A Practical Guide to 16S rRNA Microbiome Analysis in Musculoskeletal Disorders.

Microbial taxonomic assignment based on 16S marker gene amplification requires multiple data transformations, often encompassing the use of a variety of computational platforms. Bioinformatics analysis may represent a bottleneck for researchers as many tools require programmatic access in order to implement the software. Here we describe a step-by-step approach for taxonomic assignment using QIIME2 and highlight the utility of graphical-based microbiome tools for further analysis and identification of biological relevant taxa with reference to an outcome of interest.

Evidence review of physical distancing and partition screens to reduce healthcare acquired SARS-CoV-2.

We review the evidence base for two newly introduced Infection prevention and control strategies within UK hospitals. The new standard infection control precaution of 2 metres physical distancing and the use of partition screens as a means of source control of infection for SARS-CoV-2. Following review of Ovid-MEDLINE and governmental SAGE outputs there is limited evidence to support the use of 2 metres physical distancing and partition screens within healthcare.

Protecting the Microbiota.

We examine 3 different approaches to protecting the gut microbiome: highly targeted antibiotics, antibiotic destruction, and antibiotic binding. Each approach shows promise to prevent the off-target effects of antibiotics on the gut microbiome.

Dissemination of multiple carbapenem resistance genes in an in vitro gut model simulating the human colon.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) pose a major global health risk. Mobile genetic elements account for much of the increasing CPE burden. OBJECTIVES: To investigate CPE colonization and the impact of antibiotic exposure on subsequent resistance gene dissemination within the gut microbiota using a model to simulate the human colon. METHODS: Gut models seeded with CPE-negative human faeces [screened with BioMérieux chromID® CARBA-SMART (Carba-Smart), Cepheid Xpert® Carba-R assay (XCR)] were inoculated with distinct carbapenemase-producing Klebsiella pneumoniae strains (KPC, NDM) and challenged with imipenem or piperacillin/tazobactam then meropenem. Resistant populations were enumerated daily on selective agars (Carba-Smart); CPE genes were confirmed by PCR (XCR, Check-Direct CPE Screen for BD MAX™). CPE gene dissemination was tracked using PacBio long-read sequencing. RESULTS: CPE populations increased during inoculation, plateauing at ∼105 log10 cfu/mL in both models and persisting throughout the experiments (>65 days), with no evidence of CPE 'washout'. After antibiotic administration, there was evidence of interspecies plasmid transfer of blaKPC-2 (111742 bp IncFII/IncR plasmid, 99% identity to pKpQIL-D2) and blaNDM-1 (∼170 kb IncFIB/IncFII plasmid), and CPE populations rose from <0.01% to >45% of the total lactose-fermenting populations in the KPC model. Isolation of a blaNDM-1K. pneumoniae with one chromosomal single-nucleotide variant compared with the inoculated strain indicated clonal expansion within the model. Antibiotic administration exposed a previously undetected K. pneumoniae encoding blaOXA-232 (KPC model). CONCLUSIONS: CPE exposure can lead to colonization, clonal expansion and resistance gene transfer within intact human colonic microbiota. Furthermore, under antibiotic selective pressure, new resistant populations emerge, emphasizing the need to control exposure to antimicrobials.

Low fruit and vegetable consumption is associated with low knowledge of the details of the 5-a-day fruit and vegetable message in the UK: findings from two cross-sectional questionnaire studies.

