ATP-independent assembly machinery of bacterial outer membranes: BAM complex structure and function set the stage for next-generation therapeutics.

Diderm bacteria employ ß-barrel outer membrane proteins (OMPs) as their first line of communication with their environment. These OMPs are assembled efficiently in the asymmetric outer membrane by the ß-Barrel Assembly Machinery (BAM). The multi-subunit BAM complex comprises the transmembrane OMP BamA as its functional subunit, with associated lipoproteins (e.g., BamB/C/D/E/F, RmpM) varying across phyla and performing different regulatory roles. The ability of BAM complex to recognize and fold OM ß-barrels of diverse sizes, and reproducibly execute their membrane insertion, is independent of electrochemical energy. Recent atomic structures, which captured BAM-substrate complexes, show the assembly function of BamA can be tailored, with different substrate types exhibiting different folding mechanisms. Here, we highlight common and unique features of its interactome. We discuss how this conserved protein complex has evolved the ability to effectively achieve the directed assembly of diverse OMPs of wide-ranging sizes (8-36 ß-stranded monomers). Additionally, we discuss how darobactin-the first natural membrane protein inhibitor of Gram-negative bacteria identified in over five decades-selectively targets and specifically inhibits BamA. We conclude by deliberating how a detailed deduction of BAM complex-associated regulation of OMP biogenesis and OM remodeling will open avenues for the identification and development of effective next-generation therapeutics against Gram-negative pathogens.

Unintentional Child Injury in Rural and Urban Siddlagatta, Karnataka, India: A Community-Based Survey.

INTRODUCTION: Despite Indian children constituting approximately 31.4% of the population aged 0 to 14, a comprehensive exploration of childhood injuries within this demographic remains limited. This study aimed to examine the occurrence of unintentional injuries among children aged six months to 18 years in the Siddlagatta area and assess associated risk factors. METHODOLOGY: A community-based, cross-sectional study on unintentional child injuries was carried out from March 2018 to September 2020 across 11 sites in India. Employing a two-stage cluster sampling method with probability proportionate to size, 2341 urban and rural families were selected from each site. Data on unintentional injuries sustained over the past 12 months were collected using the WHO child injury questionnaire, tailored and validated for the Indian context. RESULTS: The study encompassed 10,335 individuals in households, including 2695 children aged 6 months to 18 years. Among them, 309 children experienced 390 unintentional injuries in the preceding year, excluding minor incidents. A prevalence rate of 11.5% (95% CI: 10.3-12.7) was identified for unintentional injuries among children, excluding minor cases. Falls were the most prevalent injury type (183 cases, 53.8%), while poisoning incidents were the least frequent (one case, 0.2%). More than 50% of incidents occurred within domestic settings. CONCLUSIONS: This study's outcomes underscore the prominence of fall-related injuries across all age groups and genders. Homes and schools emerged as primary settings for these injuries, highlighting the need for targeted preventive measures.

Engineering a Hyperstable Yersinia pestis Outer Membrane Protein Ail Using Thermodynamic Design.

Development of viable therapeutics to effectively combat tier I pneumopathogens such as Yersinia pestis requires a thorough understanding of proteins vital for pathogenicity. The host invasion protein Ail, although indispensable for Yersinia pathogenesis, has evaded detailed characterization, as it is an outer membrane protein with intrinsically low stability and high aggregation propensity. Here, we identify molecular elements of the metastable Ail structure that considerably alter protein-lipid and intraprotein thermodynamics. In addition, we find that four residues Q50, L88, L92, and A94 contribute additively to the lowered stability of Ail, and their conserved substitution is sufficient to re-engineer Ail to Out14, a thermodynamically hyperstable low-aggregation variant with a functional scaffold. Interestingly, Ail also shows two (parallel) folding pathways, which has not yet been reported for ß-barrel membrane proteins. Additionally, we identify the molecular mechanism of enhanced thermodynamic stability of Out14. We show that this enhanced stability of Out14 is due to a favorable change in the nonpolar accessible surface, and the accumulation of a kinetically accelerated off-pathway folding intermediate, which is absent in wild-type Ail. Such engineered hyperstable Ail ß-barrels can be harnessed for targeted drug screening and developing medical countermeasures against Yersiniae. Application of similar strategies will help design effective translational therapeutics to combat biopathogens.

NET-associated citrullinated histones promote LDL aggregation and foam cell formation in vitro.

Neutrophils have been recently identified in the atherosclerotic lesion and they can release neutrophil extracellular trap (NET) under the pro-inflammatory conditions prevailing in the lesion. Citrullinated histones (Cit-histones) are the major type of citrullinated proteins associated with NET release. Since elevated levels of citrullinated proteins have been detected in inflammatory diseases including atherosclerosis, this study analysed the role played by NET and Cit-histones in different atherogenic events in vitro. First, neutrophil recruitment and NET release in the presence of low-density lipoprotein (LDL) and oxidised LDL (Ox-LDL) were analysed by Boyden's chamber method and microscopy respectively. Then, LDL oxidation and LDL aggregation in the presence of NET and Cit-histones were analysed spectroscopically. Foam cell formation in the presence of NET or Cit-histone was studied by both microscopic and spectroscopic methods. While neutrophil recruitment was facilitated by Ox-LDL and not by LDL, the extent of NET release was significantly increased in the presence of both LDL and Ox-LDL. In the presence of NET, LDL oxidation, aggregation and foam cell formation were found to be increased. Cit-histones were found to accelerate LDL aggregation and foam cell formation at higher citrulline levels. Altogether, the results suggest that both NET and NET-associated Cit-histone released at the lesion can play major roles as pro-atherogenic mediators. Inhibiting the action of NET or Cit-histone would, therefore, be beneficial in slowing down atherosclerotic progression.

