A vertebral skeletal stem cell lineage driving metastasis.

Vertebral bone is subject to a distinct set of disease processes from long bones, including a much higher rate of solid tumour metastases1-4. The basis for this distinct biology of vertebral bone has so far remained unknown. Here we identify a vertebral skeletal stem cell (vSSC) that co-expresses ZIC1 and PAX1 together with additional cell surface markers. vSSCs display formal evidence of stemness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. vSSCs are physiologic mediators of vertebral bone formation, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and stemness features. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed in breast cancer, owing in part to increased secretion of the novel metastatic trophic factor MFGE8. Together, our results indicate that vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of vertebral metastasis.

CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc.

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.

Prognostic value of serial coronary computed tomography angiography-derived perivascular fat-attenuation index and plaque volume in patients with suspected coronary artery disease.

AIMS: To investigate the prognostic value of serial coronary computed tomography angiography (CCTA) derived plaque information, fractional flow reserve (CT-FFR), and perivascular fat-attenuation index (FAI) on major adverse cardiac events (MACE) in patients with suspected coronary artery disease. MATERIALS AND METHODS: A total of 252 patients who underwent serial CCTA between January 2018 and December 2021 and were followed until June 2022. MACE were recorded. The analysis indexes included percent diameter stenosis (%DS), lesion length, plaque volume, CT-FFR, and FAI, with an emphasis on their changes between the baseline and follow-up CCTAs. Multivariate regression analysis were employed to identify independent risk factors for MACE. RESULTS: After a median follow-up of 48-month, MACE occurred in 32 patients (12.7%). Patients with MACE displayed more severe stenosis, longer lesions, and larger plaque volumes in both baseline and follow-up CCTAs compared with no-MACE patients (all P<0.05). Patients with MACE displayed more severe stenosis, longer lesion, and larger plaque volume in both baseline and follow-up CCTAs compared with no-MACE patients. In addition, MACE patients also showed lower CT-FFR and higher △CT-FFR. Although FAI was significantly higher in MACE patients at baseline CCTA, FAI was notably increased in MACE patients, and decreased in the no-MACE patients (all P<0.05). Logistic regression analysis showed that ΔFAI, %DS, and plaque volume were independent predictors of MACE, with ΔFAI being the most significant (OR: 16.725, P<0.000). A multivariable model showed a significantly improved C-index of 0.903 (95% confidence interval: 0.836-0.970) for MACE prediction, when compared with single index alone. CONCLUSIONS: Serial CCTA-derived ΔFAI, %DS, and plaque volume are crucial independent predictors of MACE in patients with suspected coronary artery disease, highlighting the importance of CCTA in patient risk stratification and prognostic assessment.

A comparative study for the evaluation of CT-based conventional, radiomic, combined conventional and radiomic, and delta-radiomic features, and the prediction of the invasiveness of lung adenocarcinoma manifesting as ground-glass nodules.

AIM: To investigate and compare the performance of conventional, radiomic, combined, and delta-radiomic features to predict the invasiveness of lung adenocarcinoma manifesting as ground-glass nodules (GGNs). MATERIALS AND METHODS: The present retrospective study included 216 GGNs confirmed surgically as pulmonary adenocarcinomas. All the thin-section computed tomography (CT) images were imported into the software of the United Imaging Intelligence research portal, and radiomic features were extracted with three-dimensional (3D) regions of interest. Least Absolute Shrinkage and Selection Operator was used to select the optimal radiomic features. Four models were constructed, including conventional, radiomic, combined conventional and radiomic, and delta-radiomic models. The receiver operating characteristic curves were built to evaluate the validity of these. RESULTS: The type, long diameter, shape, margin, vacuole, air bronchus, vascular convergence, and pleural traction exhibited significant differences between pre-invasive lesions (PILs)/minimally invasive adenocarcinoma (MIA), and invasive adenocarcinoma (IA) groups were selected for conventional model building. Nine radiomic features were selected to build the radiomic model. The four models indicated optimal performance (AUC > 0.7). The radiomic and combined models exhibited the highest diagnostic efficiency, and their AUC were 0.89 and 0.88 in the training set, and 0.87 and 0.88 in the validation set, respectively. The delta-radiomic model indicated that the AUC was 0.83 in the training set, and 0.76 in the validation set. Finally, the conventional model exhibited an AUC in the training and validation sets of 0.78 and 0.76. CONCLUSIONS: The radiomic model and combined model, in particular, and the delta-radiomic model all demonstrated improved diagnostic efficiency in differentiating IA from PIL/MIA than that of the conventional model.

