APOE4/4 is linked to damaging lipid droplets in Alzheimer's disease microglia.

Several genetic risk factors for Alzheimer's disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimer's disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer's disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer's disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aß induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer's disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimer's disease.

Variants in autophagy genes MTMR12 and FAM134A are putative modifiers of the hepatic phenotype in α1-antitrypsin deficiency.

BACKGROUND AND AIMS: In the classical form of α1-antitrypsin deficiency, a misfolded variant α1-antitrypsin Z accumulates in the endoplasmic reticulum of liver cells and causes liver cell injury by gain-of-function proteotoxicity in a sub-group of affected homozygotes but relatively little is known about putative modifiers. Here, we carried out genomic sequencing in a uniquely affected family with an index case of liver failure and 2 homozygous siblings with minimal or no liver disease. Their sequences were compared to sequences in well-characterized cohorts of homozygotes with or without liver disease, and then candidate sequence variants were tested for changes in the kinetics of α1-antitrypsin variant Z degradation in iPS-derived hepatocyte-like cells derived from the affected siblings themselves. APPROACH AND RESULTS: Specific variants in autophagy genes MTMR12 and FAM134A could each accelerate the degradation of α1-antitrypsin variant Z in cells from the index patient, but both MTMR12 and FAM134A variants were needed to slow the degradation of α1-antitrypsin variant Z in cells from a protected sib, indicating that inheritance of both variants is needed to mediate the pathogenic effects of hepatic proteotoxicity at the cellular level. Analysis of homozygote cohorts showed that multiple patient-specific variants in proteostasis genes are likely to explain liver disease susceptibility at the population level. CONCLUSIONS: These results validate the concept that genetic variation in autophagy function can determine susceptibility to liver disease in α1-antitrypsin deficiency and provide evidence that polygenic mechanisms and multiple patient-specific variants are likely needed for proteotoxic pathology.

Switchable assembly and function of antibody complexes in vivo using a small molecule.

The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a number of synthetic formats, such as antibody-drug conjugates, bispecific antibodies, or Fc-fusion proteins to generate novel biologic drug modalities. Historically, these new therapies have been generated by covalently linking multiple molecular moieties through chemical or genetic methods. This irreversible fusion of different components means that the function of the molecule is static, as determined by the structure. Here, we report the development of a technology for switchable assembly of functional antibody complexes using chemically induced dimerization domains. This approach enables control of the antibody's intended function in vivo by modulating the dose of a small molecule. We demonstrate this switchable assembly across three therapeutically relevant functionalities in vivo, including localization of a radionuclide-conjugated antibody to an antigen-positive tumor, extension of a cytokine's half-life, and activation of bispecific, T cell-engaging antibodies.

SARS-CoV-2 nucleocapsid protein, rather than spike protein, triggers a cytokine storm originating from lung epithelial cells in patients with COVID-19.

PURPOSE: The aim of this study was to elucidate the factors associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that may initiate cytokine cascades and correlate the clinical characteristics of patients with coronavirus disease 2019 (COVID-19) with their serum cytokine profiles. METHODS: Recombinant baculoviruses displaying SARS-CoV-2 spike or nucleocapsid protein were constructed and transfected into A549 cells and THP-1-derived macrophages, to determine which protein initiate cytokine release. SARS-CoV-2-specific antibody titers and cytokine profiles of patients with COVID-19 were determined, and the results were associated with their clinical characteristics, such as development of pneumonia or length of hospital stay. RESULTS: The SARS-CoV-2 nucleocapsid protein, rather than the spike protein, triggers lung epithelial A549 cells to express IP-10, RANTES, IL-16, MIP-1α, basic FGF, eotaxin, IL-15, PDGF-BB, TRAIL, VEGF-A, and IL-5. Additionally, serum CTACK, basic FGF, GRO-α, IL-1α, IL-1RA, IL-2Rα, IL-9, IL-15, IL-16, IL-18, IP-10, M-CSF, MIF, MIG, RANTES, SCGF-ß, SDF-1α, TNF-α, TNF-ß, VEGF, PDGF-BB, TRAIL, ß-NGF, eotaxin, GM-CSF, IFN-α2, INF-γ, and MCP-1 levels were considerably increased in patients with COVID-19. Among them, patients with pneumonia had higher serum IP-10 and M-CSF levels than patients without. Patients requiring less than 3 weeks to show negative COVID-19 tests after contracting COVID-19 had higher serum IP-10 levels than the remaining patients. CONCLUSION: Our study revealed that nucleocapsid protein, lung epithelial cells, and IP-10 may be potential targets for the development of new strategies to prevent, or control, severe COVID-19.

