Identification of a novel CG307 sub-clade in third-generation-cephalosporin-resistant Klebsiella pneumoniae causing invasive infections in the USA.

Despite the notable clinical impact, recent molecular epidemiology regarding third-generation-cephalosporin-resistant (3GC-R) Klebsiella pneumoniae in the USA remains limited. We performed whole-genome sequencing of 3GC-R K. pneumoniae bacteraemia isolates collected from March 2016 to May 2022 at a tertiary care cancer centre in Houston, TX, USA, using Illumina and Oxford Nanopore Technologies platforms. A comprehensive comparative genomic analysis was performed to dissect population structure, transmission dynamics and pan-genomic signatures of our 3GC-R K. pneumoniae population. Of the 178 3GC-R K. pneumoniae bacteraemias that occurred during our study time frame, we were able to analyse 153 (86 %) bacteraemia isolates, 126 initial and 27 recurrent isolates. While isolates belonging to the widely prevalent clonal group (CG) 258 were rarely observed, the predominant CG, 307, accounted for 37 (29 %) index isolates and displayed a significant correlation (Pearson correlation test P value=0.03) with the annual frequency of 3GC-R K. pneumoniae bacteraemia. Interestingly, only 11 % (4/37) of CG307 isolates belonged to the commonly detected 'Texas-specific' clade that has been observed in previous Texas-based K. pneumoniae antimicrobial-resistance surveillance studies. We identified nearly half of our CG307 isolates (n=18) belonged to a novel, monophyletic CG307 sub-clade characterized by the chromosomally encoded bla SHV-205 and unique accessory genome content. This CG307 sub-clade was detected in various regions of the USA, with genome sequences from 24 additional strains becoming recently available in the National Center for Biotechnology Information (NCBI) SRA database. Collectively, this study underscores the emergence and dissemination of a distinct CG307 sub-clade that is a prevalent cause of 3GC-R K. pneumoniae bacteraemia among cancer patients seen in Houston, TX, and has recently been isolated throughout the USA.

Genetic determinants underlying the progressive phenotype of ß-lactam/ß-lactamase inhibitor resistance in Escherichia coli.

IMPORTANCE: The increased feasibility of whole-genome sequencing has generated significant interest in using such molecular diagnostic approaches to characterize difficult-to-treat, antimicrobial-resistant (AMR) infections. Nevertheless, there are current limitations in the accurate prediction of AMR phenotypes based on existing AMR gene database approaches, which primarily correlate a phenotype with the presence/absence of a single AMR gene. Our study utilized a large cohort of cephalosporin-susceptible Escherichia coli bacteremia samples to determine how increasing the dosage of narrow-spectrum ß-lactamase-encoding genes in conjunction with other diverse ß-lactam/ß-lactamase inhibitor (BL/BLI) genetic determinants contributes to progressively more severe BL/BLI phenotypes. We were able to characterize the complexity of the genetic mechanisms underlying progressive BL/BLI resistance including the critical role of ß-lactamase encoding gene amplification. For the diverse array of AMR phenotypes with complex mechanisms involving multiple genomic factors, our study provides an example of how composite risk scores may improve understanding of AMR genotype/phenotype correlations.

Temporal dynamics of genetically heterogeneous extended-spectrum cephalosporin-resistant Escherichia coli bloodstream infections.