Two-year outcomes of Roux-en-Y gastric bypass vs medical treatment in type 2 diabetes with a body mass index lower than 32.5 kg/m2: a multicenter propensity score-matched analysis.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) has been widely reported to be safe and feasible, and has a powerful effect on improving metabolism and weight loss in patients with a high body mass index (BMI). A few studies have focused on the comparison of RYGB with medical treatment in type 2 diabetes (T2D) patients with a lower BMI. OBJECTIVES: To compare the metabolic effects and safety of RYGB versus medical treatment during a 2 years follow-up in T2D patients with a BMI of 25 to 32.5 kg/m2. METHODS: This retrospective and multicenter cohort study participants were extracted from the T2D patients with a lower BMI (25-32.5 kg/m2) from three bariatric centers between 2009 and 2018. Propensity score matching (PSM) was used to minimize bias, and each patient in the surgical group was matched 1:2 to the patients in the medical group with the closest propensity score. Finally, 71 patients who received RYGB and 142 patients who underwent medical treatment with a 2 years follow-up were enrolled to compare the effects of RYGB and medical treatment. The primary endpoint was achievement of the triple endpoint (the simultaneous achievement of hemoglobin A1c (HbA1c) < 7.0%, fasting low-density lipoprotein cholesterol (LDL-C) < 100 mg/dL (2.6 mmol/L), and systolic blood pressure (SBP) < 130 mmHg at the year-1 visit). Changes in weight, BMI, medication usage, complications, and adverse events were assessed. RESULTS: In total, 213 patients (mean age of 47.4 ± 9.5 years, 70.4% male, mean BMI of 28.6 ± 2.2 kg/m2) were included in this study. At the end of the first year, 17 patients (23.9%) in the surgical group and 10 (7.0%) in the medical group had achieved the composite triple endpoint (OR 4.64; 95% CI 1.82-11.81; p = 0.001). Additionally, 43 patients (60.6%) in the surgical group and 11 patients (19.7%) in the medical group experienced remission of T2D. However, more complications were observed in the surgical group (36 vs. 22, p < 0.01). CONCLUSIONS: Among T2D patients with a BMI between 25.0 and 32.5 kg/m2, RYGB was more effective than medical treatment in resolving metabolic disorders and also resulted in more complications. The risk for complications should be considered in the clinical decision-making process for T2D patients with a low BMI.

Relationship between parent-child attachment and depression among migrant children and left-behind children in China.

OBJECTIVES: The objective of this study was to examine the relationship between parent-child attachment and depression in migrant children (MC), left-behind children (LBC) and non-left-behind children (NLBC) in China. STUDY DESIGN: This was a cross-sectional study. METHODS: In total, 4294 children (4th to 9th grade) participated in this study, including 677 MC, 1411 LBC and 2206 NLBC from 17 different schools. RESULTS: The results showed that (1) the prevalence of depression among MC (21.0%) and LBC (14.0%) was significantly higher than that among NLBC (10.8%); (2) the quality of parent-child attachment among MC was statistically significantly lower than among LBC and NLBC (the proportion of children whose father-child and mother-child attachments were both insecure was 55.4% among MC, 29.9% among LBC and 33.7% among NLBC); and (3) depression was affected by the interaction between the group of children and parent-child attachment; compared with NLBC whose parent-child attachments were both secure, the odds ratio of depression among MC whose parent-child attachments were both insecure was 7.39, which was significantly higher than LBC (5.34) and NLBC (4.86) whose parent-child attachments were both insecure. CONCLUSIONS: The prevalence of depression among MC and LBC was significantly higher than that among NLBC in China. The quality of parent-child attachment among MC was statistically significantly lower than that of LBC and NLBC. Secure attachment could reduce the risk of depression and insecure parent-child attachment increased the risk of depression. Depression was affected by the interaction between the group of children and parent-child attachment; migration was a significant risk factor associated with child depression.

