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The peptidoglycan (PG) sacculus is composed of long glycan strands cross-linked together by short peptides forming a covalently closed meshwork that protects the bacterial cell from osmotic lysis and specifies its shape. PG hydrolases play essential roles in remodeling this three-dimensional network during growth and division but how these autolytic enzymes are regulated remains poorly understood. The FtsEX ABC transporter-like complex has emerged as a broadly conserved regulatory module in controlling cell wall hydrolases in diverse bacterial species. In most characterized examples, this complex regulates distinct PG hydrolases involved in cell division and is intimately associated with the cytokinetic machinery called the divisome. However, in the gram-positive bacterium Bacillus subtilis the FtsEX complex is required for cell wall elongation where it regulates the PG hydrolase CwlO that acts along the lateral cell wall. To investigate whether additional factors are required for FtsEX function outside the divisome, we performed a synthetic lethal screen taking advantage of the conditional essentiality of CwlO. This screen identified two uncharacterized factors (SweD and SweC) that are required for CwlO activity. We demonstrate that these proteins reside in a membrane complex with FtsX and that amino acid substitutions in residues adjacent to the ATPase domain of FtsE partially bypass the requirement for them. Collectively our data indicate that SweD and SweC function as essential co-factors of FtsEX in controlling CwlO during cell wall elongation. We propose that factors analogous to SweDC function to support FtsEX activity outside the divisome in other bacteria.

Bacterial cells are encased in a peptidoglycan (PG) exoskeleton that protects them from osmotic lysis and specifies their distinct shapes. Cell wall hydrolases are required to enlarge this covalently closed macromolecule during growth, but how these autolytic enzymes are regulated remains poorly understood. Bacillus subtilis encodes two functionally redundant D,L-endopeptidases (CwlO and LytE) that cleave peptide crosslinks to allow expansion of the PG meshwork during growth. Here, we provide evidence that the essential and broadly conserved WalR-WalK two component regulatory system continuously monitors changes in the activity of these hydrolases by sensing the cleavage products generated by these enzymes and modulating their levels and activity in response. The WalR-WalK pathway is conserved among many Gram-positive pathogens where it controls transcription of distinct sets of PG hydrolases. Cell wall remodeling in these bacteria may be subject to homeostatic control mechanisms similar to the one reported here.

Two distinct, but rapidly converging, areas of research (the hygiene hypothesis and the study of probiotic/prebiotic effects) have emphasised the need to understand, and ultimately to manipulate, our physiological interactions with commensal flora, and with other transient but harmless organisms from the environment that affect immunoregulatory circuits. The story began with allergic disorders but now inflammatory bowel disease is increasingly involved.

Type VI secretion systems (T6SS) are macromolecular machines of the cell envelope of Gram-negative bacteria responsible for bacterial killing and/or virulence towards different host cells. Here, we characterized the regulatory mechanism underlying expression of the enteroagregative Escherichia coli sci1 T6SS gene cluster. We identified Fur as the main regulator of the sci1 cluster. A detailed analysis of the promoter region showed the presence of three GATC motifs, which are target of the DNA adenine methylase Dam. Using a combination of reporter fusion, gel shift, and in vivo and in vitro Dam methylation assays, we dissected the regulatory role of Fur and Dam-dependent methylation. We showed that the sci1 gene cluster expression is under the control of an epigenetic switch depending on methylation: fur binding prevents methylation of a GATC motif, whereas methylation at this specific site decreases the affinity of Fur for its binding box. A model is proposed in which the sci1 promoter is regulated by iron availability, adenine methylation, and DNA replication.

Integrating artificial intelligence (AI) technologies into law enforcement has become a concern of contemporary politics and public discourse. In this paper, we qualitatively examine the perspectives of AI technologies based on 20 semi-structured interviews of law enforcement professionals in North Carolina. We investigate how integrating AI technologies, such as predictive policing and autonomous vehicle (AV) technology, impacts the relationships between communities and police jurisdictions. The evidence suggests that police officers maintain that AI plays a limited role in policing but believe the technologies will continue to expand, improving public safety and increasing policing capability. Conversely, police officers believe that AI will not necessarily increase trust between police and the community, citing ethical concerns and the potential to infringe on civil rights. It is thus argued that the trends toward integrating AI technologies into law enforcement are not without risk. Policymaking guided by public consensus and collaborative discussion with law enforcement professionals must aim to promote accountability through the application of responsible design of AI in policing with an end state of providing societal benefits and mitigating harm to the populace. Society has a moral obligation to mitigate the detrimental consequences of fully integrating AI technologies into law enforcement.

We introduce here a microfluidic cell culture platform or spheroid culture chamber array (SCCA) that can synthesize, culture, and enable fluorescence imaging of 3D cell aggregates (typically spheroids) directly on-chip while specifying the flow of reagents in each chamber via the use of an array of passive magnetic valves. The SCCA valves demonstrated sufficient resistance to burst (above 100 mBar), including after receiving radiotherapy (RT) doses of up to 8 Gy combined with standard 37 °C incubation for up to 7 days, enabling the simultaneous synthesis of multiple spheroids from different cell lines on the same array. Our results suggest that SCCA would be an asset in drug discovery processes, seeking to identify combinatorial treatments.