Association of diverticulitis and potential risk of ischemic stroke: population-based matched cohort study in Taiwan.

BACKGROUND: Previous studies have suggested relationship between diverticular disease and cardiovascular disease. Since cardiovascular disease and cerebrovascular accident share a lot of pathogenesis, diverticulitis could also be a risk factor for stroke. This study tried to establish epidemiological evidence of the relationship between colon diverticulitis and ischemic stroke. METHODS: In this retrospective cohort study, patients with newly diagnosed colon diverticulitis (N = 6238) and patients without colon diverticulitis (control group; N = 24 952) were recruited between January 1, 2000, and December 31, 2017. Both groups were matched by propensity score at a 1:4 ratio by age, sex, comorbidities and medications. Cox proportional hazard regression was applied to estimate the hazard ratio (HR) and 95% confidence interval (CI) of ischemic stroke. We also conducted 4 different regression models and 2 sensitivity analyses to test the robustness of our findings. RESULTS: The diverticulitis group had a higher risk of IS than the control group (adjusted HR, 1.25; 95% CI, 1.12-1.39; P < 0.001). Serial sensitivity analyses yielded consistent positive link between diverticulitis and IS. Further subgroup analysis showed that in the study group, the risk of IS was 2.54-fold higher than the matched controls in 30-39 years. CONCLUSIONS: Our study found that colon diverticulitis was associated with a higher risk of developing subsequent ischemic stroke, especially for patients aged 30-39 years, among Asian population. This result provides us a chance to undertake preventive measures for ischemic stroke in high-risk patients.

Polystyrene microplastic-induced extracellular vesicles cause kidney-related effects in the crosstalk between tubular cells and fibroblasts.

Plastic waste accumulation and its degradation into microplastics (MPs) and nanoplastics (NPs) pose environmental concerns. Previous studies have indicated that polystyrene (PS)-MPs harm living animals. Extracellular vesicles (EVs) are associated with metabolic reprogramming and mitochondrial dysfunction in various kidney diseases. In this article, we evaluated how PS-MPs affected tubular cells and fibroblasts. The results demonstrated that PS-MPs increased EV production in human tubular cells and caused endoplasmic reticulum (ER) stress-related proteins without inducing inflammation-related proteins in human tubular cells. The uptake of PS-MPs and incubation with the conditioned medium of PS-MPs induced reactive oxygen species (ROS) production and ER stress-related proteins in fibroblast cells. The fibroblast cells treated with the conditioned medium of PS-MPs also increased the expression of fibrosis-related proteins. Our findings suggested that the expression of EV-related markers increased in tubular cells via Beclin 1 after PS-MP treatment. In addition, PS-MPs induced ROS production in vitro and in vivo. We found that PS-MPs also altered the expression of EV markers in urine, and CD63 expression was also increased in vitro and in vivo after PS-MP treatment. In conclusion, PS-MP-induced EVs lead to ER stress-related proteins, ROS production and fibrosis-related proteins in tubular cells and fibroblasts.

Learning through connections: clinical participation and interpersonal relationships in longitudinal integrated clerkships and traditional block rotations in Taiwan.

BACKGROUND: Longitudinal integrated clerkships (LICs) and traditional block rotations (TBRs) employ different designs that provide various learning experiences for students. In this study, we explored students' clinical participation and interpersonal interactions in LICs and TBRs at 2 metropolitan hospitals in Taiwan. METHODS: In April 2018, we enrolled 15 LIC and 29 TBR students. We conducted a cross-sectional survey which required the students to outline a typical daily schedule during their internal medicine rotations and draw an ecomap of the clinical team members. With the patient in the center as a reference, the size of each circle in an ecomap indicated the importance of the member; the distances and number of connecting lines between two circles represented the relationship and frequency of interaction, respectively, between the corresponding members. We analyzed the results and compared the responses of the LIC and TBR students. RESULTS: The LIC students spent more time on direct patient care and in the outpatient clinic/operation room, whereas the TBR students participated more in educational activities and in observation behind their seniors. In the ecomap analysis, the LIC students had a closer relationship with attending physicians and had better interactions with patients and preceptors than did the TBR students. Conversely, the TBR students felt closer to and interacted more frequently with interns and residents. CONCLUSIONS: The LIC students had more opportunities to care for patients directly and engaged in interactions with patients and attending physicians more frequently than did the TBR students. TRIAL REGISTRATION: Ethical approval for the study was obtained from the Institutional Review Board of Tri-Service General Hospital (TSGHIRB 2-106-05-018).