BACKGROUND: This project aimed to understand the details of the 5-a-day fruit and vegetable (FV) message (which foods are included, portion sizes, the need for variety, reasons for consumption) least known by UK consumers, and most associated with low FV consumption. METHODS: Study 1 assessed FV consumption, knowledge of the details of the message, and relationships between these, using a short questionnaire administered face-to-face to an opportunity sample of one large UK city. Study 2 assessed the same variables using a comprehensive postal questionnaire administered across the UK to a representative population sample. RESULTS: Five hundred and seven respondents completed Study 1 and 247 respondents completed Study 2. The majority of individuals in both studies were aware of the 5-a-day message and could recount this correctly. In both studies, however, knowledge of the details of the message was low, and lower knowledge was associated with lower FV consumption. Respondents had lowest knowledge of the details of the message related to portion sizes and the need for variety. However, FV consumption was not independently associated with knowledge of any one aspect of the message. CONCLUSIONS: These findings suggest that, although most of the UK population sampled were aware of the 5-a-day FV message and could recount this correctly, details of the 5-a-day FV message were not well known, and that FV consumption was related to this knowledge. These findings suggest that strategies to increase FV consumption will benefit from increasing UK consumers' knowledge of the details of the 5-a-day FV message.

Pyruvate dehydrogenase kinase 4 exhibits a novel role in the activation of mutant KRAS, regulating cell growth in lung and colorectal tumour cells.

RAS signalling is involved in the control of several metabolic pathways including glycolysis, mitochondrial respiration and glutamine metabolism. Importantly, we have found here that loss of PDHK4, a key regulator of the pyruvate dehydrogenase complex, caused a profound cell growth inhibition in tumour cells harbouring KRAS mutations. Using isogenic cells and a panel of colorectal and lung cell lines we demonstrated that KRAS mutant cells showed a dependency on PDHK4 whereas KRAS wild-type cells were significantly resistant to PDHK4 knockdown. We have found that PDHK4 plays a role in the post-translational regulation of mutant KRAS activity. Depletion of PDHK4 causes disruption of KRAS cellular localization, a reduction in KRAS activity which, in turn, results in reduced MAPK signalling. Interestingly, PDHK4 and KRAS depletion resulted in a similar metabolic phenotype consisting of a reduction of glucose and fatty acid oxidation. Moreover, stable expression of PDHK4 increased localization of activated KRAS at the plasma membrane and induced tumour cell growth in vitro and in vivo. Taken together these data support a model where PDHK4 regulates KRAS signalling and its tumorigenic properties and suggest that inhibition of PDHK4 could represent a novel therapeutic strategy to target KRAS mutant colorectal and lung cancers.

How much is '5-a-day'? A qualitative investigation into consumer understanding of fruit and vegetable intake guidelines.

BACKGROUND: Despite the known health benefits of fruit and vegetables (FV), population intakes remain low. One potential contributing factor may be a lack of understanding surrounding recommended intakes. The present study aimed to explore the understanding of FV intake guidelines among a sample of low FV consumers. METHODS: Six semi-structured focus groups were held with low FV consumers (n = 28, age range 19-55 years). Focus groups were recorded digitally, transcribed verbatim and analysed thematically using nvivo (QSR International, Melbourne, Australia) to manage the coded data. Participants also completed a short questionnaire assessing knowledge on FV intake guidelines. Descriptive statistics were used to analyse responses. RESULTS: The discussions highlighted that, although participants were aware of FV intake guidelines, they lacked clarity with regard to the meaning of the '5-a-day' message, including what foods are included in the guideline, as well as what constitutes a portion of FV. There was also a sense of confusion surrounding the concept of achieving variety with regard to FV intake. The sample highlighted a lack of previous education on FV portion sizes and put forward suggestions for improving knowledge, including increased information on food packaging and through health campaigns. Questionnaire findings were generally congruent with the qualitative findings, showing high awareness of the '5-a-day' message but a lack of knowledge surrounding FV portion sizes. CONCLUSIONS: Future public health campaigns should consider how best to address the gaps in knowledge identified in the present study, and incorporate evaluations that will allow the impact of future initiatives on knowledge, and ultimately behaviour, to be investigated.

Orthodontic therapists - has their introduction affected outcomes?