Nasopharyngeal Microbiota Profiles in Rural Venezuelan Children Are Associated With Respiratory and Gastrointestinal Infections.

BACKGROUND: Recent research suggests that the microbiota affects susceptibility to both respiratory tract infections (RTIs) and gastrointestinal infections (GIIs). In order to optimize global treatment options, it is important to characterize microbiota profiles across different niches and geographic/socioeconomic areas where RTI and GII prevalences are high. METHODS: We performed 16S sequencing of nasopharyngeal swabs from 209 Venezuelan Amerindian children aged 6 weeks-59 months who were participating in a 13-valent pneumococcal conjugate vaccine (PCV13) study. Using random forest models, differential abundance testing, and regression analysis, we determined whether specific bacteria were associated with RTIs or GIIs and variation in PCV13 response. RESULTS: Microbiota compositions differed between children with or without RTIs (P = .018) or GIIs (P = .001). Several species were associated with the absence of infections. Some of these health-associated bacteria are also observed in developed regions, such as Corynebacterium (log2(fold change [FC]) = 3.30 for RTIs and log2(FC) = 1.71 for GIIs), while others are not commonly observed in developed regions, such as Acinetobacter (log2(FC) = 2.82 and log2(FC) = 5.06, respectively). Klebsiella spp. presence was associated with both RTIs (log2(FC) = 5.48) and GIIs (log2(FC) = 7.20). CONCLUSIONS: The nasopharyngeal microbiota of rural Venezuelan children included several bacteria that thrive in tropical humid climates. Interestingly, nasopharyngeal microbiota composition not only differed in children with an RTI but also in those with a GII, which suggests a reciprocal interplay between the 2 environments. Knowledge of region-specific microbiota patterns enables tailoring of preventive and therapeutic approaches.

Development of a posterior sagittal anorectal surgical teaching model.

BACKGROUND: An Anorectal Malformation (ARM) is a rare congenital malformation, which requires proper correction to ensure the best long-term prognosis. These procedures are relatively infrequent and complex, in which a structured approach is important. Therefore, training on an affordable model could be beneficial. METHODS: A low-cost ARM model was developed. The base was reusable and the perineal body disposable. Both expert pediatric surgeons (Experts) and residents/fellows (Target group) were recruited for this study. After testing the model, they completed a questionnaire regarding the realism and didactic value of the model, using a 5-point Likert scale. RESULTS: Forty-four participants were recruited (Target group n = 20, Experts n = 24). The model has high mean scores of 3.8-4.4 for the total group and even higher on several aspects by the Target group. The experts regarded the haptics and manipulation of the fistula less realistic than the Target group (3.7 versus 4.3, p = 0.021 and 4.2 versus 4.6, p = 0.047). It was considered to be a very good training tool (mean 4.3), without significant differences between the groups. CONCLUSIONS: These results show general consensus that this model is a potent training tool for the component steps of the repair of an ARM with recto-perineal fistula by sagittal approach.

Loss of Microbial Topography between Oral and Nasopharyngeal Microbiota and Development of Respiratory Infections Early in Life.

Rationale: The respiratory microbiota is increasingly being appreciated as an important mediator in the susceptibility to childhood respiratory tract infections (RTIs). Pathogens are presumed to originate from the nasopharyngeal ecosystem.Objectives: To investigate the association between early life respiratory microbiota and development of childhood RTIs.Methods: In a prospective birth cohort (Microbiome Utrecht Infant Study: MUIS), we characterized the oral microbiota longitudinally from birth until 6 months of age of 112 infants (nine regular samples/subject) and compared them with nasopharyngeal microbiota using 16S-rRNA-based sequencing. We also characterized oral and nasopharynx samples during RTI episodes in the first half year of life.Measurements and Main Results: Oral microbiota were driven mostly by feeding type, followed by age, mode of delivery, and season of sampling. In contrast to our previously published associations between nasopharyngeal microbiota development and susceptibility to RTIs, oral microbiota development was not directly associated with susceptibility to RTI development. However, we did observe an influx of oral taxa, such as Neisseria lactamica, Streptococcus, Prevotella nanceiensis, Fusobacterium, and Janthinobacterium lividum, in the nasopharyngeal microbiota before and during RTIs, which was accompanied by reduced presence and abundance of Corynebacterium, Dolosigranulum, and Moraxella spp. Moreover, this phenomenon was accompanied by reduced niche differentiation indicating loss of ecological topography preceding confirmed RTIs. This loss of ecological topography was further augmented by start of daycare, and linked to consecutive development of symptomatic infections.Conclusions: Together, our results link the loss of topography to subsequent development of RTI episodes. This may lead to new insights for prevention of RTIs and antibiotic use in childhood.