Osteocalcin: A Potential Marker of Peripheral Arterial Stiffness in Hypertensive Patients.

Background and Objectives: Brachial-ankle pulse wave velocity (baPWV) is an established independent risk factor for cardiovascular events, cardiovascular mortality, and all-cause mortality. Osteocalcin (OC) is recognized to be associated with vascular function. The present study assessed the correlation between serum OC levels and peripheral arterial stiffness (PAS) measured through baPWV in hypertensive patients. Materials and Methods: Fasting blood samples were collected from 120 hypertensive participants. The serum total OC levels were measured using a commercial enzyme-linked immunosorbent assay kit, whereas the baPWV device was used to detect PAS. The PAS group had left or right baPWV > 18.0 m/s. Results: Among the hypertensive patients, 24 (20.0%) were classified into the PAS group. The PAS group exhibited a significantly older age (p = 0.011), higher prevalence of diabetes (p = 0.010), systolic blood pressure (p = 0.019), levels of serum fasting glucose (p = 0.003), blood urea nitrogen (p = 0.024), creatinine (p = 0.004), C-reactive protein (p = 0.007), OC (p = 0.002), and lower estimated glomerular filtration rate (p = 0.004) than the non-PAS group. Age (odds ratio [OR]: 1.076, 95% CI: 1.004-1.153, p = 0.037) and serum OC level (OR: 1.797, 95% confidence interval (CI): 1.077-3.000, p = 0.025) were independent factors linked to PAS in hypertensive patients in the multivariate logistic regression analysis. Conclusions: Serum OC levels and older age are positively associated with PAS in hypertensive patients.

Efficient Space-Time Signal Processing Scheme of Frequency Synchronization and Positioning for Sensor Networks.

The orthogonal frequency division multiple access (OFDMA) technique has been widely employed in sensor networks as the data modulation scheme. This study presents a one-dimensional (1D) space-time signal processing scheme for the joint estimation of direction of arrival (DOA) and carrier frequency offsets (CFOs) in OFDMA uplink systems. The proposed approach, initiated by a one-dimensional ESPRIT algorithm, involves estimating the DOAs of the received signal to identify subscriber positions. Spatial beamformers are then used to suppress multiple access interference and separate each subscriber's signal from the received signal. The outputs of the spatial beamformer are decimated to estimate the CFO of each subscriber. Compared with conventional two-dimensional parameter estimation algorithms, the proposed one-dimensional algorithm has a higher estimation accuracy and significantly lower computational complexity.

Network Anomaly Intrusion Detection Based on Deep Learning Approach.

The prevalence of internet usage leads to diverse internet traffic, which may contain information about various types of internet attacks. In recent years, many researchers have applied deep learning technology to intrusion detection systems and obtained fairly strong recognition results. However, most experiments have used old datasets, so they could not reflect the latest attack information. In this paper, a current state of the CSE-CIC-IDS2018 dataset and standard evaluation metrics has been employed to evaluate the proposed mechanism. After preprocessing the dataset, six models-deep neural network (DNN), convolutional neural network (CNN), recurrent neural network (RNN), long short-term memory (LSTM), CNN + RNN and CNN + LSTM-were constructed to judge whether network traffic comprised a malicious attack. In addition, multi-classification experiments were conducted to sort traffic into benign traffic and six categories of malicious attacks: BruteForce, Denial-of-service (DoS), Web Attacks, Infiltration, Botnet, and Distributed denial-of-service (DDoS). Each model showed a high accuracy in various experiments, and their multi-class classification accuracy were above 98%. Compared with the intrusion detection system (IDS) of other papers, the proposed model effectively improves the detection performance. Moreover, the inference time for the combinations of CNN + RNN and CNN + LSTM is longer than that of the individual DNN, RNN and CNN. Therefore, the DNN, RNN and CNN are better than CNN + RNN and CNN + LSTM for considering the implementation of the algorithm in the IDS device.