Extended-spectrum cephalosporin-resistant Escherichia coli (ESC-R-Ec) is an urgent public health threat with sequence type clonal complex 131 (STc131), phylogroup B2 strains being particularly concerning as the dominant cause of ESC-R-Ec infections. To address the paucity of recent ESC-R-Ec molecular epidemiology data in the United States, we used whole-genome sequencing (WGS) to fully characterize a large cohort of invasive ESC-R-Ec at a tertiary care cancer center in Houston, Texas, collected from 2016 to 2020. During the study time frame, there were 1,154 index E. coli bloodstream infections (BSIs) of which 389 (33.7%) were ESC-R-Ec. Using time series analyses, we identified a temporal dynamic of ESC-R-Ec distinct from ESC-susceptible E. coli (ESC-S-Ec), with cases peaking in the last 6 months of the calendar year. WGS of 297 ESC-R-Ec strains revealed that while STc131 strains accounted for ~45% of total BSIs, the proportion of STc131 strains remained stable across the study time frame with infection peaks driven by genetically heterogeneous ESC-R-Ec clonal complexes. bla CTX-M variants accounted for most ß-lactamases conferring the ESC-R phenotype (89%; 220/248 index ESC-R-Ec), and amplification of bla CTX-M genes was widely detected in ESC-R-Ec strains, particularly in carbapenem non-susceptible, recurrent BSI strains. Bla CTX-M-55 was significantly enriched within phylogroup A strains, and we identified bla CTX-M-55 plasmid-to-chromosome transmission occurring across non-B2 strains. Our data provide important information regarding the current molecular epidemiology of invasive ESC-R-Ec infections at a large tertiary care cancer center and provide novel insights into the genetic basis of observed temporal variability for these clinically important pathogens. IMPORTANCE Given that E. coli is the leading cause of worldwide ESC-R Enterobacterales infections, we sought to assess the current molecular epidemiology of ESC-R-Ec using a WGS analysis of many BSIs over a 5-year period. We identified fluctuating temporal dynamics of ESC-R-Ec infections, which have also recently been identified in other geographical regions such as Israel. Our WGS data allowed us to visualize the stable nature of STc131 over the study period and demonstrate a limited but genetically diverse group of ESC-R-Ec clonal complexes are detected during infection peaks. Additionally, we provide a widespread assessment of ß-lactamase gene copy number in ESC-R-Ec infections and delineate mechanisms by which such amplifications are achieved in a diverse array of ESC-R-Ec strains. These data suggest that serious ESC-R-Ec infections are driven by a diverse array of strains in our cohort and impacted by environmental factors suggesting that community-based monitoring could inform novel preventative measures.

Identification of distinct impacts of CovS inactivation on the transcriptome of acapsular group A streptococci.

Group A streptococcal (GAS) strains causing severe, invasive infections often have mutations in the control of virulence two-component regulatory system (CovRS) which represses capsule production, and high-level capsule production is considered critical to the GAS hypervirulent phenotype. Additionally, based on studies in emm1 GAS, hyperencapsulation is thought to limit transmission of CovRS-mutated strains by reducing GAS adherence to mucosal surfaces. It has recently been identified that about 30% of invasive GAS strains lacks capsule, but there are limited data regarding the impact of CovS inactivation in such acapsular strains. Using publicly available complete genomes (n = 2,455) of invasive GAS strains, we identified similar rates of CovRS inactivation and limited evidence for transmission of CovRS-mutated isolates for both encapsulated and acapsular emm types. Relative to encapsulated GAS, CovS transcriptomes of the prevalent acapsular emm types emm28, emm87, and emm89 revealed unique impacts such as increased transcript levels of genes in the emm/mga region along with decreased transcript levels of pilus operon-encoding genes and the streptokinase-encoding gene ska. CovS inactivation in emm87 and emm89 strains, but not emm28, increased GAS survival in human blood. Moreover, CovS inactivation in acapsular GAS reduced adherence to host epithelial cells. These data suggest that the hypervirulence induced by CovS inactivation in acapsular GAS follows distinct pathways from the better studied encapsulated strains and that factors other than hyperencapsulation may account for the lack of transmission of CovRS-mutated strains. IMPORTANCE Devastating infections due to group A streptococci (GAS) tend to occur sporadically and are often caused by strains that contain mutations in the control of virulence regulatory system (CovRS). In well-studied emm1 GAS, the increased production of capsule induced by CovRS mutation is considered key to both hypervirulence and limited transmissibility by interfering with proteins that mediate attachment to eukaryotic cells. Herein, we show that the rates of covRS mutations and genetic clustering of CovRS-mutated isolates are independent of capsule status. Moreover, we found that CovS inactivation in multiple acapsular GAS emm types results in dramatically altered transcript levels of a diverse array of cell-surface protein-encoding genes and a unique transcriptome relative to encapsulated GAS. These data provide new insights into how a major human pathogen achieves hypervirulence and indicate that factors other than hyperencapsulation likely account for the sporadic nature of the severe GAS disease.

