Influence of N_ε‑Protecting Groups on the Protease-Catalyzed Oligomerization of l‑Lysine Methyl Ester

The direct oligomerization of l-lys-OMe by bromelain catalysis gave oligo­(l-lys) with DP_avg ∼ 3.6 and dispersity ∼ 1.1. Since higher chain length oligo­(l-lys) with lower dispersity values and one reactive amine for selective conjugation would be beneficial, we explored protease-catalyzed oligomerization of N_ε-protected l-lys monomers where N_ε-groups included <i>tert</i>-butoxycarbonyl (Boc) or benzyloxycarbonyl (Z) groups. By using N_ε-protected l-lys monomers, oligopeptide side-chains are hydrophobic-neutral which should dramatically alter enzyme kinetic and binding constants relative to nonprotected l-lys. Schechter and Berger’s conceptual model guided our choice of papain as the protease catalyst. Papain-catalyzed oligomerization of N_ε-Boc-l-Lys-OMe gave products with DP_avg values that were pH dependent and varied from 4.7 ± 0.2 to 7.5 ± 0.1. Similarly, oligo­(N_ε-Z-l-Lys) synthesis was pH dependent, and DP_avg values varied from 4.3 ± 0.2 to 5 ± 0.2. Oligo­(N_ε-Boc/Z-l-Lys) that precipitates from reaction media had a low dispersity (∼1.01). The relatively smaller N_ε-Boc group should increase propagating chain solubility enabling oligopeptides to reach higher DP_avg values prior to their precipitation. Since papain-catalyzed oligomerizations of N_ε-Boc/Z-l-Lys proceeded slowly at 0.54 mg/mL, higher enzyme concentrations were studied. By increasing the enzyme concentration in oligomerizations from 0.54 to 1.62 mg/mL for 3 h reactions, the %-yield and DP_avg of oligo­(N_ε-Z-l-lys) increased from 24 ± 0 to 88 ± 2 and 4.1 ± 0.7 to 5.7 ± 0.1, respectively. Furthermore, at 1.89 mg/mL papain, the %-yield of oligo­(N_ε-Z-l-lys) increased with time reaching 91% in 2 h. Acetonitrile at 20%-by-volume was a useful cosolvent that increased the oligopeptide yield and DP_avg relative to reactions run in pure buffer

<b>Excessive free fatty acids sensing in pituitary lactotrophs elicits steatotic liver disease by decreasing prolactin levels</b>

Pituitary is the central endocrine gland with effects on metabolic dysfunction-associated steatotic liver disease (MASLD). However, it’s not clear whether the pituitary responds to free fatty acids (FFAs) toxicity, thus dysregulating hepatic lipid metabolism. Here, we demonstrated that elevated FFAs levels increased the risk of MASLD mediating by decreased prolactin (PRL) levels in a liver biospecimen-based cohort. Moreover, overloaded FFAs decreased serum PRL levels thus promoted liver steatosis both in mice with dynamic diets intervention and stereotactic pituitary FFAs injection. Mechanic studies showed that increased CD36 expression in pituitary lactotrophs inhibited the synthesis of PRL by enhancing FFAs sensing through lipid uptake assays and virus transfection analysis. Importantly, silencing of pituitary CD36 by stereotactic virus injection, as well as by a pituitary-targeted drug delivery system of TRH-PEG-LP-SSO, efficiently elevated PRL levels and alleviated liver steatosis. Targeted inhibition of pituitary FFAs sensing may be a potential therapeutic target for liver steatosis.</p

Image_1_Genomic insights and antimicrobial resistance profiles of CRKP and non-CRKP isolates in a Beijing geriatric medical center: emphasizing the blaKPC-2 carrying high-risk clones and their spread.TIF