A Novel Tactile Function Assessment Using a Miniature Tactile Stimulator.

Several methods for the measurement of tactile acuity have been devised previously, but unexpected nonspatial cues and intensive manual skill requirements compromise measurement accuracy. Therefore, we must urgently develop an automated, accurate, and noninvasive method for assessing tactile acuity. The present study develops a novel method applying a robotic tactile stimulator to automatically measure tactile acuity that comprises eye-opened, eye-closed training, and testing sessions. Healthy participants judge the orientation of a rotating grating ball presented on their index fingerpads in a two-alternative forced-choice task. A variable rotation speed of 5, 10, 40, or 160 mm/s was used for the tactile measurement at a variety of difficulties. All participants met the passing criteria for the training experiment. Performance in orientation identification, quantified by the proportion of trials with correct answers, differed across scanning directions, with the highest rotation speed (160 mm/s) having the worst performance. Accuracy did not differ between vertical and horizontal orientations. Our results demonstrated the utility of the pre-test training protocol and the functionality of the developed procedure for tactile acuity assessment. The novel protocol performed well when applied to the participants. Future studies will be conducted to apply this method to patients with impairment of light touch.

The Structure-Function Relationship of Human Bleomycin Hydrolase: Mutation of a Cysteine Protease into a Serine Protease.

Human bleomycin hydrolase (hBH) catalyzes deamidation of the anticancer drug bleomycins (BLM). This enzyme is involved in BLM detoxification and drug resistance. Herein, we report the putative BLM-binding site and catalytic mechanism of hBH. The crystal structures and biochemical studies suggest that hBH cleaves its C-terminal residue without significant preference for the type of amino acid, and therefore can accordingly accommodate the ß-aminoalanine amide moiety of BLM for deamidation. Interestingly, hBH is capable of switching from a cysteine protease to a serine protease that is unable to cleave the secondary amide of hBH C-terminus but reacts with the primary amide of BLMs.

Characterization and Structural Determination of CmnG-A, the Adenylation Domain That Activates the Nonproteinogenic Amino Acid Capreomycidine in Capreomycin Biosynthesis.

Capreomycidine (Cap) is a nonproteinogenic amino acid and building block of nonribosomal peptide (NRP) natural products. We report the formation and activation of Cap in capreomycin biosynthesis. CmnC and CmnD catalyzed hydroxylation and cyclization, respectively, of l-Arg to form l-Cap. l-Cap is then adenylated by CmnG-A before being incorporated into the nonribosomal peptide. The co-crystal structures of CmnG-A with l-Cap and adenosine nucleotides provide insights into the specificity and engineering opportunities of this unique adenylation domain.

Nervous Necrosis Virus Coat Protein Mediates Host Translation Shutoff through Nuclear Translocalization and Degradation of Polyadenylate Binding Protein.

Nervous necrosis virus (NNV) belongs to the Betanodavirus genus of the Nodaviridae family and is the main cause of viral nervous necrosis disease in marine fish larvae and juveniles worldwide. The NNV virion contains two positive-sense, single-stranded RNA genomes, which encode RNA-dependent RNA polymerase, coat protein, and B2 protein. Interestingly, NNV infection can shut off host translation in orange-spotted grouper (Epinephelus coioides) brain cells; however, the detailed mechanisms of this action remain unknown. In this study, we discovered that the host translation factor, polyadenylate binding protein (PABP), is a key target during NNV takeover of host translation machinery. Additionally, ectopic expression of NNV coat protein is sufficient to trigger nuclear translocalization and degradation of PABP, followed by translation shutoff. A direct interaction between NNV coat protein and PABP was demonstrated, and this binding requires the NNV coat protein N-terminal shell domain and PABP proline-rich linker region. Notably, we also showed that degradation of PABP during later stages of infection is mediated by the ubiquitin-proteasome pathway. Thus, our study reveals that the NNV coat protein hijacks host PABP, causing its relocalization to the nucleus and promoting its degradation to stimulate host translation shutoff. IMPORTANCE Globally, more than 200 species of aquacultured and wild marine fish are susceptible to NNV infection. Devastating outbreaks of this virus have been responsible for massive economic damage in the aquaculture industry, but the molecular mechanisms by which NNV affects its host remain largely unclear. In this study, we show that NNV hijacks translation in host brain cells, with the viral coat protein binding to host PABP to promote its nuclear translocalization and degradation. This previously unknown mechanism of NNV-induced host translation shutoff greatly enhances the understanding of NNV pathogenesis and provides useful insights and novel tools for development of NNV treatments, such as the use of orange-spotted grouper brain cells as an in vitro model system.