Novel regulatory mechanism of establishment genes of conjugative plasmids.

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg576. Reg576 also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg576 is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg576. Each operator is bound with extreme cooperativity by two Reg576-dimers. The X-ray structure revealed that Reg576 has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition.

Discovery of a new family of relaxases in Firmicutes bacteria.

Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named relLS20, is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the RelLS20 shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research.

Epiphrenic oesophageal diverticulum managed via laparoscopic transhiatal approach.

Epiphrenic oesophageal diverticulum is a rare disorder affecting the distal oesophagus. Surgical techniques for this condition evolve over time from open transthoracic and trans-abdominal approaches to minimally invasive surgery. We report a case of an 82-year-old male who presented with symptomatic epiphrenic oesophageal diverticulum over the last 1 year. He underwent laparoscopic transhiatal diverticulectomy, myotomy and anterior partial fundoplication and was discharged well. He remains asymptomatic after a follow-up of 6 months.

Imaging PD-L1 Expression with ImmunoPET.

High sensitivity imaging tools could provide a more holistic view of target antigen expression to improve the identification of patients who might benefit from cancer immunotherapy. We developed for immunoPET a novel recombinant human IgG1 (termed C4) that potently binds an extracellular epitope on human and mouse PD-L1 and radiolabeled the antibody with zirconium-89. Small animal PET/CT studies showed that 89Zr-C4 detected antigen levels on a patient derived xenograft (PDX) established from a non-small-cell lung cancer (NSCLC) patient before an 8-month response to anti-PD-1 and anti-CTLA4 therapy. Importantly, the concentration of antigen is beneath the detection limit of previously developed anti-PD-L1 radiotracers, including radiolabeled atezolizumab. We also show that 89Zr-C4 can specifically detect antigen in human NSCLC and prostate cancer models endogenously expressing a broad range of PD-L1. 89Zr-C4 detects mouse PD-L1 expression changes in immunocompetent mice, suggesting that endogenous PD-1/2 will not confound human imaging. Lastly, we found that 89Zr-C4 could detect acute changes in tumor expression of PD-L1 due to standard of care chemotherapies. In summary, we present evidence that low levels of PD-L1 in clinically relevant cancer models can be imaged with immunoPET using a novel recombinant human antibody.

Accelerated Step-Up Infliximab Use Is Associated with Sustained Primary Response in Pediatric Crohn's Disease.

BACKGROUND: Earlier introduction of infliximab (IFX) in Crohn's disease (CD) may be associated with a sustained remission. METHODS AND AIMS: Children on scheduled IFX therapy for predominant luminal CD after successful induction (drop in PCDAI by ≥ 15) and a minimum of 2-year IFX follow-up were included. We compared outcomes of children treated with early (within 3 months from diagnosis) versus later IFX (after failing conventional therapy ≥ 3 months) and identify clinical predictors of sustained primary response (SPR) in our cohort. SPR was defined as CS-free clinical remission without requiring IFX dose escalation and/or surgical excision and/or switch to second anti-TNFs due to LOR or allergic reaction. RESULTS: Sixty-four children received IFX therapy for CD during the study period. Forty-three children on scheduled IFX therapy for luminal CD met the inclusion criteria. During the median follow-up of 3.05 years (IQR 2.6-3.5 years), SPR was observed in 17/43 (40%). SPR was associated with shorter time from diagnosis to the initiation of IFX (5.4 vs. 18.7 months, p = 0.006). Binary logistic regression using multiple variables also confirmed that only early use of IFX is associated with SPR. CONCLUSION: Early step-up use of IFX in children with CD with inadequate clinical response to conventional therapies leads to sustained primary response over 2 years.

