity to conduct detailed metabolic and functional studies. This also permits bacterial genome sequencing which will accelerate the linkage to functionality. Obtaining new human colonic bacterial isolates can be challenging, because most of these are strict anaerobes and many have rather exact nutritional and physical requirements. Despite this many new species are being isolated and described that occupy distinct niches in the colonic microbial community. This review focuses on these under-studied yet important gut anaerobes.

Xyn11A, a multidomain multicatalytic enzyme from Pseudobutyrivibrio xylanivorans Mz5T.

The rumen bacterium Pseudobutyrivibrio xylanivorans Mz5T has a potent xylanolytic enzyme system. A small native peptide (approximately 30-kDa, designated Xyn11A) from the bacterium was first isolated and characterized by Edman degradation. The gene coding for Xyn11A was identified using PCR amplification with consensus primers. It was then fully sequenced to reveal an open reading frame of 1809 bp. The predicted N-terminal domain exhibited xylanolytic activity and was classed to the family 11 of glycosyl hydrolases; it is followed by a region with homology to a family 6 cellulose binding module. The C-terminal domain codes for a putative NodB-like polysaccharide deacetylase which is predicted to be an acetyl esterase implicated in debranching activity in the xylan backbone. As similar domain organization was also found in several other xylanases from a diverse range of bacteria, a common ancestor of such a xylanase is considered to be present and spread, possibly by horizontal gene transfer, to other microorganisms from different ecological niches.

Plant-like mating in an animal: sexual compatibility and allocation trade-offs in a simultaneous hermaphrodite with remote transfer of sperm.

The importance of sexual compatibility between mates has only recently been realized in zoological research into sexual selection, yet its study has been central to botanical research for many decades. The reproductive characteristics of remote mating, an absence of precopulatory mate screening, internal fertilization and embryonic brooding are shared between passively pollinated plants and a phylogenetically diverse group of sessile aquatic invertebrates. Here, we further characterize the sexual compatibility system of one such invertebrate, the colonial ascidian Diplosoma listerianum. All 66 reciprocal pairings of 12 genetic individuals were carried out. Fecundities of crosses varied widely and suggested a continuous scale of sexual compatibility. Of the 11 animals from the same population c. 40% of crosses were completely incompatible with a further c. 20% having obvious partial compatibility (reduced fecundity). We are unaware of other studies documenting such high levels of sexual incompatibility in unrelated individuals. RAPD fingerprinting was used to estimate relatedness among the 12 individuals after a known pedigree was successfully reconstructed to validate the technique. In contrast to previous results, no correlation between genetic similarity and sexual compatibility was detected. The blocking of many genotypes of sperm is expected to severely modify realized paternity away from 'fair raffle' expectations and probably reduce levels of intra-brood genetic diversity in this obligatorily promiscuous mating system. One adaptive benefit may be to reduce the bombardment of the female reproductive system by outcrossed sperm with conflicting evolutionary interests, so as to maintain female control of somatic : gametic investment.

The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants.

In 2000, the thematic network ENTRANSFOOD was launched to assess four different topics that are all related to the testing or assessment of food containing or produced from genetically modified organisms (GMOs). Each of the topics was linked to a European Commission (EC)-funded large shared cost action (see http://www.entransfood.com). Since the exchange of genetic information through horizontal (lateral) gene transfer (HGT) might play a more important role, in quantity and quality, than hitherto imagined, a working group dealing with HGT in the context of food and feed safety was established. This working group was linked to the GMOBILITY project (GMOBILITY, 2003) and the results of the deliberations are laid down in this review paper. HGT is reviewed in relation to the potential risks of consuming food or feed derived from transgenic crops. First, the mechanisms for obtaining transgenic crops are described. Next, HGT mechanisms and its possible evolutionary role are described. The use of marker genes is presented in detail as a special case for genes that may pose a risk. Furthermore, the exposure to GMOs and in particular to genetically modified (GM) deoxyribonucleic acid (DNA) is discussed as part of the total risk assessment. The review finishes off with a number of conclusions related to GM food and feed safety. The aim of this paper is to provide a comprehensive overview to assist risk assessors as well as regulators and the general public in understanding the safety issues related to these mechanisms.

Novel tetracycline resistance gene, tet(32), in the Clostridium-related human colonic anaerobe K10 and its transmission in vitro to the rumen anaerobe Butyrivibrio fibrisolvens.

A novel tetracycline resistance gene, designated tet(32), which confers a high level of tetracycline resistance, was identified in the Clostridium-related human colonic anaerobe K10, which also carries tet(W). tet(32) was transmissible in vitro to the rumen anaerobe Butyrivibrio fibrisolvens 2221(R). The predicted gene product of tet(32) has 76% amino acid identity with Tet(O). PCR amplification indicated that tet(32) is widely distributed in the ovine rumen and in porcine feces.

EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R. flavefaciens protein via a dockerin domain.

The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945-1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.

Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage.

A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number of Shigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.

Adaptation of Ruminococcus flavefaciens resulting in increased degradation of ryegrass cell walls.