Objective To assess the effect of the introduction of orthodontic therapists (OTs) on the quality of orthodontic treatment outcomes in two specialist orthodontic practices in the UK.Study design Retrospective cross sectional observational study.Setting Multi-centre evaluation at two specialist orthodontic practices in Yorkshire. Data collection was carried out during 2014.Materials and methods The treatment undertaken by three specialist orthodontic clinicians (A, B and C) was evaluated at two time points. The first time point (T1) was before the introduction of OTs when the specialist orthodontic clinicians were solo operators. The second time point (T2) followed the introduction of OTs. Patients at T2 had their treatment planned by a specialist orthodontist and were seen for care by both the orthodontist and an OT who had been qualified for a minimum of three years. A sample size of 30 orthodontic patients per clinician at each time point was chosen. Included participants had completed a course of fixed appliance therapy. They were consecutively selected from cases that had been completed in the specified time frame for each clinician.Main outcome measures The quality of treatment was assessed objectively using the quantitative Peer Assessment Rating index (PAR index). Data extracted from the specialist practice databases also allowed conclusions to be drawn about the length of treatment time and number of appointments in each treatment group.Results and conclusions There appears to have been no change in orthodontic treatment outcomes following the introduction of supervised OTs at two specialist orthodontic practices.

Application of a discovery to targeted LC-MS proteomics approach to identify deregulated proteins associated with idiosyncratic liver toxicity in a rat model of LPS/diclofenac co-administration.

Increasing experimental and clinical evidence suggest a contribution of non-drug related risk factors (e.g., underlying disease, bacterial/viral infection) to idiosyncratic drug reactions (IDR). Our previous work showed that co-treatment with bacterial endotoxin (LPS) and therapeutic doses of diclofenac (Dcl), an analgesic associated with drug idiosyncrasy in patients, induced severe hepatotoxicity in rats. Here, we used an integrated discovery to targeted LC-MS proteomics approach to identify mechanistically relevant liver and plasma proteins modulated by LPS/Dcl treatment, potentially applicable as early markers for IDRs. Based on pre-screening results and their role in liver toxicity, 47 liver and 15 plasma proteins were selected for targeted LC-MS analysis. LPS alone significantly changed the levels of 19 and 3 of these proteins, respectively. T-kininogen-1, previously suggested as a marker of drug-induced liver injury, was markedly elevated in plasma after repeated Dcl treatment in the absence of hepatotoxicity, possibly indicating clinically silent stress. Dcl both alone and in combination with LPS, caused up-regulation of the ATP synthase subunits (ATP5J, ATPA, and ATPB), suggesting that Dcl may sensitize cells against additional stress factors, such as LPS through generation of mitochondrial stress. Additionally, depletion of plasma fibrinogen was observed in the co-treatment group, consistent with an increased hepatic fibrin deposition and suspected contribution of the hemostatic system to IDRs. In contrast, several proteins previously suggested as liver biomarkers, such as clusterin, did not correlate with liver injury in this model. Taken together, these analyses revealed proteomic changes in a rat model of LPS/Dcl co-administration that could offer mechanistic insight and may serve as biomarkers or safety alert for a drug's potential to cause IDRs.

E-Cadherin and EpCAM expression by NSCLC tumour cells associate with normal fibroblast activation through a pathway initiated by integrin αvß6 and maintained through TGFß signalling.