Facile Solid-Phase Synthesis of Well-Defined Defect Lysine Dendrimers.

An efficient solid-phase method has been reported to prepare well-defined lysine defect dendrimers. Using orthogonally protected lysine residues, pure G2 to G4 lysine defect dendrimers were prepared with 48-95% yields within 13 h. Remarkably, high-purity products were collected via precipitation without further purification steps. This method was applied to prepare a pair of 4-carboxyphenylboronic acid-decorated defect dendrimers (16 and 17), which possessed the same number of boronic acids. The binding affinity of 16, in which the ε-amines of G1 lysine are fractured, for glucose and sorbitol was 4 times that of 17. This investigation indicated the role of allocation and distribution of peripheries for the dendrimer's properties and activity.

Combined inhibition of FGFR4 and VEGFR signaling enhances efficacy in FGF19 driven hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive liver malignancy that is difficult to treat with no approved biomarker based targeted therapies. FGF19-FGFR4 signaling blockade has been recently identified as a promising avenue for treatment of a subset of HCC patients. Using HCC relevant xenograft and PDX models, we show that Lenvatinib, an approved multi-kinase inhibitor, strongly enhanced the efficacy of FGFR4 inhibitor H3B-6527. This enhanced combination effect is not due to enhanced FGFR4 inhibition and it is likely due to cell non-autonomous VEGFR activity of Lenvatinib. This cell non-autonomous mode of action was further supported by strong in vivo combination efficacy with the mouse specific VEGFR2 antibody, DC101, which cannot cell-autonomously inhibit pathways in human xenografts. Mechanistic studies showed that the combination resulted in enhanced efficacy through increased anti-angiogenic and anti-tumorigenic activities. Overall, our results indicate that this combination can be a highly effective treatment option for FGF19 driven HCC patients, and provide preclinical validation of a combination that can be readily tested in the clinical setting.

In Vitro Comparison of Loss of Torque between Gold and Titanium Alloy Abutment Screws in Dental Implants without Any Cyclic Loads.

AIM: To find and compare the amount of screw loosening in gold and titanium alloy abutment screws without any cyclic load, this study was performed. MATERIALS AND METHODS: A total of 20 implant fixture screw samples with 10 gold abutment screws from Osstem and 10 titanium alloy abutment screws from Genesis. Implant fixtures were placed into the acrylic resin using a surveyor to maintain the same path of insertion. Using a hex driver and calibrated torque wrench, initial torque was given according to the manufacturer's recommendation. One vertical and other horizontal lines were drawn over the head of the hex driver and resin block. Acrylic block position was standardized using a putty index in a fixed table and using a tripod stand a digital single-lens reflex camera (DSLR) camera was positioned with its horizontal arm facing the floor, as well as perpendicular to the acrylic box. Photographs were taken immediately after the application of the initial torque given as per the manufacturer's recommendation and 10 minutes after the initial torque. Re-torque of 30 and 35 N cm was given to gold and titanium alloy abutment screws respectively. Photographs were taken again in that same position immediately after re-torquing and 3 hours after re-torquing. The photographs were uploaded into the Fiji-win64 analysis software and the angulations were measured in each photograph. RESULTS: Both the gold and titanium alloy abutment screws exhibited screw loosening after initial torquing. There was a significant difference in the amount of screw loosening between gold and titanium alloy abutment screws after initial torquing and no change in the abutment screw position after three hours of re-torquing. CONCLUSION: Re-torquing of both gold and titanium alloy abutment screws after 10 minutes of initial torquing should be performed routinely for retaining the preload and minimizing the screw loosening even before loading the implant fixture. CLINICAL SIGNIFICANCE: Gold abutment screws may have the ability to retain the preload better than the titanium alloy abutment screws after initial torquing, re-torquing may be necessary after 10 minutes to reduce the settling effect in a routine clinical procedure.

Simple and Rapid Synthesis of Branched Peptides through Microwave-Assisted On-Bead Ligation.

A rapid on-bead convergent method for preparing branched peptides was reported. Linear peptides were prepared on Dbz resin and ligated various branched cores, including lysine dendrons and other dendritic compounds. Alongside microwave irradiation, <1.5 equiv of peptides is sufficient to afford 50-65% yields of pure branched peptides without chromatographic purification. Remarkably, the desired compounds were prepared within hours.