This study investigated the long term adaptation of a ruminal bacterium to growth on four different plant cell wall substrates. No significant increase in degradation was detected for lucerne, barley straw or weeping lovegrass after 23 serial subcultures of the cellulolytic rumen bacterium Ruminococcus flavefaciens strain 17 on each of these substrates. Significantly increased substrate degradation by R. flavefaciens strain 17 was however observed after 23 subcultures on perennial ryegrass. The increase in dry matter solubilisation (from 24.3 to 39.5% in 24 h incubation and from 52.3 to 61% in 72 h) was at least partially due to an increase in solubilisation of xylose, glucose and arabinose. Enhanced growth of the adapted strains occurred on this substrate. Significant increases in xylanase and beta-xylosidase specific activities were detected but no effect was detected on xylanase profiles in zymogram analyses. Similar responses were observed for two cultures originally derived from single-colony re-isolates. The most likely explanation for the observed adaptation involves selection for mutations affecting the regulation of xylanolytic enzymes.

Sequence analysis of the plasmid pRRI2 from the rumen bacterium Prevotella ruminicola 223/M2/7 and the use of pRRI2 in Prevotella/Bacteroides Shuttle Vectors.

pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli. Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames. Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively. ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp. plasmid pFL1 and 30% amino acid sequence identity with BmpH from B. fragilis Tn5520. Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp. hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate. pRRI2 shows no significant similarity with the only other P. ruminicola plasmid to have been studied previously, pRAM4.

Transformation of an oral bacterium via chromosomal integration of free DNA in the presence of human saliva.

Transformation of Streptococcus gordonii DL1 by free DNA was studied in human saliva. Competent S. gordonii could be transformed in vitro with plasmid DNA that had been taken into the human mouth. Transformation also occurred with a plasmid that cannot replicate in S. gordonii, but that has a region of chromosomal homology, by integration into the bacterial chromosome, although linearised plasmid DNA gave no transformants. Linear chromosomal DNA fragments did however transform S. gordonii/Tn916 efficiently in saliva when regions of homology with the recipient chromosome flanked the marker gene. These findings are discussed in relation to the potential for acquisition of DNA sequences, including genetically modified DNA, by gut and oral bacteria.

Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens.

Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium, Ruminococcus flavefaciens. The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequences. The scaB gene was completely sequenced, and its upstream neighbor scaA was partially sequenced. The sequenced portion of scaA contains repeating cohesin modules and a C-terminal dockerin domain. ScaB contains seven relatively divergent cohesin modules, two extremely long T-rich linkers, and a C-terminal domain of unknown function. Collectively, the cohesins of ScaA and ScaB are phylogenetically distinct from the previously described type I and type II cohesins, and we propose that they define a new group, which we designated here type III cohesins. Selected modules from both genes were overexpressed in Escherichia coli, and the recombinant proteins were used as probes in affinity-blotting experiments. The results strongly indicate that ScaA serves as a cellulosomal scaffoldin-like protein for several R. flavefaciens enzymes. The data are supported by the direct interaction of a recombinant ScaA cohesin with an expressed dockerin-containing enzyme construct from the same bacterium. The evidence also demonstrates that the ScaA dockerin binds to a specialized cohesin(s) on ScaB, suggesting that ScaB may act as an anchoring protein, linked either directly or indirectly to the bacterial cell surface. This study is the first direct demonstration in a cellulolytic rumen bacterium of a cellulosome system, mediated by distinctive cohesin-dockerin interactions.

Nursing development units: progress and developments.

Nursing development units (NDUs) are centres where groups of nurses, midwives, public health nurses and health visitors use a comprehensive planned strategy to develop themselves and their practice. Some of the early defining work on NDUs is considered and a brief history is outlined in this article. The strategic approach taken by the Eastern Health Shared Services in Dublin has led to the development of many NDUs in the Dublin area over a five-year period. The NDUs are based on a strategic, supportive approach. Six now exist in the state-funded health service system, but the project has also been widened to include the private and voluntary sectors and this has stimulated several other units to emerge. Focus group work with NDU leaders and facilitators has led to a gradual redefining of the principles of NDUs and the emergence of an accreditation system run by the board. The authors summarise these findings.

Adhesion of Salmonella enterica var Enteritidis strains lacking fimbriae and flagella to rat ileal explants cultured at the air interface or submerged in tissue culture medium.

Rat ileal air interface and submerged explant models were developed and used to compare the adhesion of Salmonella enterica var Enteritidis wild-type strains with that of their isogenic single and multiple deletion mutants. The modified strains studied were defective for fimbriae, flagella, motility or chemotaxis and binding was assessed on tissues with and without an intact mucus layer. A multiple afimbriate/aflagellate (fim-/fla-) strain, a fimbriate but aflagellate (fla-) strain and a fimbriate/flagellate but non-motile (mot-) strain bound significantly less extensively to the explants than the corresponding wild-type strains. With the submerged explant model this difference was evident in tissues with or without a mucus layer, whereas in the air interface model it was observed only in tissues with an intact mucus layer. A smooth swimming chemotaxis-defective (che-) strain and single or multiple afimbriate strains bound to explants as well as their corresponding wild-type strain. This suggests that under the present experimental conditions fimbriae were not essential for attachment of S. enterica var Enteritidis to rat ileal explants. However, the possession of active flagella did appear to be an important factor in enabling salmonellae to penetrate the gastrointestinal mucus layer and attach specifically to epithelial cells.