The physicochemical properties of silver nanocomposites (MTP(BTP)/Ag NCs) were fully elucidated using spectroscopic and microscopic tools. The antibacterial activity of the nanocomposites was screened against six multidrug-resistant pathogenic strains, comparable to ampicillin and ciprofloxacin commercial drugs. The antibacterial performance of BTP was more substantial than MTP, notably with the best minimum inhibitory concentration (MIC) of 0.0781 mg/mL towards Bacillus subtilis, Salmonella typhi, and Pseudomonas aeruginosa. Among all, BTP provided the clearest zone of inhibition (ZOI) of 35 ± 1.00 mm against Salmonella typhi. After the dispersion of silver nanoparticles (AgNPs), MTP/Ag NCs offered dose-dependently distinct advantages over the same nanoparticle with BTP; a more noteworthy decline by 4098 × MIC to 0.1525 × 10−3 mg/mL was recorded for MTP/Ag-1000 against Pseudomonas aeruginosa over BTP/Ag-1000. Towards methicillin-resistant Staphylococcus aureus (MRSA), the as-prepared MTP(BTP)/Ag-1000 displayed superior bactericidal ability in 8 h. Because of the anionic surface of MTP(BTP)/Ag-1000, they could effectively resist MRSA (ATCC-43300) attachment, achieving higher antifouling rates of 42.2 and 34.4% at most optimum dose (5 mg/mL), respectively. The tunable surface work function between MTP and AgNPs promoted the antibiofilm activity of MTP/Ag-1000 by 1.7 fold over BTP/Ag-1000. Lastly, the molecular docking studies affirmed the eminent binding affinity of BTP over MTP—besides the improved binding energy of MTP/Ag NC by 37.8%—towards B. subtilis-2FQT protein. Overall, this study indicates the immense potential of TP/Ag NCs as promising nanoscale antibacterial candidates.Newly synthesized mono- and bis-thioureidophosphonate (MTP and BTP) analogues in eco-friendly conditions were employed as reducing/capping cores for 100, 500, and 1000 mg L Recently, the emergence of antibiotic-resistant microbes has been seen as a global health concern affecting many people’s lives . Regardi−1. The  nuclear magnetic resonance (NMR) spectra were consecutively recorded at 400, 101, and 162 MHz in DMSO-d6 using a Bruker Avance III HD, 600 MHz-NMR spectrometer probe, and BBFO cryoprobe . The elemental analysis of samples  was undertaken for the raw TP derivatives (MTP and BTP) by CHNS Vario EL III Elementar analyzer, Germany. The phosphorus content (%) was estimated via the chemical digestion in sulfuric/nitric solution and photometric measurements at λmax = 410 nm. Melting points (m.p) were recorded using  without correction. The surface images of nanocomposites were obtained using transmission electron microscope (TEM) by JEOL, JEM-2100 Tokyo, Japan. The crystalline properties of nanocomposites were obtained using powder X-ray diffraction (XRD) analysis using Shimadzu 6000 X-ray diffractometer with Cu Kα-radiation (λ = 1.54 nm) at 25 °C in the 2θ range of 10–80° operating at a scan rate of 1 deg/min. X-ray photoelectron spectroscopy (XPS) was conducted using a Perkin Elmer PHI 5600, Perkin Elmer Instruments, Waltham, MA, USA, with analytical zone’s diameter of 1 mm and indium sheets for sample deposition using Al Kα X-rays radiation source (200 W). The photometric assays of antibacterial results were measured by (Infinite F50 Robotic absorbance microplate reader) in the wavelength range of 400 to 750 nm. The zeta potential charge of silver nanocomposites was determined via dynamic light scattering using Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, UK. Scanning electron microscopy (SEM) was performed using JSM 6390 LA, JEOL, Tokyo, Japan, with a 15 KV accelerating voltage.The Fourier transform infrared spectroscopy (FT-IR) analysis has been accomplished for all samples on Bruker Alpha II spectrometer, Bremen, Germany, in the wavenumber range 4000–400 cm3) were purchased from Aladdin . Solvents, such as acetonitrile (CH3CN), ethanol, and methanol were obtained from a commercial supplier  and used without further drying and purification. Deionized water was used to prepare all solutions and suspensions.Thiourea  and triphenyl phosphite  were obtained from Macklin . Terephthalaldehyde , pyridinium trifluoromethanesulfonate  (as protic ionic liquid), and silver nitrate   with the aid of a pyridinium trifluoromethanesulfonate (pyridinium triflate) as a Lewis acid protic ionic liquid-based catalyst. In the meantime, thiourea /,  terephthalaldehyde, triphenyl phosphite /, and pyridinium triflate / in 5 and 10 mL of CH3CN were progressively incorporated, affording MTP and BTP in a molar ratio of 1:1:1 and 2:1:2, respectively. Further, both solutions were magnetically stirred overnight under ambient conditions and the reaction was monitored via qualitative thin layer chromatography (TLC) using hexane–methylene chloride (3:1) as eluent mixture. Eventually, the final products were filtered off under vacuum, washed with methanol, dried, and kept in a desiccator for 2 days giving MTP and BTP in good yields.The synthesis process included preparation of two different systems of mono and bis thioureidophosphonate (TP) products, where the involved components  were dissolved in acetonitrile (CHDiphenyl ((4-formylphenyl) (thioureido)methyl) phosphonate, denoted as mono-thioureidophosphonate (MTP):−1 (NH)str. + (NH2) str. overlapped, 3057.58 cm−1 , 2894.63 cm−1 , 1698.02 cm−1 , 1590.99 cm−1, (NH+NH2) bending, 1525.42 cm−1 (>C=C<) phenyl rings, 1488.78 cm−1 (>C=S) asym., 1194.69 cm−1 (-P=O), 944.95 cm−1 (P-O-C), 760.78 cm−1 (P–C), and 684.61 cm−1 (>C=S) sym. 1H-NMR; δ ppm: 5.50 , 6.63–7.99 , 9.40–9.52 , 9.76 , and 10.04 . 13C-NMR ; δ ppm: 54.59 and 56.16 ppm (P-C–H), 115.24, 120.22, 120.53, 125.39, 127.96, 128.92, 129.90, 149.80 (Ar-C), 183.84 (>C=S), and 192.73 (-CHO). 31P-NMR ; δ 14.08 ppm. Elemental analysis calc. (%) for C21H19N2O4PS: C, 59.15; H, 4.49; N, 6.57; P, 7.26; S, 7.52; and O, 15.01; meas. (%): C, 57.85; H, 4.27; N, 6.34; P, 6.97; and S, 6.99.Light-beige solid, yield: , m.p: 160–163 °C, FT-IR: 3457.74–3311.18 cmTetraphenyl ) bis(phosphonate) denoted as bis-thioureidophosphonate (BTP):−1 [(NH)str. + (NH2) str. overlapped], 3057.58 cm−1 , 2895.59 cm−1 , 1591.95 (>NH + -NH2) bending, 1527.35 cm−1 (>C=C<) phenyl rings, 1489.74 cm−1 (>C=S) asym., 1195.65 (-P=O), 947.84 (P-O-C), 761.74 cm−1 (P–C), and 761.74 cm−1 (>C=S) sym. 1H-NMR; δ ppm: 5.93 , 6.54–7.61 , 9.21–9.33 , and 9.38 ppm . 13C-NMR ; δ ppm: 54.50 and 56.06 (P-C–H), 115.26, 120.29, 120.86, 125.40, 127.50, 129.33, 129.84, and 149.94 (Ar-C), and 183.83 (>C=S). 31P-NMR ; δ ppm: 14.85 ppm. Elemental analysis calc. (%) for C34H32N4O6P2S2: C, 56.82; H, 4.49; N, 7.82; P, 8.62; S, 8.92; and O, 13.36; found: C, 54.97; H,4.36; N, 8.18; P, 8.83; and S, 7.91.Light-yellow solid, yield: , m.p: 135–137 °C, FTIR: 3310.21 cm3 solution of 100, 500, and 1000 mg L−1 concentrations. Then, the prepared solutions were magnetically stirred (200 rpm) for 12 h, at ambient conditions. Afterward, the attained colloidal solutions were centrifuged, and the settled nanoparticles were washed with ethanol/deionized water and then oven-dried at 50 °C for further characterization ..36].Well dispersed AgNPs were produced via one-pot synthesis method using thioureidophosphonates (TPs) as dual-functioning reducing and capping agents. This affords spherical AgNPs different dispersion patterns based on the reaction conditions of AgNPs precursor concentrations and the structure of TPs .−1) illustrated meaningful changes in the characteristic absorption bands of capping agent (MTP)  was also involved in the interaction and reduction of Ag+ ions in all concentrations of 100, 500, and 1000 mg Ag L−1, respectively. Further, the aliphatic (H-C-P) bond was dramatically shifted from ν = 2895 cm−1 to ν = 2914 cm−1 in MTP/Ag-1000. On the other side, the FT-IR spectra of BTP and its  AgNP dispersions displayed similar changes, with a substantial shifting of amine functional groups from ν = 3310 cm−1 to ν = 3306, 3305, and (3320–3428) cm−1, consecutively  a. Notablcutively b. Hence,cutively . Basicalg Ag L−1 .+ ions, while the higher density of chelating centers in BTP significantly improved the reduction rate of Ag+ ions (with deeper color); thus, some smaller AgNPs were attained with the BTP surface (The morphology and dispersion nature of developed silver nanoparticles (AgNPs) were scrutinized using microscopic techniques. Accordingly, the TEM imaging for MTP/Ag-500 displayed good dispersion of spherical AgNPs with an average mean size range of 6.8–20.1 nm a–c. On t surface d–f. The Moreover, the crystallinity of the as-synthesized TPs after AgNPs functionalization was investigated compared to that of raw capping agents using XRD analysis. First, broad XRD patterns were individually centered at 2 theta (2θ) = 21.7° with (110) diffraction phases, reflecting the amorphous structure of both raw MTP and BTP analogues. The emergence of broad patterns stemmed from the strong inter- and intra-molecular hydrogen bonding in raw TP cores . Further+ ions incorporation and formation of AgNPs, X-ray photoelectron spectroscopy analysis (XPS) was carried out for MTP/Ag-500. The XPS results of MTP/Ag-500 were studied based on evaluating the changes in binding energies (BEs), atomic percentages (%), electronic properties, and chemical composition of MTP before and after Ag+ ion interactions. Five elemental signals of C 1s (at BE: 285.84–287.23 eV), N 1s (400.72–401.63 eV), O 1s (533.02–534.81 eV), P 2p (134.32–134.57 eV), and S 2p (163.19–164.19 eV) were mainly evolved, consecutively  eV, besides considerable changes in the atomic percentages (%) of raw elemental functional groups that were estimated from 0.72–25.57%. Basically, these notably affected functional groups were ordered, respectively, in a descending mode according to changes in BEs as follows: C–S (2p1/2) > C–C, C–H, and C=C > HC=O and -OH (H2O) > C–S (2p3/2) > P–C and P–O (2p1/2) > >NH and -NH2 > C=S > C–N > π–π* sat, benzene rings, and C=S > C-OH, P–O, and C–O–C > C–O, C–N, C–P, and C=O  on the MTP, the main signal deconvolution assessed that Ag+ ions may be sorbed in their hydrated form . In this regard, the changes that occurred in BEs after AgNPs decoration were consistent with the results discussed before.To validate the most significant changes that occurred to raw functional groups after Agcutively . Regardiectively b 62]. H. H+ ions and C=O . FurtherStaphylococcus aureus (MRSA), Streptococcus mutans, and Bacillus subtilis) and Gram-negative  bacteria as well. Moreover, zone of inhibition diameters, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time–kill kinetics, antiadhesion activity, and antibiofilm assay with morphological investigation were assessed for all developed compounds.The antibacterial potency of synthesized thioureidophosphonates (TPs) and their fabricated AgNPs-based composites was evaluated against several strains. The antibacterial activity using the agar well diffusion method was conducted against six clinical bacterial strains and their ATCC references of Gram-positive (methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus mutans (S. mutans), and Bacillus subtilis (B. subtilis), compared to Ampicillin and Ciprofloxacin as reference antibiotics  resulted in higher MIC values = 1.25 mg/mL for both analogues. MTP and BTP recorded MICs at lower concentrations of 0.625 and 0.3125 mg/mL against S. mutans (ATCC-35668) and B. subtilis (ATCC-6633), progressively. The zone of inhibition (ZOI) was measured for each strain with DMSO as a negative control. Including the diameter of each well (6 mm) before treatment, MTP gave ZOIs in a range of 28 ± 1.0, 30 ± 1.00, and 24 ± 1.00 mm, whereas the treatment with BTP increased the ZOIs width towards MRSA, S. mutans, and B. subtilis strains, respectively. Both TPs with MBC/MIC ratio of 2(+) have bactericidal properties, unlike Ampicillin and Ciprofloxacin. TPs loaded with silver nanoparticles (AgNPs)  were used to investigate the antibacterial properties towards three strains accompanied with a dose-dependent improvement in susceptibility. A clear MIC decline to very minute concentrations was appreciated from 0.156 to 0.1525 × 10−3 mg/mL with the three strains. The MIC of MTP significantly dropped by (4 × MIC) after treating MRSA and S. mutans with MTP/Ag-100. Afterward, MTP/Ag-500 and MTP/Ag-1000 showed increased bactericidal properties against both strains, i.e., 16 and 2049 times sequentially. The