Fibroblasts in the tumour stroma (cancer-associated fibroblasts) influence tumour progression and response to therapeutics; little is known about the mechanisms through which the tumour cell co-opts a normal fibroblast. To study the activation of fibroblasts by tumour cells, a panel of non-small cell lung cancer (NSCLC) cell lines and normal human dermal fibroblasts were co-cultured. A subset of the NSCLC cells induced an activated cancer-associated fibroblast-like fibroblast phenotype defined by induction of fibroblast α-smooth muscle actin expression. Tumour cells that activated fibroblasts were associated with E-Cadherin and EpCAM expression and expression of integrin αvß6. Co-culture of activating tumour cells with fibroblasts resulted in induction of transcripts associated with tumour cell invasion and growth, TGFß1 and TGFBR1, SERPINE-1, BMP6, SPHK1 and MMP9. Fibroblast activation was inhibited by an αvß6/8 integrin blocking antibody (264RAD) and a small molecule inhibitor of the TGF-beta type I receptor activin-like kinase (ALK5) (SB431542), demonstrating that transactivation of the TGFß pathway initiates fibroblast activation. Both integrin and ALK5 antagonists inhibited initiation. Only ALK5 was effective when added after 3 days of co-culture. This suggests that although activation is αvß6-dependent, once fibroblasts are activated alternative TGFß pathway regulators maintain an activation loop. In co-culture activating cells had reduced sensitivity to selumetinib, AZD8931 and afatinib compared with mono-culture. In contrast, non-activating cells were insensitive to selumetinib and AZD8931 in both mono-culture and co-culture. In conclusion NSCLC cell lines, positive for E-Cadherin, EpCAM and αvß6 expression, activate normal fibroblasts through avß6/TGFß signalling in vitro, and influence both gene expression and response to therapeutic agents.

Robust T cell responses to aspergillosis in chronic granulomatous disease: implications for immunotherapy.

Chronic granulomatous disease (CGD) patients are highly susceptible to invasive aspergillosis and might benefit from aspergillus-specific T cell immunotherapy, which has shown promise in treating those with known T cell defects such as haematopoietic stem cell transplant (HSCT) recipients. But whether such T cell defects contribute to increased risks for aspergillus infection in CGD is unclear. Hence, we set out to characterize the aspergillus-specific T cell response in CGD. In murine CGD models and in patients with CGD we showed that the CD4(+) T cell responses to aspergillus were unimpaired: aspergillus-specific T cell frequencies were even elevated in CGD mice (P < 0·01) and humans (P = 0·02), compared to their healthy counterparts. CD4-depleted murine models suggested that the role of T cells might be redundant because resistance to aspergillus infection was conserved in CD4(+) T cell-depleted mice, similar to wild-type animals. In contrast, mice depleted of neutrophils alone or neutrophils and CD4(+) T cells developed clinical and pathological evidence of pulmonary aspergillosis and increased mortality (P < 0·05 compared to non-depleted animals). Our findings that T cells in CGD have a robust aspergillus CD4(+) T cell response suggest that CD4(+) T cell-based immunotherapy for this disease is unlikely to be beneficial.

Combining T-cell immunotherapy and anti-androgen therapy for prostate cancer.

BACKGROUND: Prostate cancer remains a significant health problem for men in the Western world. Although treatment modalities are available, these do not confer long-term benefit and are accompanied by substantial side effects. Adoptive immunotherapy represents an attractive alternative to conventional treatments as a means to control tumor growth. METHODS: To selectively target the tumor-expressed form of Muc1 we constructed a retroviral vector encoding a chimeric antigen receptor (CAR) directed against the aberrantly-expressed extracellular portion of Muc1 called the 'variable number of tandem repeats'. RESULTS: We now demonstrate that T cells can be genetically engineered to express a CAR targeting the tumor-associated antigen Muc1. CAR-Muc1 T cells were able to selectively kill Muc1-expressing human prostate cancer cells. However, we noted that heterogeneous expression of the Muc1 antigen on tumor cells facilitated immune escape and the outgrowth of target-antigen loss variants of the tumor. Given the importance of androgen ablation therapy in the management of metastatic prostate cancer, we therefore also tested the value of combining conventional (anti-androgen) and experimental (CAR-Muc1 T cells) approaches. We show that CAR-Muc1 T cells were not adversely impacted by anti-androgen therapy and subsequently demonstrate the feasibility of combining the approaches to produce additive anti-tumor effects in vitro. CONCLUSIONS: Adoptive transfer of CAR-Muc1 T cells alone or in combination with other luteinizing hormone-releasing hormone analogs or antagonists should be tested in human clinical trials.