BackgroundThe escalating resistance of Klebsiella pneumoniae, a prevalent pathogen in healthcare settings, especially its carbapenem-resistant K. pneumoniae (CRKP), to a wide array of antibiotics, notably β-lactams, constitutes a formidable challenge for healthcare and global public health management.MethodsThis research compared the resistance phenotypes and genomic profiles of CRKP and Non-CRKP isolates in a Beijing hospital, focusing on high-risk blaKPC-2 gene-bearing CRKP clones and the structure of mobile genetic elements facilitating their spread across hospital departments. Forty K. pneumoniae isolates were collected from various departments of the hospital and subjected to antimicrobial susceptibility testing and whole-genome sequencing to analyze their resistance phenotypes and genomic features.ResultsThe study revealed that among the 31 CRKP isolates, ST11 is the most common sequence type, with K47 and OL101 being the dominant capsule types, primarily observed in the respiratory department. In terms of antimicrobial susceptibility: 87.5% of the isolates exhibited multidrug resistance (MDR), with a high resistance rate of 30% against tigecycline. All CRKP isolates demonstrated resistance to multiple drug classes (≥5 CLSI classes). Non-CRKP isolates also showed high resistance rates to minocycline and doxycycline (77.8%). the ST11-KL47-OL101 type emerged as the predominant clone among the CRKP isolates carrying the blaKPC-2 gene. This dominance appears to be mediated by the pKpnR03_2 plasmid, which harbors not only blaKPC-2 and rmtb but also gene clusters pertinent to iron transport and arsenic resistance. These isolates, clustering in the C3 clade of the phylogenetic tree, exhibited minor genetic variations and close evolutionary relationships, suggesting a plasmid-driven spread across various hospital departments.ConclusionIn summary, our study highlights the extensive spread of antibiotic-resistant K. pneumoniae across various departments in our hospital, with a particular emphasis on the dominant clonal proliferation of the ST11-KL47-OL101 CRKP strain. This finding underscores the significant role of plasmid-mediated gene transfer in the evolution and dissemination of resistant strains within hospital environments. The study emphasizes the necessity for ongoing surveillance of antibiotic resistance and genomic analysis in hospital settings to effectively monitor and manage these challenges.</p

Table_1_Genomic insights and antimicrobial resistance profiles of CRKP and non-CRKP isolates in a Beijing geriatric medical center: emphasizing the blaKPC-2 carrying high-risk clones and their spread.XLSX

BackgroundThe escalating resistance of Klebsiella pneumoniae, a prevalent pathogen in healthcare settings, especially its carbapenem-resistant K. pneumoniae (CRKP), to a wide array of antibiotics, notably β-lactams, constitutes a formidable challenge for healthcare and global public health management.MethodsThis research compared the resistance phenotypes and genomic profiles of CRKP and Non-CRKP isolates in a Beijing hospital, focusing on high-risk blaKPC-2 gene-bearing CRKP clones and the structure of mobile genetic elements facilitating their spread across hospital departments. Forty K. pneumoniae isolates were collected from various departments of the hospital and subjected to antimicrobial susceptibility testing and whole-genome sequencing to analyze their resistance phenotypes and genomic features.ResultsThe study revealed that among the 31 CRKP isolates, ST11 is the most common sequence type, with K47 and OL101 being the dominant capsule types, primarily observed in the respiratory department. In terms of antimicrobial susceptibility: 87.5% of the isolates exhibited multidrug resistance (MDR), with a high resistance rate of 30% against tigecycline. All CRKP isolates demonstrated resistance to multiple drug classes (≥5 CLSI classes). Non-CRKP isolates also showed high resistance rates to minocycline and doxycycline (77.8%). the ST11-KL47-OL101 type emerged as the predominant clone among the CRKP isolates carrying the blaKPC-2 gene. This dominance appears to be mediated by the pKpnR03_2 plasmid, which harbors not only blaKPC-2 and rmtb but also gene clusters pertinent to iron transport and arsenic resistance. These isolates, clustering in the C3 clade of the phylogenetic tree, exhibited minor genetic variations and close evolutionary relationships, suggesting a plasmid-driven spread across various hospital departments.ConclusionIn summary, our study highlights the extensive spread of antibiotic-resistant K. pneumoniae across various departments in our hospital, with a particular emphasis on the dominant clonal proliferation of the ST11-KL47-OL101 CRKP strain. This finding underscores the significant role of plasmid-mediated gene transfer in the evolution and dissemination of resistant strains within hospital environments. The study emphasizes the necessity for ongoing surveillance of antibiotic resistance and genomic analysis in hospital settings to effectively monitor and manage these challenges.</p