treatment of MTP/Ag- to B. subtilis increased the potential bactericidal activity by 16, 128, and 1024 times, respectively. BTP/Ag-100 showed a more significant decline in MIC values by two and eight times against MRSA and B. subtilis, compared to MTP/Ag NCs. BTP/Ag-500 had less biocidal effects by 4 and 16 times that of MTP/Ag-500 on MRSA and B. subtilis, consecutively. This reflected that BTP and AgNPs have less synergistic interactions than MTP. However, BTP/AgNPs had the same bactericidal activity as MTP/AgNPs against S. mutans in 100 and 500 dispersions. Noteworthy, MTP and BTP loaded with 1000 mg L−1 resulted in a decrease in MICs by 2049 times against MRSA while BTP/Ag-1000 showed higher MIC against S. mutans and B. subtilis by only 1023 and 512 times, respectively , 38 ± 1.00 (MTP/Ag-500), and 42 ± 1.00 mm for MTP/Ag-1000, respectively. Likewise, ZOIs exhibited a gradual increase from 37 ± 1.00 in BTP to 40 ± 1.00 (BTP/Ag-100), 43 ± 0.60 (BTP/Ag-500), and 49 ± 0.100 mm (BTP/Ag-1000). MTP and BTP gave higher ZOIs with diameters of 35 ± 1.00, 38 ± 1.00, and 40 ± 1.00 mm and 36 ± 1.00, 41 ± 1.00, and 41 ± 1.00 mm, respectively, for B. subtilis , Salmonella typhi (S. typhi), and Serratia marcescens (S. marcescens) than previously screened Gram-positive bacteria and reference antibiotics  against P. aeruginosa than MTP, indicating higher susceptibility towards Gram-negative strains. Both MTP and BTP compounds had excellent inhibitory properties against S. typhi and S. marcescens, as their MIC ratio was reduced fourfold. Regarding the Ampicillin and Ciprofloxacin, 5× and 20× MIC reductions by MTP were recorded against S. typhi, while 5.0 and 1.2× MIC were dropped with S. marcescens. On the contrary, BTP decreased the MICs of Ampicillin and Ciprofloxacin by 20 and 80 times towards S. typhi, and to 20 and 5 times against S. marcescens, respectively. Furthermore, the selectivity of these TP derivatives was determined against P. aeruginosa (ATCC-27853), S. typhi (ATCC-6539), and S. marcescens (ATCC-13880). Meanwhile, the MICs obtained by inoculating both ATCC isolates of P. aeruginosa and S. marcescens with MTP were 1.25 mg/mL for S. typhi (ATCC-6539). The selectivity of MTP was more effective, affording an MIC value of 0.625 mg/mL. BTP had two lowered MIC values against P. aeruginosa (ATCC-27853), S. typhi (ATCC-6539), and S. marcescens, with 0.3125 mg/mL for P. aeruginosa and 0.625 mg/mL for S. marcescens (ATCC-13880). On the other hand, ZOI results of MTP and BTP against P. aeruginosa, S. typhi, and S. marcescens were 28 ± 1.00, 28 ± 1.00, and 35 ± 1.00 mm for MTP, while 33 ± 0.60, 35 ± 1.00, and 38 ± 1.00 mm for BTP, consecutively. Therefore, MBC values ranged from 1.25 to 0.156 mg/mL, resulting in a significant bactericidal efficacy against all bacterial strains with MBC/MIC ratio = 2(+). Notably, AgNPs improved the antibacterial susceptibility of TP cores, lowering MIC values from 0.01953 mg/mL to 7.629 × 10−5 mg/mL. Basically, MTP/Ag-100 minimized the MIC concentration of raw MTP by around 32 times towards P. aeruginosa, and 16 times for both S. typhi and S. marcescens, successively. MTP/Ag-500 reduced MICs by 256 times for P. aeruginosa and 128 times for S. typhi and S. marcescens compared to raw MTP. For MTP/Ag-1000, MTP displayed an elevated antibacterial activity towards P. aeruginosa, S. typhi, and S. marcescens. Nevertheless, the net antibacterial activity of BTP was higher than MTP, and the combinatorial interactions of AgNPs with MTP were more tunable, unlike BTP. In the meantime, BTP/Ag-100 lessened the MIC ratio to raw BTP by fourfold towards P. aeruginosa and eightfold against both S. typhi and S. marcescens. BTP/Ag-500 had a remarkable bactericidal effect on S. typhi up to 128 times. Then, the BTP/Ag-1000 reduced the dose of MIC to 512 times with P. aeruginosa and 1023 times with both S. typhi and S. marcescens, respectively. Pertinently, BTP loaded with AgNPs manifested less powerful bactericidal potency than MTP capped AgNPs  started to dramatically decline after 1 h of treatment (10) and (1.8 log10) CFU/mL after 18 h of incubation, respectively. However, once MRSA was treated with TP/Ag NCs, the picture was changed where the MTP/Ag-100 induced the killing potential with cell count reductions to (2.2 log10) and (0.9 log10) CFU/mL for MTP/Ag-500, consecutively. More significant reductions of the cultural population of MRSA were achieved to (1.1 log10) and (0.3 log10) CFU/mL for BTP/Ag-100 and BTP/Ag-500, progressively. Despite the higher killing efficacy of BTP/Ag NCs than MTP/Ag NCs, the combination between AgNPs and MTP brought more bactericidal advantages over BTP, which was observed from the gradual improvement of killing (%) between dose 100 and 500 for both AgNPs formulations. Remarkably, the entire population was reduced and killed within 8 h, for both MTP (BTP)/Ag-1000 composites. Interestingly, the bactericidal rates towards MRSA were previously confirmed by both TP/Ag-1000; where both reduced MBC values to the same degree of 2049 times  of MRSA (ATCC-43300) suspensions for 18–24 h, using vancomycin hydrochloride (VAN) as a positive control, and the negative control was tryptic soy broth (TSB). Antiadhesion activity was quantitatively estimated against MRSA (ATCC-43300) on the microplate reader at λmax = 570 nm, validating an increase in MRSA (ATCC-43300) detachment in a dose-dependent manner within 1.25, 2.5, 5.0, and 10 mg/mL  than MTP, which could be attributed to its superior hydrophobic characteristics as the non-polar parts (aromatic rings) exceeded the polar ones, based on its molecular formula (M.F), namely, C34H32N4O6P2S2, comparable to MTP with M.F of C21H19N2O4PS. Moreover, the electrostatic repulsion forces assessed between the negatively charged MTP/Ag NCs and MRSA (ATCC-43300) surface played a critical role . Ad. Ad6 CFUactivity . On thisStaphylococcus aureus (MRSA) is one of the most opportunistic and resistant pathogens, being able to produce an extracellular polymeric substance (EPS) inducing biofilm growth prior to the occurrence of cross infection [MRSA (ATCC-43300) as a Gram-positive bacterium with reference to vancomycin hydrochloride (VAN) as a positive control. The biofilm disintegration activity (%) was validated by the microtiter plate assay using a semi-quantitative analysis based on crystal violet staining. Regarding 96-well plates treated with raw MTP and BTP, the biofilm (violet color circle) was slightly eliminated, while the gradual removal was notably viewed by six TP/Ag NCs, (max ≈ 570 nm with respect to the negative control of tryptic soy broth (TSB). The results showed a significant reduction in biofilm growth when treated with raw TPs, even after 24 h of incubation. The biofilm inhibition (%) was promoted with the gradual increase in the concentrations of tested compounds in the 1.25, 2.5, 5.0, and 10.0 mg/mL (MRSA (ATCC-43300). The studied TP cores (α-aminophosphonate derivatives) are considered structural bio-isosteres of α-amino acids (peptides) with a common amine functional group, but the carboxylic groups in α-amino acids are replaced with related phosphorous-containing moieties such as phosphonic acid. However, the reported data about their antibiofilm mechanism of action towards MRSA biofilm are directed for hybrid α-APs; thus, the inhibitory actions of mentioned α-APs analogues were proposed [Methicillin-resistant nfection . The ant/Ag NCs, . Therefo.0 mg/mL . Yet, frproposed ,73. Firsproposed . Moreoveproposed . It was proposed ,76.+ ions and reactive oxygen species (ROS), which increased the toxic effects on the integrity of bacterial cell wall. Additionally, ROS can cause the total impairment of envelope-bound proteins, deoxyribonucleic acid (DNA), respiratory coenzymes, and antioxidants such as glutathione (GSH) [MRSA (ATCC-43300) in the next section [After AgNPs functionalization, biofilm inhibition capability (%) was promoted for both MTP and BTP in all studied concentrations , consecutively. The most effective and synergistic concentration needed for the disruption of MRSA (ATCC-43300) biofilm was observed at 5.0 mg/mL for both TP/Ag NCs. For 10.0 mg/mL, the biofilm eradicating ability for both MTP(BTP)/Ag-1000 reached maximum values at 79.5 and 83.6%, progressively . Nonethene (GSH) . RemarkaMRSA (ATCC-43300) was also qualitatively verified through scanning electron microscopy (SEM) after incubation by 100 µg/mL of raw compounds and their TP/Ag-1000. The two upper images (MRSA (ATCC-43300) surface on which aggregating grape-like colonies were developed to form the biofilm. On the contrary, low percentages of biofilm clusters were present on the surfaces of MRSA (ATCC-43300) after treatment with raw MTP and BTP, indicating that these compounds could prevent bacterial biofilm formation and induce cell death. Moreover, almost no biofilm matrices could be observed on MRSA treated with MTP and BTP decorated with AgNPs, validating the potent biofilm disintegration ability of TP/Ag NCs. In summary, these observations were in line with antibiofilm investigation results and other studies referring to the ability of synthesized AgNPs to severely interfere with cell wall (biofilm) components, thus hampering metabolic functions, and thereby, MRSA (ATCC-43300) cells are killed [The biofilm inhibition of r images  represene killed .MRSA and B. subtilis) and Gram-negative (P. aeruginosa and S. typhi) bacterial proteins with respective codes of PDB: 4DKI, 2FQT, 5ZHN, and 3ZQE, in addition to confirming their illegibility as antibacterial agents with the experimental work. Furthermore, the docking results for the prepared raw compounds (MTP and BTP) in all downloaded proteins, along with type of chemical bonding and the amino acids involved in the interaction with the protein, were estimated. Additionally, values recorded for binding affinity and root mean square deviation (RMSD) were tabulated. There are different types of interactions, including hydrogen and hydrophobic H-pi interactions, with the binding sites of all the investigated proteins  for MRSA, with values of −7.9776 kcal/mol and −7.9074 kcal/mol, respectively. Amino acids involved in the interaction between BTP and the B. subtilis protein were GLY127 , GLY127 , and GLU57 , which are the same amino acids involved in the interaction of the co-crystallized ligand and the protein  and TYR446   and thereby for biofilm growth [Molecular docking studies were conducted for the prepared TP compounds to investigate their binding affinity for Gram-positive  , while Tcutively . It was 1.2593 Å . Interesproteins . These bm growth ,82; thusIt is pertinent to note that in this study, the structural and functional diversity of raw TP surfaces afforded highly tunable surface interactions and synergy, with AgNPs giving remarkable antibacterial activity. Additionally, the molecular docking studies were in good coherence with most experimental results for TPs and TP/Ag NCs. Based on the above considerations, some structural modifications may be applied in the future to exploit these compounds in various applications such as antibacterial textile fabrics, antibacterial food packaging films, and antibacterial thermoplastic polymer nanocomposites ,84,85.p > −2.5 [50 value was 0.335 and 0.285log μg/L for MTP-AgNPs and BTP-AgNPs, respectively. The anticipated scores are summarized below.Absorption, distribution, metabolism, and excretion (ADME) analysis is very helpful in simplifying clinical trials, especially in the early stage of drug design. Intestinal absorption, skin sensitization, and oral bioavailability are absorption parameters considered in drug discovery . An intep > −2.5 ; howeverp > −2.5 ; it measp > −2.5 , MTP-AgNp > −2.5 . Regardip > −2.5 ,88. Comp+ ions affording TP/Ag NCs, the physicochemical properties of which were thoroughly investigated through FT-IR, -NMR, elemental analysis, and TEM, XRD, and XPS analyses, respectively. The antibacterial properties of the as-prepared raw cores were studied via tuning different concentrations of silver nitrate, i.e., 100, 500, and 1000 mg L−1. Both raw Mono-TP and Bis-TP analogues proved to be bactericidal towards all isolates. In a dose-increasing manner, time–kill assay results manifested the record-breaking bactericidal effect of both MTP(BTP)/Ag-1000 towards all MRSA cells within 8 h. At high concentrations, the antifouling characteristics of evaluated compounds were influenced by the surface electrostatic repulsion with MRSA (ATCC-43300), while hydrophobicity played a critical role at lower concentrations. Targeting the biofilm of MRSA (ATCC-43300) was confirmed, with net activity of 79.5 and 83.6% for MTP/Ag-1000 and BTP/Ag-1000, progressively, at highest used concentration. Nonetheless, higher net antibiofilm activity was observed in BTP/Ag NCs; the simpler MTP structure tuned the surface work function with Ag+ ions, giving better colloidal AgNPs and a gradual improvement in the biofilm disruption % after the nanoloading. The docking results were in line with the experimental work, where the best results were estimated for raw BTP towards B. subtilis-2FQT protein, recording binding energy (BE) of −7.9776 Kcal/mol. Interestingly, the progress in BE of MTP/AgNPs to −7.6668 Kcal/mol could stem from the exerted Ag-acceptor bonds with both amino acids (SER80 and GLU57) of B. subtilis-2FQT protein. Further, in silico pharmacokinetics assessment was conducted as a step to predict the potential safety and toxicity of the TP/Ag NCs, which revealed AMES toxicity for one of the compounds with neither hepatic toxicity nor skin sensitization. In summary, this study has provided a good design for the synthesis and development of a new generation of nano-based aminophosphonate composites as promising antibacterial candidates, although their safety should be carefully evaluated for each specific application; further studies are needed to fully understand the toxicity mechanisms and the optimal conditions for their pharmaceutical application.Two potent antibacterial agents were facilely synthesized in a one-pot green Kabachnik–Fields reaction and then exploited as reducing/capping surfaces for Ag