Allergic asthma is a chronic inflammatory disease and despite the introduction of potent and effective drugs, the prevalence has increased substantially over the past few decades 1 . The explanation that has attracted the most attention is the 'hygiene hypothesis', which suggests that the increase in allergic diseases is caused by a cleaner environment and fewer childhood infections 2 , 3 , 4 . Indeed, certain mycobacterial strains can cause a shift from T-helper cell 2 (Th2) to Th1 immune responses, which may subsequently prevent the development of allergy in mice 5 , 6 , 7 . Although the reconstitution of the balance between Th1 and Th2 is an attractive theory, it is unlikely to explain the whole story, as autoimmune diseases characterized by Th1 responses can also benefit from treatment with mycobacteria and their prevalence has also increased in parallel to allergies 8 . Here we show that treatment of mice with SRP299, a killed Mycobacterium vaccae -suspension, gives rise to allergen-specific CD4 + CD45RB Lo regulatory T cells, which confer protection against airway inflammation. This specific inhibition was mediated through interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), as antibodies against IL-10 and TGF-β completely reversed the inhibitory effect of CD4 + CD45RB Lo T cells. Thus, regulatory T cells generated by mycobacteria treatment may have an essential role in restoring the balance of the immune system to prevent and treat allergic diseases.

This work addresses aspects of industrial noise produced by the facilities of oil extraction near Yasuní National Park, located in the Ecuadorian Amazon region. The acoustic sources within this kind of facilities could influence in the wildlife behavior, which could impact negatively on the species. The acoustic wave radial propagation model in an open field is proposed through a geometric divergence attenuation, atmospheric absorption effect, dispersion effects due to obstacles, and soil effects. The initial model without obstacles makes predictions based on an algorithm considering that the data are associated to a GPS position, the first propagation model without obstacles determines the level of sound pressure for each quasi-horizontal measurement ( x, y ). Then, dispersion factors are incorporated through the NORD2000 model to consider the local flora that introduces reflection and dispersion phenomena which are in the environment considering geometric measurements estimated in situ. The pressure level of the model without obstacles decreases as the frequency increases, but with less intensity compared to the pressure level of the model with obstacles. As the frequency increases, there are important fluctuations since the attenuating factors influence more than the correction factors.

The type VI secretion system (T6SS) is a widespread machine used by bacteria to control their environment and kill or disable bacterial species or eukaryotes through toxin injection. The T6SS comprises a central tube formed of stacked hexamers of hemolysin co-regulated proteins (Hcp) and terminated by a trimeric valine-glycine repeat protein G (VgrG) component, the cell puncturing device. A contractile tail sheath, formed by the TssB and TssC proteins, surrounds this tube. This syringe-like machine has been compared to an inverted phage, as both Hcp and VgrG share structural homology with tail components of Caudovirales. Here we solved the crystal structure of a tryptophan-substituted double mutant of Hcp1 from enteroaggregative Escherichia coli and compared it to the structures of other Hcps. Interestingly, we observed that the purified Hcp native protein is unable to form tubes in vitro. To better understand the rationale for observation, we measured the affinity of Hcp1 hexamers with themselves by surface plasmon resonance. The intra-hexamer interaction is weak, with a KD value of 7.2 µM. However, by engineering double cysteine mutants at defined positions, tubes of Hcp1 gathering up to 15 stacked hexamers formed in oxidative conditions. These results, together with those available in the literature regarding TssB and TssC, suggest that assembly of the T6SS tube differs significantly from that of Sipho- or Myoviridae.

The Type VI secretion system (T6SS) is a versatile machine that delivers toxins into either eukaryotic or bacterial cells. At a molecular level, the T6SS is composed of a membrane complex that anchors a long cytoplasmic tubular structure to the cell envelope. This structure is thought to resemble the tail of contractile bacteriophages. It is composed of the Hcp protein that assembles into hexameric rings stacked onto each other to form a tube similar to the phage tail tube. This tube is proposed to be wrapped by a structure called the sheath, composed of two proteins, TssB and TssC. It has been shown using fluorescence microscopy that the TssB and TssC proteins assemble into a tubular structure that cycles between long and short conformations suggesting that, similarly to the bacteriophage sheath, the T6SS sheath undergoes elongation and contraction events. The TssB and TssC proteins have been shown to interact and a specific α-helix of TssB is required for this interaction. Here, we confirm that the TssB and TssC proteins interact in enteroaggregative E. coli. We further show that this interaction requires the N-terminal region of TssC and the conserved α-helix of TssB. Using site-directed mutagenesis coupled to phenotypic analyses, we demonstrate that an hydrophobic motif located in the N-terminal region of this helix is required for interaction with TssC, sheath assembly and T6SS function.