Maturation of the Infant Respiratory Microbiota, Environmental Drivers, and Health Consequences. A Prospective Cohort Study.

RATIONALE: Perinatal and postnatal influences are presumed important drivers of the early-life respiratory microbiota composition. We hypothesized that the respiratory microbiota composition and development in infancy is affecting microbiota stability and thereby resistance against respiratory tract infections (RTIs) over time. OBJECTIVES: To investigate common environmental drivers, including birth mode, feeding type, antibiotic exposure, and crowding conditions, in relation to respiratory tract microbiota maturation and stability, and consecutive risk of RTIs over the first year of life. METHODS: In a prospectively followed cohort of 112 infants, we characterized the nasopharyngeal microbiota longitudinally from birth on (11 consecutive sample moments and the maximum three RTI samples per subject; in total, n = 1,121 samples) by 16S-rRNA gene amplicon sequencing. MEASUREMENTS AND MAIN RESULTS: Using a microbiota-based machine-learning algorithm, we found that children experiencing a higher number of RTIs in the first year of life already demonstrate an aberrant microbial developmental trajectory from the first month of life on as compared with the reference group (0-2 RTIs/yr). The altered microbiota maturation process coincided with decreased microbial community stability, prolonged reduction of Corynebacterium and Dolosigranulum, enrichment of Moraxella very early in life, followed by later enrichment of Neisseria and Prevotella spp. Independent drivers of these aberrant developmental trajectories of respiratory microbiota members were mode of delivery, infant feeding, crowding, and recent antibiotic use. CONCLUSIONS: Our results suggest that environmental drivers impact microbiota development and, consequently, resistance against development of RTIs. This supports the idea that microbiota form the mediator between early-life environmental risk factors for and susceptibility to RTIs over the first year of life.

An audit of antimicrobial prescribing by dental practitioners in the north east of England and Cumbria.

BACKGROUND: Inappropriate prescribing of antimicrobials is a significant threat to global public health. In England, approximately 5% of all antimicrobial items are prescribed by dentists, despite the limited indications for their use in the treatment of oral infections in published clinical guidelines. The objective of this study was to survey antimicrobial prescribing by dental practitioners in North East England and Cumbria, identify educational and training needs and develop a self-assessment tool that can be used for Continued Professional Development by individual practitioners. METHODS: During October 2016, 275 dental practitioners used a standardised form to record anonymous information about patients who had been prescribed antimicrobials. Clinical information and prescribing details were compared against clinical guidelines published by the Faculty of General Dental Practitioners UK. RESULTS: Dental practitioners provided data on 1893 antimicrobial prescriptions. There was documented evidence of systemic spread, such as pyrexia in 18% of patients. Dentists recorded patients' pain (91.1% of patients), local lymph gland involvement (41.5%) gross diffuse swelling (55.5%) dysphagia (7.2%) and trismus (13.6%). Reasons for prescribing antimicrobials included patient expectations (25.8%), patient preference (24.8%), time pressures (10.9%), and patients uncooperative with other treatments (10.4%). The most commonly prescribed antimicrobials were amoxicillin, accounting for 61.2% of prescriptions, followed by metronidazole (29.9%). Most prescriptions for amoxicillin were for either 5 days (66.8%) or 7 days (29.6%) and most prescriptions for metronidazole were for a 5-day course (65.2%) or 7-day (18.6%) course. CONCLUSION: In most cases, when an antimicrobial was prescribed, practitioners used the correct choice of agents and usually prescribed these at the correct dose. However, some evidence of suboptimal prescribing practices when compared to the Faculty of General Dental Practitioner guidelines were identified. The audit has identified training needs across the region and aided the development of Continued Professional Development sessions. Further work to identify barriers and facilitators for improving antimicrobial prescribing and determining appropriate methods to improve clinical practice are required.

[The safety of decitabine as bridging pretreatment regimen before hematopoietic stem cell transplantation in pediatric hematological malignancies].