Weight gain associated with integrase strand transfer inhibitors (INSTI) has been well documented among patients with HIV. However, recent reports suggest that the observed weight gain among patients who switch to INSTIs may be associated in part with their pre-switch regimen.We conducted retrospective analyses of a 50% sample of all treatment-experienced patients who switched to second-generation INSTI containing regimen (bictegravir/dolutegravir) at the Duke Adult Infectious Diseases Clinic  between 1 January 2014 and 31 December 2021. Covariates included sex at birth, age, race, ethnicity, pre-switch regimen, body mass index (BMI), CD4 count and HIV-1 viral load. All weights documented within 24 months from regimen switch were included. Outcomes of interest were percent change in weight and absolute weight change. Ordinary least squares was used to obtain average percent weight change from baseline, adjusted for covariates.Overall, 339 persons were included in the analyses. Cohort demographics were: median age 51.2 , 73% male sex at birth, 56% Black, 4% Hispanic ethnicity. At regimen switch, median CD4 count was 671 (IQR 460-935), and 78% had a viral load < 200 copies/cc. Mean weight at regimen switch was 87.8 kg (standard deviation [SD] 22.1kg), and mean total weight change over 24 months post-switch was +1.3 kg (SD 7.1 kg). Overall, 28% of cohort members gained >5% of baseline body weight within 24 months of switch. Hispanic ethnicity  and BMI < 20  were associated with post-switch weight gain. Only efavirenz-containing pre-switch regimens were significantly associated with weight gain over 24 months post-switch; EFV/FTC/TDF , all other EFV-containing regimens (6.3% [95% 0.1-12.4]).Pre-switch regimens containing efavirenz were significantly associated with weight gain after switch to second-generation INSTI-based regimens, consistent with findings from non-US based cohorts.Nwora Lance Okeke, MD MPH, Gilead Sciences: Advisor/Consultant


PurposeMarijuana use has been increasing in the adolescent population. Our objective was to examine the prevalence of marijuana use among a sample of adolescents and young adults, determine an association with risk-taking behaviors, identify reported medical symptoms, and delineate common beliefs about marijuana use.MethodsA questionnaire was administered to a sample of patients aged between 12 and 23 years old presenting to the emergency department of Penn State Hershey Medical Center, Hershey, Pennsylvania. Data were stratified by marijuana users and non-users, and further stratified by traditional  and non-traditional  use.ResultsThe analysis was based on 200 questionnaires. Thirty-nine percent (n=78) reported marijuana use. Marijuana users were more likely to report previous sexual intercourse , as well as the use of alcohol , cigarettes , prescription pain medications , and cocaine . Users more likely reported texting while driving  and experienced physical or electronic victimization due to bullying . Users were more likely to report gastroesophageal reflux disease (GERD), attention deficit disorder (ADD), anxiety, and depression. The most common symptoms associated with marijuana use were anxiety (65.4%), headache (61.6%), nausea/vomiting (53.8%), cough (51.3%), and abdominal pain (47.4%). Sixty-nine percent of respondents believed marijuana was “safer than other drugs”.ConclusionBased on our sample, we identified risk-taking behaviors, medical symptoms, and beliefs associated with marijuana use. Healthcare professionals may use these data to provide screening and anticipatory guidance to adolescents who use marijuana and consider marijuana use in their differential diagnosis. According to the 2021 Monitoring the Future study, marijuana use by adolescents declined from the late 1990s to the late 2000s but has since begun to rise again, peaking in 2020 with legalization both recreationally and medically in many states [Despite these negative consequences, many benefits of marijuana use have been cited. Clinical indications for medical marijuana vary by state but can include epilepsy and seizure disorders, cancer, HIV/AIDS, neurodegenerative disease, chronic pain, nausea, and post-traumatic stress disorder .In recent years, CBD has quickly grown in popularity in the form of oils, tinctures, and vapes as a treatment for a wide variety of diseases ranging from cancer to epilepsy, anxiety, pain control, and palliative care, with forms of epilepsy being the only FDA-approved indication to date . Many ofWhile there are many studies documenting different adverse effects associated with marijuana use -12, therWe conducted an observational, self-administered questionnaire-based study between August 2019 and September 2020, utilizing a sample of 200 patients presenting to the emergency department at the Penn State Hershey Medical Center, Hershey, Pennsylvania, aged 12 and 23 years. We excluded patients presenting with altered mental status, severe pain, developmental delay, acute psychiatric illness, or respiratory distress. Patients were also excluded if they were under 18 years of age, did not have a guardian present, were non-English speakers, or had previously completed the questionnaire.Once potential subjects were identified, the study team approached the patient and their guardian (for those less than 18 years old), obtained verbal consent, and provided the questionnaire and writing utensil. The questionnaire included 35 questions, focusing on demographics , risk-taking behaviors , self-esteem  , medicalData were stratified by whether participants were marijuana users or non-users, and marijuana users were further stratified by traditional and non-traditional use. Marijuana users were those who reported marijuana use; non-users were those who did not report marijuana use. Traditional marijuana users were classified as those reporting using vapes, pipes, or edibles, and non-traditional marijuana users were those using oils or concentrates, topical creams, or others.Descriptive statistics were generated, including means and standard deviations for continuous variables. This was an exploratory survey to find out more information about marijuana, and therefore, no formal sample size calculation was performed. Comparisons between categorical variables were analyzed using contingency table analysis; significance levels were determined by Chi-square tests. Data were stratified by marijuana users and nonusers, and marijuana users were further stratified by traditional and nontraditional use. The internal consistency of Likert scale questions was assessed by Cronbach's alpha, which was found to be 0.89. No adjustments for multiple tests were made. Tests were two-sided and based on a significance criterion of p<0.05. A multivariable logistic regression was then run, including all variables that were significant in the univariate analyses. All analyses were performed with the use of the SAS statistical package, version 9.4 . The Pennsylvania State University Institutional Review Board approved this study .Data analysis was performed on 200 completed questionnaires. The mean age of respondents was 17.5 years; 62.5% were female, and 66% identified as White (Table Marijuana use was reported in 39% (n=78) of respondents: 59% (n=46) were traditional, and 41% (n=32) were non-traditional. Marijuana users were less likely to live in a two-parent household than non-users (30.8% (95% CI: 22-42) vs. 54.1% (95% CI: 45-63); p= 0.0055).When stratified by frequency of use, compared to non-users, users were also more likely to have had previous sexual intercourse (79.5% (95% CI: 69-87) vs. 32.8% (95% CI: 25-41.6); p=<0.0001), and have had coitarche before age 18 (53.8% (95% CI: 43-64.5) vs. 21.3% (95% CI: 15-29.5); p=<0.0001)  vs. 10.7%% (95% CI: 6-17.5); p=<0.0001), cigarettes use (41% (95% CI: 30-52) vs. 8.2% (95% CI: 4-14); p=<0.0001), cigars use (30.8% (95% CI: 22-42) vs. 3.3% (95% CI: 1-8); p=<0.0001), chewing tobacco use (18% (95% CI: 11-28) vs. 0.8% (95% CI: 0-4); p<0.0001), prescription pain medications without a prescription (20.5% (95% CI: 13-31) vs. 4.1% (95% CI: 1-9); p=0.002), and cocaine use (14.1% (95% CI: 8-24) vs. 0.8% (95% CI: 0.01-5); p=0.0017)  (24.4% (95% CI: 16-35) vs. 9.0% (95% CI: 5-15.6); p=0.0164), anxiety (57.7% (95% CI: 47-68) vs. 35.2% (95% CI: 27-44); p=0.007), attention deficit disorder (ADD) (29.5% (95% CI: 20-40) vs. 10.7% (95% CI: 6-17.5); p=0.003), and depression (51.3% (95% CI: 40-62) vs. 27.9% (95% CI: 21-36); p=0.002). There were no significant differences between users and non-users who reported the following symptoms during the previous six months: chest pain, racing heart, difficulty breathing or coughing, dizziness, abdominal discomfort, nausea or vomiting, headache, tremors, sleep disturbances, or dehydration. However, there was a statistically significant increase in reports of anxiety in users as compared to non-users (66% (95% CI: 55-76) vs. 47% (CI: 38-56); p=0.009) (Table The most common symptoms associated with marijuana use were anxiety (65.4%), headache (61.6%), nausea/vomiting (53.8%), cough (51.3%), and abdominal pain (47.4%); there was no significant difference in symptom reports between traditional and nontraditional users.Vapes (25.6%) and pipes (25.6%) were the most common forms of marijuana use reported, and 61.6% of respondents reported using marijuana recreationally Table .With respect to perceived beliefs, 64% of respondents believed cannabis products are “less addictive than other drugs,” 69% believed they are “safer than other drugs," 48% believed they “help to treat my medical illness” and 33% believed that marijuana use is “just for fun." When asked to report a myth regarding cannabis products, respondents reported many common phrases, including “it is a gateway drug” (n=11), “it is addictive” (n=9), and “it can kill you” (n=7).After a multivariable logistic regression of all variables that were significant in the univariate analyses, the variables that ended up being significant in the final model were: bullying, sex, cigars, weakness, ADD, and anxiety , and anxiety complaints. More research is necessary to ensure the safe and appropriate use of marijuana as a plant-derived medication for adolescents and adults alike. Discovering key clinical symptomatology will aid physicians in identifying adolescents using marijuana. Furthermore, physicians should consider marijuana use in adolescents and young adults with high-risk behaviors presenting with anxiety-related complaints or other common symptoms associated with marijuana use, such as headache, nausea/vomiting, cough, and abdominal pain.