Generation and characterization of a bivalent protein boost for future clinical trials: HIV-1 subtypes CR01_AE and B gp120 antigens with a potent adjuvant

<div><p>The RV144 Phase III clinical trial with ALVAC-HIV prime and AIDSVAX B/E subtypes CRF01_AE (A244) and B (MN) gp120 boost vaccine regime in Thailand provided a foundation for the future development of improved vaccine strategies that may afford protection against the human immunodeficiency virus type 1 (HIV-1). Results from this trial showed that immune responses directed against specific regions V1V2 of the viral envelope (Env) glycoprotein gp120 of HIV-1, were inversely correlated to the risk of HIV-1 infection. Due to the low production of gp120 proteins in CHO cells (2–20 mg/L), cleavage sites in V1V2 loops (A244) and V3 loop (MN) causing heterogeneous antigen products, it was an urgent need to generate CHO cells harboring A244 gp120 with high production yields and an additional, homogenous and uncleaved subtype B gp120 protein to replace MN used in RV144 for the future clinical trials. Here we describe the generation of Chinese Hamster Ovary (CHO) cell lines stably expressing vaccine HIV-1 Env antigens for these purposes: one expressing an HIV-1 subtype CRF01_AE A244 Env gp120 protein (A244.AE) and one expressing an HIV-1 subtype B 6240 Env gp120 protein (6240.B) suitable for possible future manufacturing of Phase I clinical trial materials with cell culture expression levels of over 100 mg/L. The antigenic profiles of the molecules were elucidated by comprehensive approaches including analysis with a panel of well-characterized monoclonal antibodies recognizing critical epitopes using Biacore and ELISA, and glycosylation analysis by mass spectrometry, which confirmed previously identified glycosylation sites and revealed unknown sites of O-linked and N-linked glycosylations at non-consensus motifs. Overall, the vaccines given with MF59 adjuvant induced higher and more rapid antibody (Ab) responses as well as higher Ab avidity than groups given with aluminum hydroxide. Also, bivalent proteins (A244.AE and 6240.B) formulated with MF59 elicited distinct V2-specific Abs to the epitope previously shown to correlate with decreased risk of HIV-1 infection in the RV144 trial. All together, these results provide critical information allowing the consideration of these candidate gp120 proteins for future clinical evaluations in combination with a potent adjuvant.</p></div

Antigenicity analyses of antigens and mAbs targeted to CD4bs, V1V2, V2 and V3 regions.

<p>Antigenicity analyses of antigens and mAbs targeted to CD4bs, V1V2, V2 and V3 regions.</p

gp70-V1V2 protein and cyclic V2- and V3-peptide-specific Ab responses in guinea pigs.

<p><b>(A-C)</b> Ab responses to gp70-V1V2 AE.92TH023 and gp70-V1V2 B.Case A2 proteins were evaluated by ELISA. Ab responses for the cyclic V2 (<b>D-F</b>) and V3 peptides (<b>G-I</b>) were measured by SPR. In each panel, comparisons of epitope-specific Ab responses elicited in animals immunized with vaccines formulated with MF59 or AH are shown: <b>(A, D, G)</b> monovalent 6240.B (Grp 1 vs. Grp4); <b>(B, E, H)</b> A244.AE (Grp2 vs. Grp5) and <b>(C, F, I)</b> bivalent (Grp3 vs. Grp6). <b>(A-C)</b> Ab responses to gp70-V1V2 by ELISA; (<b>D-F</b>) Ab responses to cyclic V2 peptides by SPR; (<b>G-I</b>) Ab responses to cyclic V3 peptides by SPR. Each symbol for the ELISA represents the mean end-point titer for an individual animal analyzed in triplicate. The SPR assays were conducted with 4 replicates and are represented as the response units. The values shown in each panel represent the geometric mean response units for each group. Differences between groups (MF59 vs. AH) were compared using Wilcoxon rank-sum test with confidence level of p < 0.05. The p values are provided above groups that showed statistically significant differences.</p