The safety of decitabine as bridging treatment before allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children with refractory hematological malignancies was evaluated. All 11 cases succeeded in hematopoietic reconstitution. The main adverse reaction was hematological toxicity. Neither did infections occur, nor drug-induced liver damage and renal impairment during decitabine administration. Most cases showed grade Ⅰ-Ⅱgastrointestinal adverse events. One case was diagnosed as severe acute graft versus host disease and died of intracranial hemorrhage on day 61 after allo-HSCT. The other 10 patients survived. Decitabine bridge is a safe regimen before allo-HSCT in children with refractory hematological malignancies.

Complex polar machinery required for proper chromosome segregation in vegetative and sporulating cells of Bacillus subtilis.

Chromosome segregation is an essential process of cell multiplication. In prokaryotes, segregation starts with the newly replicated sister origins of replication, oriCs, which move apart to defined positions in the cell. We have developed a genetic screen to identify mutants defective in placement of oriC during spore development in the Gram-positive bacterium Bacillus subtilis. In addition to the previously identified proteins Soj and DivIVA, our screen identified several new factors involved in polar recruitment of oriC: a reported regulator of competence ComN, and the regulators of division site selection MinD and MinJ. Previous work implicated Soj as an important regulator of oriC positioning in the cell. Our results suggest a model in which the DivIVA-interacting proteins ComN and MinJ recruit MinD to the cell pole, and that these proteins work upstream of Soj to enable oriC placement. We show that these proteins form a polar complex, which acts in parallel with but distinct from the sporulation-specific RacA pathway of oriC placement, and also functions during vegetative growth. Our study further shows that MinD has two distinct cell cycle roles, in cell division and chromosome segregation, and highlights that cell probably use multiple parallel mechanisms to ensure accurate chromosome segregation.

Concordance between upper and lower airway microbiota in infants with cystic fibrosis.

Nasopharyngeal and oropharyngeal samples are commonly used to direct therapy for lower respiratory tract infections in non-expectorating infants with cystic fibrosis (CF).We aimed to investigate the concordance between the bacterial community compositions of 25 sets of nasopharyngeal, oropharyngeal and bronchoalveolar lavage (BAL) samples from 17 infants with CF aged ∼5 months (n=13) and ∼12 months (n=12) using conventional culturing and 16S-rRNA sequencing.Clustering analyses demonstrated that BAL microbiota profiles were in general characterised by a mixture of oral and nasopharyngeal bacteria, including commensals like Streptococcus, Neisseria, Veillonella and Rothia spp. and potential pathogens like Staphylococcus aureus, Haemophilus influenzae and Moraxella spp. Within each individual, however, the degree of concordance differed between microbiota of both upper respiratory tract niches and the corresponding BAL.The inconsistent intra-individual concordance between microbiota of the upper and lower respiratory niches suggests that the lungs of infants with CF may have their own microbiome that seems seeded by, but is not identical to, the upper respiratory tract microbiome.

A complex genetic switch involving overlapping divergent promoters and DNA looping regulates expression of conjugation genes of a gram-positive plasmid.

Plasmid conjugation plays a significant role in the dissemination of antibiotic resistance and pathogenicity determinants. Understanding how conjugation is regulated is important to gain insights into these features. Little is known about regulation of conjugation systems present on plasmids from Gram-positive bacteria. pLS20 is a native conjugative plasmid from the Gram-positive bacterium Bacillus subtilis. Recently the key players that repress and activate pLS20 conjugation have been identified. Here we studied in detail the molecular mechanism regulating the pLS20 conjugation genes using both in vivo and in vitro approaches. Our results show that conjugation is subject to the control of a complex genetic switch where at least three levels of regulation are integrated. The first of the three layers involves overlapping divergent promoters of different strengths regulating expression of the conjugation genes and the key transcriptional regulator RcoLS20. The second layer involves a triple function of RcoLS20 being a repressor of the main conjugation promoter and an activator and repressor of its own promoter at low and high concentrations, respectively. The third level of regulation concerns formation of a DNA loop mediated by simultaneous binding of tetrameric RcoLS20 to two operators, one of which overlaps with the divergent promoters. The combination of these three layers of regulation in the same switch allows the main conjugation promoter to be tightly repressed during conditions unfavorable to conjugation while maintaining the sensitivity to accurately switch on the conjugation genes when appropriate conditions occur. The implications of the regulatory switch and comparison with other genetic switches involving DNA looping are discussed.