The last few years have witnessed dramatic advances in our understanding of the structure and function of the mammalian mito-ribosome. At the same time, the first attempts to elucidate the effects of mito-ribosomal fidelity (decoding accuracy) in disease have been made. Hence, the time is right to push an important frontier in our understanding of mitochondrial genetics, that is, the elucidation of the phenotypic effects of mtDNA variants affecting the functioning of the mito-ribosome. Here, we have assessed the structural and functional role of 93 mitochondrial (mt-) rRNA variants thought to be associated with deafness, including those located at non-conserved positions. Our analysis has used the structural description of the human mito-ribosome of the highest quality currently available, together with a new understanding of the phenotypic manifestation of mito-ribosomal-associated variants. Basically, any base change capable of inducing a fidelity phenotype may be considered non-silent. Under this light, out of 92 previously reported mt-rRNA variants thought to be associated with deafness, we found that 49 were potentially non-silent. We also dismissed a large number of reportedly pathogenic mtDNA variants, 41, as polymorphisms. These results drastically update our view on the implication of the primary sequence of mt-rRNA in the etiology of deafness and mitochondrial disease in general. Our data sheds much-needed light on the question of how mt-rRNA variants located at non-conserved positions may lead to mitochondrial disease and, most notably, provide evidence of the effect of haplotype context in the manifestation of some mt-rRNA variants. Aminoglycoside antibiotics (AGs) are some of the most commonly prescribed antibacterials, despite their well-known capacity to cause toxic side effects to the kidneys and inner ear . AlthougOur understanding of the mito-ribosome has advanced much more slowly than that of other ribosomal systems. In particular, the issue of the maintenance of mito-ribosomal fidelity, defined as the accuracy of decoding during mitochondrial translation, could not be tackled until very recently. Despite this, evidence obtained mostly from studies with chimeric mito-bacterial ribosomes, as well as in yeast, has been successfully used to ascertain that decreased mito-ribosomal fidelity was behind the deleterious effect caused by the two known ototoxic variants . The recIn this analysis, we used a total of 83 and 9 variants, identified in deafness patients, that mapped to the mito-ribosomal small and large subunit (SSU and LSU) mt-rRNAs, respectively. The variants originated from 35 studies reported in the literature (80 variants), MITOMAP (2 variants), and this work (1 variant)  Ruiz-Pe. Figure The low conservation of most variants made the comparison to heterologous structures largely useless, implying that the type of evidence used in our previous heterologous inferential analysis (HIA) studies with exceptionally rare mt-rRNA variants could not be raised in this case . In the More than half of the 92 variants  could cause structural distortions, possibly resulting in some degree of defective mito-ribosomal function and constituting valid candidates for deafness-inducing variants under the new theoretical framework for the evaluation of the pathogenic potential of mt-rRNA variants. Forty-one variants, including the nine mapping to 16S mt-rRNA, were deemed likely silent and two unclear . TertiarThis section contains the structural description of all 49 variants regarded as potentially non-silent. Given the number of variants analyzed, most accompanying figures are provided as The existence of pathogenic mutations in several mt-SSU mito-ribosomal proteins is a good indication that variants in mt-rRNA residues interacting with these proteins may also lead to deleterious phenotypes. Early binding proteins are also of particular interest, as they constitute key nucleation sites for the assembly of the mt-SSU. Hence, mt-rRNA variants that interfere with the binding of these proteins could lead to defective mito-ribosomal assembly and other functional defects. All these variants will be discussed in this section.Escherichia coli S12 and mitochondrial MRPS12/uS12m  variant has been detected in several studies targeting hearing-impaired patients (22U (m.669U) base pairs with 281A (m.928A) in h3 of the SSU  and 4 (m.651) of h1 is visible (22U>C (m.669U>C) variant would replace a Watson:Crick U:A base pair with a C•A mismatch at the junction between helices h1 and h3 and in an rRNA region that is surrounded by proteins MRPS12/uS12m and MRPS5/uS5m, the potential for a fidelity mutation is high.The patients . Positio the SSU . A hydro the SSU , in clos the SSU . Positio the SSU , with tw the SSU  is also  the SSU , one of  visible . As the 283G>A (m.930G>A) has been found to be associated with deafness in three studies (283G (m.930G) lies in the single-stranded stretch linking h3 and the functionally important central pseudoknot (helices h1 and h2) to h19 (283G (m.930G) has been modeled 2.39 Å away from Arg 47 of MRPS12/uS12m (283G (m.930G) to MRPS12/uS12m makes the 283G>A (m.930G>A) variant constitute a good candidate for a pathogenic residue. However, in light of its abundance in the population  to h19  (Brink e) to h19 . In the ) to h19 , the O6 12/uS12m , whose e12/uS12m . However12/uS12m . Despite139G>A (m.786G>A) rare variant was detected by The 178U>A (m.825U>A) and 180A>G (m.827A>G) are both haplotype markers that were found in association with deafness in several studies (178U (m.825U) and 180A (m.827A) are located in the single-stranded RNA stretch linking helices h5 and h15 (178U (m.825U) and 179A (m.826A) (178U (m.825U) and the N7 of 180A (m.827A) (178U (m.825U) is located ∼3 Å away from the factor, indicating that this region might have an important role during translocation. In addition to this, 180A (m.827A) is an almost universally conserved residue (position 364 in E. coli 16S rRNA) (2 group at position N3 due to the 180A>G (m.827A>G) base change might result in a slight structural distortion of the region, perhaps by interfering with its interaction with helix h3. A similar structural argument could be made for the 178U>A (m.825U>A) variant in that the insertion of a bulkier adenosine at this position could also slightly induce the distortion of the local structure. Hence, the potential for a fidelity variant exists in both cases.The variants  studies . In the  and h15 , a regio and h15 . Several and h15 . Additio(m.826A) . These M(m.826A)   . Notably(m.827A) . The bas(m.827A) , thus ph(m.827A) . Positio(m.827A) . Since t(m.827A) , as judg(m.827A) . Specifi6S rRNA) , althoug295A>G (m.942A>G) variant was found in two patients with hearing loss (295A (m.942A) is located at the 5′ end of h20 (295A>G (m.942A>G), namely, 400A (m.1047A) and 401C (m.1048C), where an A>G and a C>U transition were, respectively, identified in patients with hearing loss (401C (m.1048C) is modeled as part of a water-mediated, hydrogen bonding interaction with one of the phosphate oxygens of NAD (401C (m.1048C) O2′ to NAD O2′ and a hydrogen bond between spermine N1 and the RNA backbone at position 400A (m.1047A) (not shown). 400A (m.1047A) is unpaired and its base stacks onto that of 401C (m.1048C) (295A (m.942A) and 400A (m.1047A) OP1 (295A (m.942A) also forms water-mediated hydrogen bonds with the backbone of h19 at positions 487–88 (m.1134–35). Notably, position 487G (m.1134G) is within 3 Å of MRPS12/uS12m (295A (m.942A) is the Watson:Crick base pairing formed by positions 294G (m.941G) and 486C (m.1133C)  . A hydro47A) OP1 . The sam12/uS12m . Adjacen295 (m.942) is involved in the stabilization of mito-ribosomal structure via direct (and water-mediated) hydrogen bonds, namely, in the interaction between helices h19, h20 in the neighborhood of protein MRPS12/uS12m. Guaran et al. found the 295A>G (m.942A>G) variant together with the 35delG/35delG allele in the gene GJB2, encoding the protein connexin 26, which is the most common cause of genetic sensorineural deafness in Caucasian patients (295A>G (m.942A>G) variant to the observed symptoms is unclear in this patient. No GJB2 mutations were reported together with 295A>G (m.942A>G) in the other study (400A>G (m.1047A>G) and 401C>U (m. 1048C>U) variants, the situation is uncertain. The base of 401C (m.1048C) only interacts via a water-mediated hydrogen bond with NAD (401C (m.1048C) and NAD is of any relevance to mitochondrial translation is unknown. The abundance of the 401C>U (m. 1048C>U) variant, itself a haplotype marker, clearly indicates that the C>U base change is well-tolerated at this position. In the case of 400A (m.1047A), its base is not involved in any hydrogen-bonding interaction, prompting us to regard it as a silent polymorphism. Despite all this, due to the proximity of 401C (m.1048C) and 400A (m.1047A) to MRPS12, a potentially pathogenic role cannot be completely ruled out for these residues.Of the three variant bases, only patients . Hence, er study . As for with NAD . Whether304G>A (m.951G>A) variant is a haplotype marker that is associated with deafness in several studies (304G (m.951G) is base-paired to 396C (m.1043C) in the distal part of h20 (304G (m.951G). As the G>A base change at position 304 (m.951) would disrupt a base pair interaction next to sites of MRPS12/uS12 that are heterologous equivalents of known fidelity mutations, the potential of the variant to cause a fidelity phenotype is considered high.The  studies . Positiot of h20 , a regiot of h20 , near tht of h20   variant was found in heteroplasmy in a patient with non-syndromic hearing loss while the haplotype marker 460U>C (m.1107U>C) was identified as a potential deafness-causing variant in three subjects from a cohort of aminoglycoside-induced and non-syndromic hearing-impaired Chinese pediatric patients (459C (m.1106C) displays a potential contact with the backbone of protein MRPS37/mS37 (459C>U (m.1106C>U) variant, the results of this loss are uncertain.The patients . In the patients . The basS37/mS37 , which hS37/mS37 . Althoug460U (m.1107U) with the N6 of 478A (m.1125A), two nucleotides away from the central domain pseudoknot that connects helices h19 and h25 of 12S mt-rRNA (shown in red in 460U (m.1107U) O2′ to 290U (m.937U) OP1 is 3.217 Å, possibly indicating a direct tertiary interaction with the central domain pseudoknot (E. coli and were shown to be deleterious (460U (m.1107U) is not involved in any direct interactions with other residues except for a water-mediated hydrogen bond with position 947A (m.1594G) near the 3′end of the molecule  suggests that the 460U>C (m.1107U>C) variant could be a simple polymorphism (but see below).A tertiary interaction is observed between the backbone at position eudoknot . Mutatioeterious . Despitemolecule . The imp459C>U (m.1106C>U) and 460U>C (m.1107U>C) cannot be ruled out.Considering all of this, as the 5’ side of the central domain pseudoknot is adjacent to h19, which, in turn, wraps around MRPS12/uS12m, it is possible that even small distortions in this region could be transmitted through this protein and result in fidelity defects. As a result, the potential for the creation of slight fidelity phenotypes by the variants During the early assembly, mito-ribosomal proteins MRPS16/bS16m and MRPS18B/mS40 interact with the nascent 12S mt-rRNA transcript  base change was found in two patients with non-syndromic hearing loss (76A (m.723A) is involved in a triple base interaction with the 144G:150C (m.791G:797C) base pair, thus bringing together helices h7 and h12 (145C (m.792C) of h12, where a C>U base change was identified in two patients with non-syndromic hearing loss (145C (m.792C) is canonically base-paired to 149G (m.796G), closing the terminal loop of h12 (The ing loss . Positio and h12 . A seconing loss . Positiop of h12 .76A>C (m.723A>C) variant, the base change would prevent the base triple interaction, while the C>U base change at position 145C (m.792C) would result in a structurally disfavored C:G/U•G exchange at a terminal base pair  and the 98A>G (m.745A>G) variants were identified in a patient with idiopathic, sensorineural hearing loss and a patient with profound hearing impairment, respectively (95U (m.742U) base-pairs to 85A (m.732A) in h7 (98A (m.745A) is involved in a base quadruple interaction involving the 82U:97A (m.729U:744A) base pair and position 83A (m.730A), both of them in h7 (95U (m.742U) and 98A (m.745A) are structurally