Generation and characterization of a bivalent protein boost for future clinical trials: HIV-1 subtypes CR01_AE and B gp120 antigens with a potent adjuvant - Fig 4

<p>gp120-specific Ab responses in guinea pigs following immunization with A244.AE and 6240.B gp120s. Six groups of ten guinea pigs each were immunized with monovalent gp120 vaccine formulations [Grp1 (6240.B + MF59), Grp4 (6240.B + AH), Grp2 (A244.AE + MF59), Grp5 (A244.AE + AH)], or bivalent gp120 vaccines {Grp3 (A244.AE + 6240.B + MF59), Grp6 (A244.AE + 6240.B + AH)} as described in Methods. Sera were conducted at four weeks following each immunization (4wp1, 4wp2, 4wp3 and 4wp4). (<b>A—C</b>) A244.AE gp120 protein was coated onto the 96-well plates. (<b>D—F</b>) 6240.B gp120 protein was coated onto the 96-well plates. Each symbol represents the end-point-titer for an individual animal with geometric mean titers indicated for each animal within a group. Differences between groups (MF59 vs. AH) were compared using Wilcoxon rank-sum test with confidence level of p < 0.05.</p

N-linked and O-linked glycosylation of the A244.AE and 6240.B gp120 proteins.

<p>N-linked and O-linked glycosylation sites of the A244.AE gp120 (upper panel) and 6240.B gp120 (lower panel) were identified by LC-MS/MS analysis after enzymatic deglycosylations (PNGase F, Endo H or Endo F3) and proteolytic digestions (trypsin or pepsin) of the proteins. The identified N-linked glycosylation sites are represented by the following: circles correspond to high mannose/hybrid glycans; triangles, to complex glycans; squares, to complex high mannose/hybrid glycans; double underscore, undetermined glycan type (asparagine residues identified as aspartic acid residues after treatment with PNGase F). Non-glycosylated asparagine residues in a consensus motif are reported with a star. Glycosylated asparagine residues in a non-consensus motif (N165 of A244.AE) is indicated with an asterisk. The “T” in the figure indicates the O-linked glycosylations identified for both proteins. The figure also reports the variable loops (V1-V5), conserved sequences (C1-C5) and the glycan epitopes mapped by PG9/16, 2G12 and PGT128 mAbs.</p

Evaluation of the avidities (kd off-rate) of Abs-specific to A244.AE and 6240.B gp120s by SPR.

<p>Either A244.AE (A, B, C) or 6240.B (D, E, F) gp120 protein was immobilized on CM5 chip and the diluted sera were injected onto the immobilized CM5 chip followed by extended dissociation– 30 min. The SPR assay was conducted using different immobilized densities with more than 3 replicates and arithmetic mean was used for each group. The dissociation fitting was conducted using 1:1 binding model. Avidity for A244.AE gp120 for (<b>A</b>) monovalent Grp1 and Grp4. (<b>B</b>) monovalent Grp2 and Grp5. (<b>C</b>) bivalent Grp3 and Grp6 and Avidity for to 6240.B gp120 for (<b>D</b>) monovalent Grp1 and Grp4. (<b>E</b>) monovalent Grp2 and Grp5. (<b>F</b>) bivalent Grp3 and Grp6. Differences between groups (MF59 vs. AH) were compared using Wilcoxon rank-sum test with confidence level of p < 0.05. The p-values indicated show comparisons of 4wp1 vs. 4wp2, 4wp1 vs. 4wp3, 4wp1 vs. 4wp4 within the same group. Multiple comparisons were shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194266#pone.0194266.s012" target="_blank">S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194266#pone.0194266.s013" target="_blank">S5</a> Tables.</p