N-Heterocyclic silylene/germylene ligands in Au(i) catalysis.

Cationic Au(i) complexes (2, 5 and 8) supported by N-heterocyclic carbene, silylene and germylene ligands were prepared and their potential as catalysts in glycosidation chemistry has been evaluated. Insights into the mechanism are provided using DFT studies. Practical application of them as catalysts was achieved by the synthesis of the branched pentamannan core of the HIV-gp120 envelope under mild conditions.

Molecular Switch between Structural Compaction and Thermodynamic Stability by the Xxx-Pro Interface in Transmembrane ß-Barrels.

Transmembrane ß-barrel scaffolds found in outer membrane proteins are formed and stabilized by a defined pattern of interstrand intraprotein H-bonds, in hydrophobic lipid bilayers. Introducing the conformationally constrained proline in ß-barrels can cause significant destabilization of these structural regions that require H-bonding, with proline additionally acting as a secondary structure breaker. Membrane protein ß-barrels are therefore expected to show poor tolerance to the presence of a transmembrane proline. Here, we assign a thermodynamic measure for the extent to which a single proline can be tolerated at the C-terminal interface of the model transmembrane ß-barrel PagP. We find that proline drastically destabilizes PagP by 7.0 kcal mol-1 with respect to wild-type PagP (F161 → P161). Interestingly, strategic modulation of the preceding residue can modify the measured energetics. Placing a hydrophobic or bulky side chain as the preceding residue increases the thermodynamic stability by ≤8.0 kcal mol-1. While polar substituents at the preceding residue decrease the PagP stability, these residues demonstrate stronger tertiary packing interactions in the barrel and retain the catalytic activity of native PagP. This biophysical interplay between enhanced thermodynamic stability and attaining a structurally compact functional ß-barrel scaffold highlights the detrimental effect caused by proline incorporation. Our findings also reveal alternative mechanisms that protein sequences can employ to salvage the structural integrity of transmembrane protein structures.

N-heterocyclic silylene stabilized monocordinated copper(i)-arene cationic complexes and their application in click chemistry.

Herein for the first time we report monocoordinated cationic Cu(i) complexes with unsymmetrical arenes (toluene and m-xylene) [LCu(η3-C7H8)]+[SbF6]- and [{LCu(η2-Me2C6H4)}]+[SbF6]- [L = {PhC(NtBu)2SiN(SiMe3)2}], [IPr (1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene)], their reactivity and catalytic applications in CuAAC reactions (12 examples). The bonding analysis was performed in both silylene and carbene complexes using the EDA-NOCV method at the BP86/TZ2P level of theory.

Treatment of Silylene-Phosphinidene with Chalcogens Resulted Exclusively in the Formation of Silicon-Bonded Chalcogens.

Chalcogen-bonded silicon phosphinidenes LSi(E)-P-Me cAAC (E=S (1); Se (2); Te (3); L=PhC(NtBu)2 ; Me cAAC=C(CH2 )(CMe2 )2 N-2,6-iPr2 C6 H3 )) were synthesized from the reactions of silylene-phosphinidene LSi-P-Me cAAC (A) with elemental chalcogens. All the compounds reported herein have been characterized by multinuclear NMR, elemental analyses, LIFDI-MS, and single-crystal X-ray diffraction techniques. Furthermore, the regeneration of silylene-phosphinidene (A) was achieved from the reactions of 2-3 with L'Al (L'=HC{(CMe)(2,6-iPr2 C6 H3 N)}2 ). Theoretical studies on chalcogen-bonded silicon phosphinidenes indicate that the Si-E (E=S, Se, Te) bond can be best represented as charge-separated electron-sharing σ-bonding interaction between [LSi-P-Me cAAC]+ and E- . The partial double-bond character of Si-E is attributed to significant hyperconjugative donation from the lone pair on E- to the Si-N and Si-P σ*-molecular orbitals.

Two stage surgical procedure for root coverage.

Gingival recession may present problems that include root sensitivity, esthetic concern, and predilection to root caries, cervical abrasion and compromising of a restorative effort. When marginal tissue health cannot be maintained and recession is deep, the need for treatment arises. This literature has documented that recession can be successfully treated by means of a two stage surgical approach, the first stage consisting of creation of attached gingiva by means of free gingival graft, and in the second stage, a lateral sliding flap of grafted tissue to cover the recession. This indirect technique ensures development of an adequate width of attached gingiva. The outcome of this technique suggests that two stage surgical procedures are highly predictable for root coverage in case of isolated deep recession and lack of attached gingiva.

Fluorescent coronene monoimide gels via H-bonding induced frustrated dipolar assembly.

Two stage self-assembly of novel coronene monoimide (CMI) based gels that results in resurfacing of monomer emission in the aggregated state is reported. This process is attributed to a frustrated head-head dipolar assembly forced by hydrogen bonding.