important in the context of h7 and that this helix is part of the MRPS16/bS16 binding site, a disruptive effect beyond the local structure of h7 cannot be ruled out.The ectively . PositioA) in h7 . Positioem in h7 . Althougem in h7 , known tem in h7 . Given t209A>G (m.856A>G) variant was identified in four patients with non-syndromic hearing loss (209A (m.856A) is base-paired to 183U (m.830U) at the proximal end of helix h15 (209A>G (m.856A>G) variant is considerable.The ing loss . The varing loss  and Lebeing loss . Positioelix h15 . Hydrogeelix h15 . In addielix h15 . The 209During assembly, the 5′ and 3′ domains are brought together by the strong interactions established by the 5′-domain binders MRPS16/bS16m, MRPS18B/mS40, and MRPS22/mS22 and the 3′-domain binders MRPS27/mS27 and MRPS34/mS34 . The imp62G>A (m.709G>A), which has been frequently associated with hearing impairment (63U>C (m.710U>C) and 65C>A (m.712C>A) variants (62G (m.709G) is modeled, making two hydrogen bonds to Arg 48 of MRPS34/mS34 , 53A (m.700A), and 65C (m.712C) stabilizes the structure of the RNA stretch separating helices h6 and h6a and provides a recognition surface for the late-binding protein MRPS26/mS26 (pink in 62G>A (m.709G>A) clearly implies that the variant must be easily accommodated in the SSU structure. Despite this, the G>A base change is expected to disrupt the hydrogen bonding network observed in the structure, involving tertiary and quaternary contacts and likely leading to some degree of distortion. Similarly, the pyrimidine-to-purine replacement caused by the 65C>A (m.712C>A) variant is expected to disrupt the non-canonical base pair between 65C (m.712C) and 53A (m.700A). With this in mind, both variants must be regarded as potentially pathogenic.Three deafness-associated variants have been identified in the single-stranded stretch connecting helices h6 and h6a in the secondary structure map of the mt-SSU, namely, the haplotype defining variant pairment , as wellvariants  (Koningsvariants . The bas pink in  (Bogenha pink in . The abu62G (m.709G) is 63U (m.710U), whose base is stacked between 62G (m.709G) and Arg 30 of MRPS26/mS26 (63U (m.710U) is involved in a water-mediated hydrogen bonding interaction with 62G (m.709G) N7 (U>C base change at position 63 (m.710) is not expected to bring about any disruptive effect, as shown by the fact that a C is found at the equivalent position in the S. scrofa mito-ribosome (not shown) (Adjacent to S26/mS26 . 63U (m.709G) N7 . Despitet shown) .878C>G (m.1525C>G) variation was found by our group in a patient with profound hearing loss (878C (m.1525C) is located at the very distal end of h44 (878C (m.1525C) has been modeled, making a hydrogen bond to the backbone of protein MRPS27/mS27 (878C (m.1525C) would, in principle, argue for a silent phenotype, the rarity of the C>G variant, together with the existence of pathogenic mutations in MRPS34/mS34  (545C>A/U (m.1192C>A/U) (546U>C (m.1193U>C) (815G>A (m.1462G>A) (545C (m.1192C) and 815G (m.1462G) form a Watson:Crick base pair at the distal end of h28 (546U (m.1193U) (545C (m.1192C) and the functionally important β-hairpin of protein MRPS7/uS7m is less than 3 Å (not shown)  , 545C>A/92C>A/U) , 546U>C 1193U>C) , and 8151462G>A) . Positiod of h28 . This red of h28 , which id of h28 . Contactm.1193U) . Notablym.1193U) , adjacenm.1193U) . Additiot shown) .542U>C (m.1189U>C) variant was identified in several independent studies (542U (m.1189U) maps to the single-stranded stretch of h28 (542U (m.1189U) is nearly on the same plane as the 545C:815G (m.1192C:1462G) base pair but slightly tilted, allowing it to use its O2 to form a hydrogen bond with the neighboring 544C:816G (m.1191C:1463G) base pair (542U (m.1189U) O4 to the NH2 of 782C (m.1429C) (542U>C (m.1189 U>C) variant. 542U>C (m.1189U>C) was found together with the 141A>G (m.1811A>G) variant in 16S mt-rRNA and the R127H allele in gene GJB2 (present in heterozygosity) (The  studies . In the h of h28 . In the ase pair . The tilm.1429C) , a contaygosity) , both ofygosity) .In addition to elongation, this region of the mito-ribosome is also implicated in translation initiation. 545C:815G (m.1192C:1462G) base pair and the tertiary interactions of 542U (m.1189U) are important to stabilize the structurally complex and dynamic junction between helices h28 and h29. Of the three variants affecting the 545C:815G (m.1192C:m.1462G) base pair, 545C>A (m.1192C>A) and 815G>A (m.1462G>A) would replace a Watson:Crick base pair with either an A•G or C•A mismatch, while 545C>U (m.1192C>U) would result in a disruptive U•G wobble at the end of a helical segment (546U (m.1193U) makes no hydrogen bonds which, together with the fact that a C is found in the S. scrofa structure at this position (position 544 in structure 5AJ4), strongly argues for a silent phenotype associated with the 546U>C (m.1193U>C) variant.All this evidence strongly suggests that the  segment . For all596U>C (m.1243U>C) was found in association with deafness in several studies (596U (m.1243U) maps to helix h32 of 12S mt-rRNA, where it base pairs to 699A (m.1346A) (596U:699A (m.1243U:1346A) base pair by the U>C base change at position 596U (m.1243T) could affect the interaction of h32 and MRPS14/uS14m in this region. Although the abundance of the 596U>C (m.1243U>C) variation implies that the base change is well-tolerated, whether it could lead to slight phenotypic effects remains uncertain.The haplotype marker  studies . Positiom.1346A) . Arg 36 m.1346A)  forms a 663C>U (m.1310C>U) variant has been identified in a Japanese patient with inherited sensorineural hearing loss (663C (m.1310C) maps to h38 of 12S mt-rRNA, where it base pairs with 668G (m.1315G) (663C (m.1310C) would create a disfavored U•G wobble base pair at the end of a helical segment (The ing loss . Positiom.1315G) . Its O2′m.1315G) . Late-bim.1315G)  also conm.1315G)  and the  segment  with the735A>C (m.1382A>C) variant was identified in two families with hearing loss (735A (m.1382A) is base-paired to 723U (m.1370U) at the proximal end of h41 in the head of the mt-SSU (735A (m.1370U:m.1382A) base pair, the pathogenic potential of this variant must be considered.The ing loss . Positioe mt-SSU . A contae mt-SSU  to the Re mt-SSU  establise mt-SSU . As the 796U>C (m.1443U>C) variant was identified in a non-syndromic hearing-impaired Chinese pediatric subject (796U (m.1443U) maps to the loop that caps h43 of 12S mt-rRNA (796U (m.1443U) establishes a hydrogen bond with the RNA backbone at position 799 (m.1446) (7 (m.1443-4) are visible in the structure. The adjacent position 797A (m.1444A) makes a tertiary interaction by forming a triple base interaction with 585A (m.1232A) of h31 and 753G (m.1401G) of h42 (796U (m.1443U) (not shown) (796U>C (m.1443U>C) must be considered significant.The highly rare  subject . Positio mt-rRNA . The N3 (m.1446) , an inte(m.1446)  and the ) of h42 . This ant shown) . Given a805U (m.1452U) and 806A (m.1453A), presumably associated with hearing impairment, map to h43 of 12S mt-rRNA (805U (m.1452U), while MRPS9/bS9 and MRPS14/uS14m (beige in 806A (m.1453A). Position 805U (m.1452U) forms a wobble base pair with 791G (m.1438G) (791 (m.1438), which is present in only 5% of the GenBank sequences (791/805 (m.1438/1452) base pair. As a result, both the 805U>C (m.1452U>C) and the 791G>A (m.1438G>A) variants should be considered silent, as they would result in the exchange of the wobble configuration seen in the structure for a Watson:Crick base pair. Preceding the 791/805 (m.1438/1452) base pair lies position 806A (m.1453A), which forms a Watson:Crick base pair with 790U (m.1437U) (806A (m.1453A) (The sites of variation  mt-rRNA  (Padma a mt-rRNA . Protein mt-rRNA  contact beige in  do the sm.1438G) , anotherm.1438G) . In factequences . These dm.1437U) . A deafnm.1453A)  that wouThe early binding proteins MRPS2/uS2m and MRPS23/uS23, together with MRPS28/bS1m, bind as a cluster to the solvent side of the 12S mt-rRNA molecule . Pathoge469A>G (m.1116A>G), 471A>G (m.1118A>G), and 472U>C (m.1119U>C) variants were found in four hearing-impaired patients (471A (m.1118A) and 472U (m.1119U) are located in the loop capping h25 (471A (m.1118A) has been modeled within 3 Å of Arg 297 of protein MRPS5/uS5m (beige in ram residue V336 within the same protein (not shown) (472U (m.1119U), its base has been modeled, making a water-mediated hydrogen bond, through its O2, to the base and the sugar of 479A (m.1126A) and 480A (m.1127A), respectively (472U (m.1119U) would be unaffected by a U>C base change, thus making 472U>C (m.1119U>C) a good candidate for a silent variant. In this particular patient, the presence of 62G>A (m.709G>A) together with 472U (m.1119U) could offer a better explanation for the observed deafness, as explained previously (469A>G (m.1116A>G) variant is different in that its base is part of a base triple with the 467U:480A (m.1114U:1127A) that would be likely affected by the base change. Additionally, Arg 100 of MRPS2/uS2m (orange in the structure) is within hydrogen bonding distance of the RNA backbone at position 469A (m.1116A). The density around 469A (m.1116A) supports these interactions. Hence, despite the peripheral location of the 469A (m.1116A) variant, the structural evidence does not permit its scoring as a silent mutation.The patients . Positioping h25  toward tbeige in  and ∼13-t shown) . Despitet shown)  suggestsectively . In addiectively . All theeviously . The sit103 (m.750) is an A in the Cambridge mtDNA reference sequence (103 (m.750) is a G that forms a Watson:Crick base pair to 77C (m.724C) in h7 (103G (m.750G) is visible (103G (m.750G), the introduction of an A•C mismatch at this position in h7 is expected to cause some level of structural distortion whose overall consequences cannot be predicted. Hence, the 103G>A (m.750G>A) base change is another example of a haplotype marker with a potentially pathogenic role.Position sequence . Howeversequence . Base chsequence . In the C) in h7 . A conta visible . Althoug122G>A (m.769G>A) and the 123C>U (m.770C>U) variant were described in the context of deafness in two studies (122G (m.769G) and 123C (m.770C) map to the GNRA tetraloop capping h11 (123C>U (m.770C>U) variant would not change the GNRA consensus, 122G>A (m.769G>A) would do so, likely disrupting the structure of the tetraloop and, perhaps, the binding of MRPS17/uS17m. Given the role of MRPS17/uS17m as an early binding protein, the possibility of disruptive potential in the case of the 122G>A (m.769G>A) variant must be considered.The haplotype marker  studies . Positioping h11  , 358U (m.1005U), 360G (m.1007G), and 361A (m.1008A) map to h23 of 12S mt-rRNA (358U (m.1005U) is involved in a water-mediated, non-canonical base pair with 336C (m.983C) and a quaternary contact with Asn 104 of MRPS11/uS11m (pink in 360G (m.1007G) and 361A (m.1008A) form Watson:Crick base pairs with residues 334C (m.981C) and 333U (m.980U), respectively. Lys 6 of MRPS21/bS21 contacts the RNA backbone at positions 359U (m.1006U) and 360G (m.1007G) (light green in 333U>C (m.980U>C) and 360G>A (m.1007G>A) would both result in a disruptive C•A mismatch, while the 361A>G (m.1008A>G) would give rise to a less damaging U•G wobble. In the case of 358U>C (m.1005U>C), the hydrogen bonding pattern observed in the structure would not be maintained, possibly causing some degree of structural disruption. All variants have been identified in MITOMAP with moderate-to-high frequency (two are haplotype markers) (Positions  mt-rRNA  (Li et a mt-rRNA . The bas pink in . Positiomarkers)  (Ruiz-Pemarkers) , indicat341G (m.988G) and 343U (m.990U) of h23 were found in the studies of 341G>A (m.988G>A) and 343U>C (m.990U>C) would disrupt the base hydrogen bonding pattern observed in the two high-resolution structures used in this analysis  variant was found in several patients with sensorineural and non-syndromic hearing loss (448U (m.1095U) base-pairs to 413A (m.1060A) in h24 of 12S mt-rRNA (448U (m.1060A:1095U) base pair by the U>C transition has the potential to affect the functioning of bridge B7b and cause translational defects. Results of biochemical analyses with cybrids carrying this variant in heteroplasmy were consistent with decreased mitochondrial function and increased AG susceptibility  variant was identified by Rydzanicz et al. in a patient with non-syndromic and aminoglycoside-induced hearing loss and by Chen et al. in a Uyghur patient with non-syndromic deafness (856G (m.1503G) is