Discovery of Aminopyrazole Derivatives as Potent Inhibitors of Wild-Type and Gatekeeper Mutant FGFR2 and 3.

Fibroblast growth factor receptors (FGFR) 2 and 3 have been established as drivers of numerous types of cancer with multiple drugs approved or entering late stage clinical trials. A limitation of current inhibitors is vulnerability to gatekeeper resistance mutations. Using a combination of targeted high-throughput screening and structure-based drug design, we have developed a series of aminopyrazole based FGFR inhibitors that covalently target a cysteine residue on the P-loop of the kinase. The inhibitors show excellent activity against the wild-type and gatekeeper mutant versions of the enzymes. Further optimization using SAR analysis and structure-based drug design led to analogues with improved potency and drug metabolism and pharmacokinetics properties.

Small Molecule Inhibition of CPS1 Activity through an Allosteric Pocket.

Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.

The R882H DNMT3A hot spot mutation stabilizes the formation of large DNMT3A oligomers with low DNA methyltransferase activity.

DNMT3A (DNA methyltransferase 3A) is a de novo DNA methyltransferase responsible for establishing CpG methylation patterns within the genome. DNMT3A activity is essential for normal development, and its dysfunction has been linked to developmental disorders and cancer. DNMT3A is frequently mutated in myeloid malignancies with the majority of mutations occurring at Arg-882, where R882H mutations are most frequent. The R882H mutation causes a reduction in DNA methyltransferase activity and hypomethylation at differentially-methylated regions within the genome, ultimately preventing hematopoietic stem cell differentiation and leading to leukemogenesis. Although the means by which the R882H DNMT3A mutation reduces enzymatic activity has been the subject of several studies, the precise mechanism by which this occurs has been elusive. Herein, we demonstrate that in the context of the full-length DNMT3A protein, the R882H mutation stabilizes the formation of large oligomeric DNMT3A species to reduce the overall DNA methyltransferase activity of the mutant protein as well as the WT-R882H complex in a dominant-negative manner. This shift in the DNMT3A oligomeric equilibrium and the resulting reduced enzymatic activity can be partially rescued in the presence of oligomer-disrupting DNMT3L, as well as DNMT3A point mutations along the oligomer-forming interface of the catalytic domain. In addition to modulating the oligomeric state of DNMT3A, the R882H mutation also leads to a DNA-binding defect, which may further reduce enzymatic activity. These findings provide a mechanistic explanation for the observed loss of DNMT3A activity associated with the R882H hot spot mutation in cancer.

Author Correction: Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Expedient on-resin modification of a peptide C-terminus through a benzotriazole linker.

A convenient and efficient chemical toolbox was developed for the on-resin C-terminal functionalization of various peptides. By transforming resin-bound 3,4-diaminobenzoic acid species with isoamyl nitrite, the resulting resin-bound benzotriazole entity can be efficiently displaced by nucleophiles during cleavage of the peptide-resin connection in a short reaction time. The resin cleavage step allowed for the use of various nucleophiles including water, EtOH, amines, thiol, and G5 poly(amidoamino) dendrimers with yields ranging from 66% to 82% within 5 h. This method was successfully applied to prepare the elastin sequence (VPGVG)4 through on-resin ligation in 77% yield in one day and a head-to-tail cyclic peptide, sunflower trypsin inhibitor-1, in 42% yield.

H3B-6527 Is a Potent and Selective Inhibitor of FGFR4 in FGF19-Driven Hepatocellular Carcinoma.

Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1-3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19. Cancer Res; 77(24); 6999-7013. ©2017 AACR.

Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer.

Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. Although immunotherapies are approved for MIBC, the majority of patients fail to respond, suggesting existence of complementary immune evasion mechanisms. Here, we report that the PPARγ/RXRα pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC. Recurrent mutations in RXRα at serine 427 (S427F/Y), through conformational activation of the PPARγ/RXRα heterodimer, and focal amplification/overexpression of PPARγ converge to modulate PPARγ/RXRα-dependent transcription programs. Immune cell-infiltration is controlled by activated PPARγ/RXRα that inhibits expression/secretion of inflammatory cytokines. Clinical data sets and an in vivo tumor model indicate that PPARγHigh/RXRαS427F/Y impairs CD8+ T-cell infiltration and confers partial resistance to immunotherapies. Knockdown of PPARγ or RXRα and pharmacological inhibition of PPARγ significantly increase cytokine expression suggesting therapeutic approaches to reviving immunosurveillance and sensitivity to immunotherapies. Our study reveals a class of tumor cell-intrinsic "immuno-oncogenes" that modulate the immune microenvironment of cancer.Muscle-invasive bladder cancer (MIBC) is a potentially lethal disease. Here the authors characterize diverse genetic alterations in MIBC that convergently lead to constitutive activation of PPARgamma/RXRalpha and result in immunosurveillance escape by inhibiting CD8+ T-cell recruitment.

Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3' Splice Site Selection through Use of a Different Branch Point.

Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.

Evaluation of the influence of blood glucose level on oral candidal colonization in complete denture wearers with Type-II Diabetes Mellitus: An in vivo Study.

BACKGROUND: Candidal colonization in complete denture wearers is a commonly encountered condition that worsens in the presence of untreated Diabetes Mellitus. The aim of this study was to evaluate the correlation between oral candidiasis in denture-bearing mucosa and elevated blood glucose levels in complete denture wearers and to evaluate the effect of oral hypoglycemic drug therapy in controlling oral candidal colonization in denture-bearing mucosa of complete denture wearers with Type II Diabetes Mellitus. MATERIALS AND METHODS: This prospective observational study involved the participation of 15 complete denture wearers with Type II Diabetes Mellitus. The sample collection was made prior and after oral hypoglycaemic drug intervention, by swabbing the rugal surfaces of palatal mucosa, cultured and the density of the candidal colony formed was analyzed and interpreted as colony forming units (CFU) per mL. The candidal samples CFU and corresponding pre- and post-prandial blood glucose levels were estimated, analyzed and compared using Karl Pearson correlation analysis and paired t-test (α = 0.05). RESULTS: The Karl Pearson correlation analysis showed that there was a positive correlation between the blood glucose levels (PPS and FBS) and the candidal colonization (CFU) (P < 0.05). The mean values of all the variables were analyzed using the paired t-test. There was significant reduction in the mean values of blood glucose levels (P < 0.001) and the mean values of the CFU (P < 0.001) following oral hypoglycemic drug therapy. CONCLUSION: Positive correlation was observed between oral candidiasis in complete denture-bearing mucosa and elevated blood glucose levels and oral hypoglycemic drug therapy has a positive effect in controlling oral candidal colonization in complete denture wearers with Type II Diabetes Mellitus.

Sublingual drug dispensing prosthesis.

Sublingual mode of drug administration is one of the fastest and predictable methods of drug delivery to the patient. Some patients, due to the pathological or psychological reasons, experience considerable difficulty in retaining the sub-lingual tablets. Therefore, often they either swallow or chew the sublingual tablets, thus reducing the efficacy of the medicines. Sublingual drug dispensing prosthesis is a special type of appliance that helps such patients to house the sublingual tablets in special slots in an intra-oral appliance, and thus enhances proposed drug delivery and improves the patient's health.

Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner.

Dysfunctional mTORC1 signaling is associated with a number of human pathologies owing to its central role in controlling cell growth, proliferation, and metabolism. Regulation of mTORC1 is achieved by the integration of multiple inputs, including those of mitogens, nutrients, and energy. It is thought that agents that increase the cellular AMP/ATP ratio, such as the antidiabetic biguanides metformin and phenformin, inhibit mTORC1 through AMPK activation of TSC1/2-dependent or -independent mechanisms. Unexpectedly, we found that biguanides inhibit mTORC1 signaling, not only in the absence of TSC1/2 but also in the absence of AMPK. Consistent with these observations, in two distinct preclinical models of cancer and diabetes, metformin acts to suppress mTORC1 signaling in an AMPK-independent manner. We found that the ability of biguanides to inhibit mTORC1 activation and signaling is, instead, dependent on the Rag GTPases.