near the highly conserved bridge mB5, which according to lower-resolution structures of the human mito-ribosome, involves positions 858A (m.1505A) and 892C (m.1539C) in h44 of 12S mt-rRNA and MRPL14/uL14m in the LSU  and 890C>U (m.1537C>U) also map to h44 of 12S mt-rRNA and the neighborhood of mB5 (861C>U (m.1508C>U) has been identified in a patient with profound hearing loss without previous exposure to AGs (890C>U (m.1537C>U) variant, it has been identified in two patients with non-syndromic hearing impairment (890C (m.1537C) establishes two hydrogen bonds, one with the N3 of the adjacent 889A (m.1536A) and the other with the O2′ of 864C (m.1511C) (Variants d of mB5 . Positioe to AGs . This ree to AGs . As for pairment . The amim.1511C) .861C>U (m.1508C>U) or 890C>U (m.1537C>U) variants could affect the proper functioning of mB5. Regarding 856G>A (m.1503G>A), its high frequency in the population clearly indicates that an A must be tolerated at this position. However, the loss of a quaternary interaction involving 856G (m.1503G) and 941U (m.2611T) of 16S mt-rRNA observed in the 2.2-Å cryo-EM human mito-ribosomal structure  variant was found in homoplasmy in pediatric subjects with non-syndromic hearing loss (533U>G (m.1180U>G) and 827G (m.1474G) of h28 form a wobble base pair (533U•827G (m.1180U•m.1474G) wobble lies position 826C (m.1473C), where a C>U transition was found in a patient with severe hearing loss (826C (m.1473C) forms a Watson:Crick base pair with 534G (m.1181G) (533U (m.1180U) lies the universally conserved 532G (m.1179G)  , and 534G (m.1181G)  in the cytoplasmic ribosome confer lethal phenotypes (533U (m.1180U) and 534G (m.1181G) , compensatory mutations restoring base pairing potential also restored growth (533U>G (m.1180U>G) variant would replace a U•G wobble with a G•G mismatch, its disruptive potential must be considered high. Hence, the use of the new structural data corroborates our previous assignment as an expectedly disruptive mutation. In the case of 826C>U (m.1473C>U), the transition would replace a Watson:Crick G:C with a G•U wobble, creating a highly non-isosteric U•G/G•U tandem wobble combination (826C>U (m.1473C>U) variant is well tolerated, as judged by its high frequency (The very rare ing loss . We haveing loss  and descase pair . Next toing loss . Positiom.1181G) . On the nd 100%) , which ind 100%)   . Strikinl growth , but witin vitro . Mutagenenotypes . In the d growth . As the bination . Althougrequency , subtle 579C>G (m.1226C>G) variant was found in a pediatric subject with non-syndromic hearing loss (579C (m.1226C) is universally conserved (579C (m.1226C) is located at the single-stranded junction connecting helices h31 and h32 (579C (m.1226C) canonically base-pairs with 570G (m.1217G), also universally conserved  and adjacent residues were mutated, they resulted in moderate-to-strong growth defects and elicited fidelity phenotypes (579C (m.1226C)  , we cons579C (m.1226C). The 684A>G (m.1331A>G) base change was identified in one Han Chinese pediatric subject with aminoglycoside-induced and non-syndromic hearing loss (684A (m.1331A) maps to h34, it is brought into the neighborhood of 579C (m.1226C) by a tertiary interaction in the form of a trans-Watson:Crick base pair, with the base of 568U (m.1215U) (684A>G (m.1331A>G) must be considered a potentially pathogenic variant.A second variant maps to the neighborhood of position ing loss . Althougm.1215U)  (Leontism.1215U) . This in910A>C (m.1557A>C) variation was identified in a Russian individual suffering from deafness . Position 910A (m.1557A) maps to a single-stranded stretch of h44 (910A>C (m.1557A>C) variant in our previous analysis of highly rare variants mapping to 12S mt-rRNA, performed in the absence of high-resolution mito-ribosomal structures (910A (m.1557A) and 911A (m.1558A) in monitoring the geometry of A-site decoding is evident from the 2.2-Å structure of the mito-ribosome  was found in two aminoglycoside-induced hearing-impaired subjects (951G (m.1598G) maps to the single-stranded stretch near the 3′end of the molecule (951G (m.1598G) would impact its interaction with 921U (m.1568U), possibly resulting in a distortion of the mRNA channel and leading to phenotypic effects. These effects, however, must not be drastic, as the 951G>A (m.1598G>A) base change has been found frequently in the population  and 908A>G (m.1555A>G) of 12S mt-rRNA. Positions 847C (m.1494C) and 908A (m.1555A) normally form a C•A mismatch at the penultimate helix of human mt-12S rRNA , which ie trauma . However (76A>C (m.723A>C) variant. Position 76A (m.723A) is involved in a triple base interaction that brings together helices h7 and h12 near the recognition sites for MRPS16/bS16 and MRPS25/mS25, both known to harbor pathogenic mutations (76A>C (m.723A>C) is expected to disrupt this triple base pair interaction, possibly affecting the helical arrangement of part of 12S mt-rRNA and the binding of MRPS16/bS16 and MRPS25/mS25. However, the triple base interaction is not conserved, as it is absent in the S. scrofa mito-ribosome  and 908A>G (m.1555A>G) affect structurally non-conserved positions, forming a mismatch in the human mito-ribosome t-rRNAs) . This tyutations  (Miller utations . The potribosome . This exThe possibility that haplotype context could affect the manifestation of mt-rRNA variants was recently proposed by our group  and has 401C (m.1048C) (401C (m.1048C) and the NAD molecule is of any relevance to mitochondrial translation remains unknown.As mentioned in the introduction, AGs antibiotics are an important class of external agents capable of affecting the penetrance of mt-rRNA variants. HIA studies conducted by our group with highly rare mt-rRNA variants reportedly in association with diseases showed that an important number of them mapped to the vicinity of the binding sites of known ribosomal antibiotics . As a rem.1048C)  (Itoh etm.1048C) . The prem.1048C) . Despite910A>C (m.1557A>C) is a good example, as it has the strongest disruptive potential. Base changes at position 910A (m.1557A) are expected to completely abolish mito-ribosomal translation by directly interfering with genetic decoding (The growing realization in the field that mtDNA disease and, more specifically, deafness can be brought about by variants with low-profile phenotypes, such as those affecting mito-ribosomal fidelity (reviewed in accompanying study by decoding . Unfortudecoding .In summary, we have provided a much-needed assessment of the structural and functional role of suspect mt-rRNA variants identified in the context of deafness. Besides highlighting the key role of the primary sequence of mt-rRNA in the etiology of deafness, this research provides a new framework to understand how mt-rRNA variants, particularly those located at non-conserved positions, may lead to deafness. Hence, the results presented here constitute an important push toward the final elucidation of the role of mt-rRNA in mitochondrial deafness and mitochondrial disease. Such elucidation will eventually require biochemical evidence obtained with mutant mitochondria carrying mito-ribosomes with altered mt-rRNAs.Structural analysis was performed with Chimera X , essenti


The Supporting Information file legends are incorrect. Please view the correct Supporting Information file legends below.S1 Fig. CFTR presence and localization in the normal human pancreas. CFTR immunostaining using CFF-217  of normal pancreas from donors of different ages: 2 months (A-B), 6 months (C-D), 2 months (E-F), 7 months (G-H), 30 years (I-J), and 39 years (K-L). CFTR immunoreactivity is shown in ductal and islet endocrine cells. Shown in B, D, F, H and J are consecutive sections immunolabeled by using the pan-endocrine marker synaptophysin (SYN) to confirm CFTR expression in the pancreatic islets. A larger magnification of the section area in the inset in K is shown in L. Scale bars represent 50μm.https://doi.org/10.1371/journal.pone.0242749.s001(TIF)S2 Fig. Immunohistochemistry for CFTR. Normal human pancreas tissues obtained from three donors aged 6 months , 11 months  and 4 years  are immunostained by using anti-human CFTR antibodies CFF-432 (A-C), CFF-412 (D-F) and CFF-570 (G-i). CFTR expression (brown) occurs in both duct (green squares) and islet endocrine cells (red squares). Higher magnifications of the areas noted by green and white inserts i.e., ductal and islet areas, respectively, are shown under each image. Scale bars represent 100μm.https://doi.org/10.1371/journal.pone.0242749.s002(TIF)S3 Fig. CFTR antibodies not-suitable for immunohistochemistry. Normal human pancreatic tissues stained with the indicated CFTR antibodies. A. CFF-596 antibody generates non-specific immunostaining in the CFTR-negative control human tissue with the G542X truncation mutation. B-C. CFF-660 (B) and CFF-450 (C) antibodies do not generate specific immunostaining in the positive control ductal cells. D. Antibody Ab4067 does not generate immunostaining signal in the normal human pancreas. E-J. Pancreatic tissue from CftrWT (E-G) and CftrG542X mice (H-J) co-immunolabeled by using the mouse anti CFTR-specific antibody MrPink (J and H), insulin  and glucagon .https://doi.org/10.1371/journal.pone.0242749.s003(TIF)S4 Fig. Original blots used in Fig 4.https://doi.org/10.1371/journal.pone.0242749.s004(TIF)


Pseudomonas aeruginosa has not been fully elucidated. This study evaluated the in vitro activity of novel β-lactam/β-lactamase inhibitor combinations against Pseudomonas aeruginosa clinical isolates, determined how avibactam restored ceftazidime activity, and compared the activity of ceftazidime-avibactam (CZA) and imipenem-relebactam (IMR) against KPC-producing P. aeruginosa. Similar high susceptibility rates for CZA, IMR, and ceftolozane-tazobactam (88.9% to 89.8%) were found for 596 P. aeruginosa clinical isolates from 11 hospitals in China, and a higher susceptibility rate to ceftazidime than imipenem was observed (73.5% versus 63.1%). For CAZ-NS and IPM-NS isolates, susceptibility rates for CZA, ceftolozane-tazobactam, and IMR were 61.5% (75/122), 54.9% (67/122), and 51.6% (63/122), respectively. For CAZ-NS, IPM-NS but CZA-susceptible isolates, 34.7% (26/75) harbored acquired β-lactamases with KPC-2 predominant (n = 19), and 45.3% (34/75) presented overexpression of chromosomal β-lactamase ampC. Among 22 isolates carrying KPC-2 carbapenemase alone, susceptibility rates to CZA and IMR were 86.4% (19/22) and 9.1% (2/22), respectively. Notably, 95% (19/20) of IMR-nonsusceptible isolates had an inactivating mutation of oprD gene. In conclusion, CZA, ceftolozane-tazobactam, and IMR exhibit high activity against P. aeruginosa, and CZA is more active than IMR against CAZ-NS and IPM-NS isolates as well as KPC-producing P. aeruginosa. Avibactam overcomes ceftazidime resistance engendered by KPC-2 enzyme and overexpressed AmpC.The role of novel β-lactam/β-lactamase inhibitor combinations in ceftazidime-nonsusceptible (CAZ-NS) and imipenem-nonsusceptible (IPM-NS) IMPORTANCE The emergence of antimicrobial resistance poses a particular challenge globally, and the concept of P. aeruginosa with “difficult-to-treat” resistance (DTR-P. aeruginosa) was proposed. Here, P. aeruginosa clinical isolates were highly susceptible to three β-lactamase inhibitor combinations, CZA, IMR, and ceftolozane-tazobactam. The combination of KPC-2 enzyme and nonfunctional porin OprD contributed to IMR resistance in P. aeruginosa, and CZA was more active than IMR in fighting against KPC-2-producing P. aeruginosa. CZA also showed good activity against CAZ-NS and IPM-NS P. aeruginosa, primarily by inhibiting KPC-2 enzyme and overproduced AmpC, supporting the clinical use of CZA in the treatment of infections caused by DTR-P. aeruginosa. Pseudomonas aeruginosa is a major cause of serious infections in humans, which often become notoriously refractory to treatment and lead to considerable morbidity, mortality, and socioeconomic impacts worldwide. In this scenario, β-lactam antibiotics remain one of the mainstays of therapy. One interesting phenomenon is the higher in vitro susceptibility rate of this pathogen to ceftazidime than to carbapenems  was proposed in 2018  and fluoroquinolones (ciprofloxacin and levofloxacin)  is commonly responsible for resistance to ceftazidime via structural mutations or overexpression. The intrinsic resistance-nodulation-division family of efflux systems  are also critical features associated with β-lactam resistance  . Mechani diverse . Mutatio diverse . Chromossistance , and theP. aeruginosa strains with inhibitory Ambler class A, C, and some D β-lactamases. IMR exhibits an antimicrobial spectrum similar to that of CZA except for inhibition activity against OXA-48 enzyme. Ceftolozane, a novel oxyimino-aminothiazolyl cephalosporin, is characterized by its stability in PDC hydrolysis. In previous studies, CZA and CT showed excellent in vitro activity against P. aeruginosa with susceptibility rates of 86.5% and 88.5%, respectively, and IMR demonstrated a susceptibility rate of 90.8% (P. aeruginosa according to the guidance of the Infectious Diseases Society of America (IDSA) , ceftolozane-tazobactam (CT), and imipenem-cilastatin-relebactam (IMR), have recently been approved and cast a new spotlight on the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections. CZA expands the therapeutic coverage of ceftazidime against of 90.8% . These cP. aeruginosa isolates are emerging in China. The resistant gene blaKPC-2 is commonly located in plasmids with mobile elements and is frequently detected in sequence type 463 (ST463) isolates  to 100% (16/16)  , 12. Howin vitro activities of CZA, CT, and IMR against clinical P. aeruginosa isolates collected from multiple medical centers in China and uncovered the extent to which avibactam restored the activity of ceftazidime in ceftazidime-nonsusceptible (CAZ-NS) and imipenem-nonsusceptible (IPM-NS) P. aeruginosa. This study found high in vitro activities of CZA, CT, and IMR against P. aeruginosa clinical isolates. The combination of KPC-2 carbapenemase and nonfunctional porin OprD led to IMR resistance in P. aeruginosa, and CZA was more active than IMR in fighting against KPC-2-producing P. aeruginosa. CZA also retained good activity against CAZ-NS and IPM-NS P. aeruginosa isolates, as avibactam overcame the ceftazidime resistance engendered by acquired β-lactamase KPC-2 and overproduced chromosomal β-lactamase AmpC.This study investigated the P. aeruginosa isolates. The MIC50 and MIC90 for CZA were 2 mg/L and 16 mg/L, respectively, and avibactam restored the susceptibility of ceftazidime in 61.4% (97/158) of CAZ-NS isolates. Moreover, higher susceptibility to CAZ than to IPM was observed . The most active agents against 596 P. aeruginosa isolates were colistin  and amikacin (90.8%), and susceptibility rates to the remaining six agents ranged from 58.2% (aztreonam) to 78.2% (cefepime) (P. aeruginosa accounted for 11.9% (71/596).Overall, susceptibility rates to CZA, IMR, and CT were 89.8%, 88.9%, and 88.9%, respectively, among the 596 efepime) . DTR-P. P < 0.001) but relatively weaker correlations between the CZA and imipenem MICs  among the 596 P. aeruginosa clinical isolates  ceftazidime-susceptible (CAZ-S) and imipenem-susceptible (IPM-S) isolates, 98 (16.4%) CAZ-S but IPM-NS isolates, 36 (6.0%) IPM-S but CAZ-NS isolates, and 122 (20.5%) CAZ-NS and IPM-NS isolates  of these were susceptible to CZA  of e to CZA . Among te to CZA . In addiP. aeruginosa isolates displayed nonsusceptibility to imipenem, 78.6% (173/220) of IPM-NS isolates were susceptible to CZA, including 98 (44.5%) CAZ-S isolates and 75 (34.1%) CAZ-NS isolates (While 36.9% (220/596) of isolates .For 122 CAZ-NS and IPM-NS isolates, susceptibility rates for CZA, CT, and IMR were 61.5% (75/122), 54.9% (67/122), and 51.6% (63/122), respectively.To illuminate how avibactam overcomes ceftazidime resistance, mechanisms of ceftazidime nonsusceptibility were investigated using whole-genome sequencing (WGS) and quantitative real-time PCR (qRT-PCR) among 75 CAZ-NS, IPM-NS but CZA-S isolates .n = 19), VIM-2 (n = 1), and GES-5 (n = 1), as well as coexistence of GES-5 and PER-1 (n = 1). The VIM-2-carrying isolate had a CZA MIC of 8 mg/L. Extended-spectrum β-lactamases (ESBL) were detected in four isolates (excluded the one coexisting with GES-5): PER-1 (n = 2) and OXA-101 (n = 1), as well as coexistence of PER-1 and OXA-101 (n = 1) (Table S2).Among these 75 isolates, 26 (34.7%) carried horizontally acquired β-lactamases  and maniampC overexpression phenotype  were witnessed in 21 isolates, including a premature stop codon (n = 6) and amino acid substitution G154R (n = 3) in ampR, a frameshift mutation (n = 7) in ampD, and a premature stop codon (n = 1), frameshift mutation (n = 3), and amino acid substitution T428P (n = 1) in dacB. A total of 17 subtypes of PDC were present in the 75 isolates (Table S3), with PDC-8 (n = 18) being the most common, followed by PDC-5 (n = 16), PDC-1 (n = 8), and PDC-3 (n = 6). Additionally, overexpression of the efflux pump gene mexAB-oprM was detected in 35 isolates  isolates, including frameshift mutations (n = 53) and premature stop codons (n = 12) (Inactivating mutations in (n = 12) .blaPER-1. These isolates did not harbor carbapenemases, nor did they show inactivating mutations in oprD. In total, 59.1% (13/22) of isolates displayed ampC overexpression, and 50.0% (11/22) of isolates exhibited alteration of regulatory factors contributing to ampC overproduction, including a premature stop codon (n = 1), frameshift mutation (n = 1), and amino acid substitution D135N/Y (n = 4) in ampR, a premature stop codon (n = 4) and frameshift mutation (n = 1) in ampD, and a premature stop codon (n = 1) in dacB. A total of 10 subtypes of PDC were detected in these 22 isolates (Table S3), with PDC-3 (n = 10) being the most prevalent, followed by PDC-8 (n = 2), PDC-16 (n = 2), and PDC-63 (n = 2).Among the 22 CAZ-NS, IPM-S, and CZA-S strains, only one isolate carried an ESBL-encoding gene, n = 17), ST16 (n = 2), ST238 (n = 1), ST244 (n = 1), and an undefined ST (n = 1). Notably, their susceptibility rates to CZA, CT, and IMR were 86.4% (19/22), 0%, and 9.1% (2/22), respectively.Among the 158 CAZ-NS isolates, 22 carried KPC-2 carbapenemase and did not carry metallo-β-lactamase. They were assigned to five STs, ST463  of IMR-nonsusceptible isolates had an inactivating mutation of the imipenem . The oneimipenem .P. aeruginosa clinical isolates were highly susceptible to three β-lactam/β-lactamase inhibitor combinations, CZA, IMR, and CT. In total, 89.8% of P. aeruginosa clinical isolates from China were susceptible to CZA with MIC50/MIC90 of 2/16 mg/L, and avibactam restored the susceptibility to ceftazidime in 61.4% of CAZ-NS isolates. Similar results were found in a study in Spain, in which antimicrobial activity of CZA was demonstrated against 1,445 P. aeruginosa isolates, with a susceptibility rate of 94.2% and MIC50/MIC90 of 2/8 mg/L  was found for 2,623 P. aeruginosa isolates from Asia, and relebactam restored imipenem susceptibility from 0% to 64.6% for 805 IPM-NS isolates (P. aeruginosa (in vitro activity (88.9%) was also observed for CT in the 596 isolates, with an MIC50/MIC90 of 0.5/8 mg/L. Nevertheless, variation was noted in the susceptibility to CT in different regions, which ranged from 89.1% in Europe to 98.2% in North America  of IPM-NS isolates being susceptible to ceftazidime. Susceptibility  to ceftazidime was also reported for carbapenem-resistant P. aeruginosa (CRPA), as evidenced by data from the Antimicrobial Testing Leadership and Surveillance (ATLAS) program in the Asia-Pacific region from 2015 to 2019 . A global study reported a susceptibility rate to CZA of 75.9% for 29 KPC-2-positive clinical P. aeruginosa isolates, while another group from China reported a CZA susceptibility rate of 49.7% for 151 KPC-2-positive clinical P. aeruginosa isolates . Therefore, the combination of KPC-2 enzyme and nonfunctional porin OprD leads to IMR resistance in P. aeruginosa , and CZA is more active than IMR in fighting against KPC-2-producing P. aeruginosa.Among 22 isolates carrying KPC-2 carbapenemases alone, 95% (19/20) of IMR-nonsusceptible isolates had an inactivating mutation of the imipenem  and furtimipenem . AdditioP. aeruginosa, supporting the clinical use of CZA in the treatment of infections caused by DTR-P. aeruginosa. We also found that 45.3% (34/75) of isolates demonstrated overexpression of AmpC in CAZ-NS and IPM-NS but CZA-S isolates, indicating that overexpressed AmpC could be inhibited by avibactam. Consistently, the addition of avibactam increased ceftazidime susceptibility for 46 AmpC-hyperproducing clinical isolates from 10.9% to 76.1%  , few of which could contribute to CZA resistance  were active against the majority of P. aeruginosa isolates were collected from 11 hospitals across China from July 2018 and February 2019 (Table S4) (P. aeruginosa isolates were predominantly from respiratory tract specimens (61.7%), followed by urinary tract (12.6%) and blood specimens (4.0%).A total of 596 nonduplicate clinical able S4) . The 596P. aeruginosa  (www.eucast.org). P. aeruginosa ATCC 27853 served as a quality control strain.Antimicrobial susceptibility was tested by the agar dilution method, except that antimicrobial susceptibility to colistin was tested by the broth microdilution method. Susceptibility to all antimicrobial agents was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints , except https://github.com/ablab/spades) after trimming. The presence of resistance genes was explored using the Comprehensive Antibiotic Resistance Database (CARD) webtool (https://card.mcmaster.ca/home). Multilocus sequence typing (MLST) was ascertained according to the guideline on P. aeruginosa on the MLST website (https://pubmlst.org/organisms/pseudomonas-aeruginosa). A core genome phylogenetic tree was generated using kSNP and visualized using iTOL  was performed after extraction from a fasta file.The CAZ-NS isolates were sent for WGS performed with an Illumina NovaSeq system. The raw reads were assembled using SPAdes  were determined by qRT-PCR for clinical isolates as described previously (rpsL as the internal reference. Isolates were considered to overexpress AmpC, MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM when the transcriptional levels of ampC, mexB, mexC, mexE, and mexY were at least 10-, 3-, 10-, 10-, and 10-fold higher, respectively, than those of the PAO1 strain (The transcription levels of the gene eviously . Briefly1 strain .Data were analyzed and visualized using R software v4.1.3. Spearman rank-order correlation was used to evaluate associations between antibiotic MICs.PRJNA891673.The genomic sequence data have been submitted to the NCBI GenBank database with BioProject number


A recent review indicated that physical activity (PA), facilitated by organisations/clubs, may reduce alcohol consumption in early to mid-adolescence. Our study aims were to examine these factors, and identify how health determinants may influence association.2 tests achieved p<.10.Cross-sectional secondary data analysis using UK cohort data from ALSPAC. Ages and sample-size: Time-point (TP) 1: 13-14 (n = 1824), TP2: 15-16 (n = 1334). Variables: minutes per day spent in moderate to vigorous PA > =3 days of accelerometer wear. Ancillary PA variables: Club-type (CT); and frequency of attending club (FAC) collected age 15 only. Risk of alcohol-related harm (RARH) categorised as: no current risk, increasing risk, and at risk (AR). Ordinal regression was conducted at each TP, using PA as a covariate. Explanatory variables (EVs): Psychosocial health (PSH) - Cluster membership numbers generated through K-means cluster analysis (uniquely represented at each TP); Socioeconomic status (SES); Educational attainment; BMI; Smoking status, and Gender. EVs entered into regression model if preliminary Xp<0.002). At TP2: OR 1.24 . TP1: CT (age 15) was not statistically significant at age 13. However, ‘sports club only’ (SCO), had greatest RARH  vs. no CT. All EVs retained statistical significance at p<.05, excepting IDACI (p = .854) and gender (p = .917). TP2: Although CT was not statistically significant, SCO had the greatest RARH . RARH was reduced if attending club most evenings vs. no attendance . Statistically significant EVs were PSH  and smoking . Model fit χ2 331.010, df 11, p<0.001. Pearson .455. Deviance .218. Nagelkerke .251. -2LL test of parallel lines with significance .321.Regression showed a positive association between PA and RARH at both TPs. Odds of being AR at TP1 were 1.31 greater for each 30 minute increase in PA (95%CI 1.10-1.57; The relationship between PA and alcohol consumption is complex. While facilitated PA can provide many benefits for adolescents, potential unwanted consequences may be an increase in risk-behaviours like alcohol consumption. Further research is needed for greater comprehension of this association.


Kalamiella piersonii clinical isolate URMC-2103A041 from human bacteremia was determined using the hybrid assembly of short- and long-read sequencing chemistry. The genome contains a 3.93 Mb chromosome, three circular plasmids, and one linear plasmid.The complete genome of  Kalamiella piersonii is a Gram-negative Enterobacterales in the Erwiniaceae family. First described from the International Space Station environment, a recent phylogenomic analysis of Erwiniaceae has proposed reclassification as Pantoea piersonii comb. nov ]. Biochemical identification  produced Pantoea spp. (98.2% identification). The BD Phoenix NID panel identification was Shigella flexneri. Identification was resolved by 16S rRNA V1-V3 sequencing (MicroSeq 500 16S rDNA Sequening Kit) . Susceptibility was determined using zone diameter breakpoints for Enterobacterales   identified the organism as Pantoea spp., URMC-2103A041 was Voges-Proskauer- and ONPG-positive, fermented mannitol, arabinose, and rhamnose, but was arginine dihydrolase positive . DNA was extracted following the protocol for “Pretreatment for Gram-Negative Bacteria” . DNA shearing and size selection were not performed. DNA was quantified with the QuantiFluor dsDNA system (Promega).For short reads, paired-end sequencing following library preparation  was performed on a MiSeq instrument . Unless otherwise specified, sequences were analyzed with tools available on the Galaxy server , using default parameters . Reads w50: 11,125 bp) for hybrid assembly  and polishing (Quast v5.2.0) , SQK-RBK004]. Long-read libraries were sequenced , and base calling was performed with Guppy v6.1.5 (MinKNOW v22.05.5). Filtering  generated 93,023 reads (read N v5.2.0) , 14.CP115895.1), three circular plasmids , and one linear plasmid . Annotation was added by the NCBI Prokaryotic Genome Annotation Pipeline (The hybrid assembly genome was 4,990,348 bp with 57.02% GC content; it consisted of one circular chromosome (3.93 Mb, GenBank: Pipeline 17.


We conducted surveillance of respiratory syncytial virus (RSV) genomic sequences for 100 RSV-A and 27 RSV-B specimens collected during November 2022–April 2023 in Arizona, USA. We identified mutations within prefusion F-protein antigenic sites in both subtypes. Continued genomic surveillance will be critical to ensure RSV vaccine effectiveness. Respiratory syncytial virus (RSV), an RNA virus of the family Pneumoviridae, causes acute respiratory infections, primarily in children, adults with severe lung disease, and elderly persons , panel AThe 2 major RSV subtypes, RSV-A and RSV-B, have distinct antigenic characteristics in the P, N, F, and G proteins , panel Bhttps://www.illumina.com). We used Trim Galore version 0.6.10  to quality filter and adaptor trim sequencing reads, mapped the reads to RSV-A and RSV-B reference sequences (GISAID accession nos. EPI_ISL_412866 and EPI_ISL_165399) using Burrows-Wheeler Aligner version 0.7.17-r1188 (https://bio-bwa.sourceforge.net), and generated consensus sequences using SAMtools version 1.17 (https://github.com/samtools/samtools/releases). We assembled 92 RSV-A (GA2.3.5 genotype) and 24 RSV-B (GB5.0.5a genotype) complete genome sequences and 8 RSV-A (GA2.3.5) and 3 RSV-B (GB5.0.5a) partial genome sequences  . We performed genomic surveillance to determine RSV strains circulating in Arizona during the 2022–2023 season. We performed 2 × 150-bp paired-end next-generation sequencing using a hybrid capture enrichment panel  , panel Ahttps://clades.nextstrain.org) showed that RSV-A sequences from Arizona clustered in clade GA2.3.5, indicating >3 independent introductions of RSV-A into Arizona  is monovalent and Abrysvo , bivalent (The US Food and Drug Administration has approved 2 RSV vaccines for persons bivalent . RSV-B Sbivalent  Figure 2Although RSV remains a substantial clinical burden, approved RSV vaccines reduce the risk of lower respiratory tract illness. By tracking RSV evolution, we can improve design of vaccine formulations to improve effectiveness. Our study was limited because we lacked understanding of potential functional consequences from mutations in F-protein antigenic sites. Although the G protein is under greater selective pressure and has higher mutation rates (Additional information about a study of respiratory syncytial virus in Arizona, USA, 2022–2023


Escherichia coli strains isolated from the recto-anal junction of slaughter-age Irish sheep. In silico serotyping and genome analysis determined that each of the strains harbored a Shiga-toxin subtype, a complete locus of enterocyte effacement, and a rare O-island 122.This study describes the hybrid genome assemblies of four Shiga toxin-producing  Escherichia coli (STEC) are significant foodborne zoonotic pathogens with primary virulence determinants being the bacteriophage encoded Shiga toxins (Stx) and the intimin adhesion protein  and trimmed using Porechop (v0.2.4) for ONT or Fastp (v0.20.1) (de novo hybrid assemblies were constructed with Unicycler (v0.4.9)  with 16 mg/L of novobiocin (Oxoid) and incubated at 41.5°C for 24 h. RT-PCR was performed on DNA extracted using InstaGene matrix (Bio-Rad) as described by Macori et al.  to confiv0.20.1)  for Illu(v0.4.9)  and rang(v0.4.9) .https://github.com/phac-nml/ecoli_vf) and a customized database containing nle gene sequences (https://github.com/macgenomegue/nle_database). Protein-coding sequences, tRNAs, and insertion sequence elements were predicted using Prokka (1.14.6) (Genome annotation was conducted using the ABRIcate tool (v1.0.1) , and vir(1.14.6) , tRNA-sc(1.14.6) , and ISE(1.14.6) , respect(1.14.6)  webtool.


Staphylococcus aureus infections.As daptomycin (DAP) CL does not scale proportionally with weight, fixed-dose DAP has been proposed to optimize efficacy and mitigate toxicity. This study evaluated systemic exposure of a fixed dose (750 mg) of DAP in patients with S. aureus infection were eligible for enrollment. DAP 750 mg was infused over 30 minutes every 24 hours. At steady state, concentrations were obtained pre-dose (0.5 hours) and 1, 12, and 24 hours after the start of infusion. Concentrations were measured using a validated LC-MS assay. Pharmacokinetic (PK) analysis was performed using first-order equations and stratified by weight: low (< 65 kg), normal (65-85 kg), and high ( > 85 kg). Therapeutic AUC24 (≥ 666 mg*hr/L) and elevated trough (Cmin > 24.3 mg/L) were the efficacy and safety targets, respectively.Adult non-ICU patients with CrCl ≥ 30 mL/min admitted to our academic medical center warranting DAP for a e 0.078 h-1 , CL 0.87 L/h , AUC24 861 mg*hr/L , and Cmin 10.5 mg/L . Therapeutic AUC24 was achieved in 14/22 (64%) and elevated Cmin occurred in 7/22 (32%), with no differences seen among weight groups. However, differences were noted between males (n=10) and females (n=12) with significantly higher CL (median [IQR]) noted in males  vs. females  (p=0.027). Hence, median [IQR] AUC24 was significantly lower in males at 594 mg*hr/L  vs. 1513 mg*hr/L  in females (p=0.021). Therapeutic AUC24 was achieved in 40% of males versus 83% of females, while elevated Cmin was seen in 20% of males and 32% of females.Overall, 22 patients  were enrolled with no differences observed in baseline characteristics . PK parameters (median [IQR]) did not differ by weight with Vd 9.9 L , K24 and Cmin) was not impacted by weight. These results support fixed, rather than weight-based, dosing of DAP in non-ICU patients with normal renal function. Notably, gender differences were observed with males having significantly higher CL, likely warranting higher doses of DAP to ensure optimal exposure.DAP PK and exposure : Grant/Research Support


Streptomyces sp. strain ICN988, an actinomycete isolated from Gorgonia. The assembled genome consists of 6,122,654 bp with a GC content of 73%. A comprehensive analysis revealed 19 biosynthetic gene clusters.This study elucidates the draft genomic sequence of  Actinomycetes produce many secondary metabolites, including antibiotics, antitumor agents, and immunosuppressive compounds, and are accountable for over half of the antibiotics used clinically . Marine Streptomyces sp. strain ICN988 was isolated from a Gorgonia sea-fan by-catch collected from Colachel coastal area, Tamil Nadu, India (8°10'22.0"N 77°14'55.2"E) on 28 December 2018. Initially washed with seawater to remove surface deposits, Gorgonia’s holdfast was scraped aseptically, and the sand was suspended in 2% NaCl to prepare serial dilutions, then spread on Actinomycete Isolation Media (AIM) agar plates (supplemented with filter-sterilized 10 µg/mL nystatin and 50 µg/mL cycloheximide). Streptomyces sp. strain ICN988 (S5K) was one of the actinomycetes found after 7 days of incubation at 30°C. Genomic DNA was isolated from 5-day-old axenic ICN988 aerial mycelia using the NucleoSpin Tissue Kit (Macherey-Nagel). The 16S rRNA was PCR amplified with 63F forward primer and 1387R reverse primer. The actinomycete’s 16S rRNA gene (GenBank: OP585661) was closely related (98.34% similarity) to Streptomyces ardesiacus NBRC 15402 (GenBank: NR_112454) via NCBI-BLAST and (96.60% similarity) to Streptomyces coelicoflavus NBRC 15399 (GenBank: AB184650) via EzBioCloud database . Sequence libraries were created using Nextera XT DNA Library Preparation Kit (Illumina). The whole-genome sequencing was obtained using Illumina HiSeq 4000 System (paired-end 150 bp). A total of 18,237,528 bp reads were obtained with 2,753,866,728 total read bases. The GC content was 71.01%, and Q30 was 88.73%. The tools such as Trimmomatic (v 0.36) for adapter trimming  (The genomic DNA was extracted from a 7-day-old AIM agar plate culture using HiPurA trimming  utilized(v3.0.2)  revealed(v3.0.2)  identifiStreptomyces sp. strain ICN988 was antagonistic to methicillin-resistant Staphylococcus aureus ATCC 33591 in agar-plug and metabolic extract disc diffusion assays.Through antiSMASH v7.0.0beta1-67b538a9 , 13, 30


Correction:J Exp Clin Cancer Res 42, 169 (2023)https://doi.org/10.1186/s13046-023-02723-zFollowing publication of the original article , an erro6*, Youtao Xu1* and Rong Yin1,4,5,7*Guoren ZhouThe correction do not affect the overall Conclusion of the article. The original article has been corrected.