In Vivo Profiling with 18F-YJH08 Reveals Diverse Tissue Patterns of Antagonist/Glucocorticoid Receptor Interactions.

Demonstrating target engagement in vivo is an important milestone in drug development, both to establish on target, on tissue interactions but also to identify potentially undesirable off tissue binding. The glucocorticoid receptor (GR) is a long-studied yet vexing drug target that has recently re-emerged as a potential druggable driver of many solid tumor types including breast and prostate cancer, and several antagonists are currently in early phase clinical trials. Since GR is also ubiquitously expressed in normal tissues, understanding antagonist/GR interactions in normal tissues and tumor is crucial to defining a therapeutic index. Herein, we demonstrate that the GR radioligand 18F-YJH08 can map drug/GR engagement in vivo. Profiling target engagement in vivo showed that the GR antagonists RU486 (mifepristone) and CORT125281 engaged GR in fewer normal tissues compared to ORIC-101 or the agonist dexamethasone. Furthermore, 18F-YJH08 detected GR in human prostate cancer tumor models and measured receptor binding by RU486. In summary, these data show for the first time that antagonist/GR interactions can be measured in vivo with 18F-YJH08, a finding with clinical relevance as GR antagonists and 11C-YJH08 are currently in clinical trials.

Janus Kinase 3 Deficiency Promotes Vascular Reendothelialization-Brief Report.

[Figure: see text].

Neutral Ligands as Potential 64Cu Chelators for Positron Emission Tomography Imaging Applications in Alzheimer's Disease.

Positron emission tomography (PET), which uses positron-emitting radionuclides to visualize and measure processes in the human body, is a useful noninvasive diagnostic tool for Alzheimer's disease (AD). The development of longer-lived radiolabeled compounds is essential for further expansion of the use of PET imaging in healthcare, and diagnostic agents employing longer-lived radionuclides such as 64Cu (t1/2 = 12.7 h, ß+ = 17%, ß- = 39%, electron capture EC = 43%, and Emax = 0.656 MeV) can accomplish this task. One limitation of 64Cu PET agents for neuroimaging applications is their limited lipophilicity due to the presence of several anionic groups needed to ensure strong Cu chelation. Herein, we evaluate a series of neutral chelators containing the 1,4,7-triazacyclononane or 2,11-diaza[3.3](2,6)pyridinophane macrocycles that have pyridyl-containing arms incorporating Aß-peptide-interacting fragments. The crystal structures of the corresponding Cu complexes confirm that the pyridyl N atoms are involved in binding to Cu. Radiolabeling and autoradiography studies show that the compounds efficiently chelate 64Cu, and the resulting complexes exhibit specific binding to the amyloid plaques in the AD mouse brain sections versus wild-type controls.

Shared decision-making and patient decision aids in choosing first-line targeted therapy for metastatic colorectal cancer in Taiwan.

INTRODUCTION: In Taiwan, given the discrepancy between current treatment guidelines and reimbursement options, patients might require a tool to support their decision-making process when selecting a regimen for metastatic colorectal cancer, especially therapeutic strategies, and subsequent costs, along with efficacy and safety outcomes. Therefore, we developed a patient decision aid (PDA) to support patients in choosing between treatment options recommended based on the current evidence and those reimbursed by the Taiwanese National Health Insurance. METHODS: By carefully reviewing the updated data and then interpreting the clinical tool, we conducted a needs assessment using a serial questionnaire to test for a step-by-step adjustment of the PDA. RESULTS: Patients, their relatives, and medical team members were most concerned about outcomes, such as overall survival, progression-free survival, objective response rate, tumor shrinkage to resectable status, total medical cost, severe gastrointestinal perforation, and severe skin reaction. After a serial alpha test for quality, we performed quantitative evaluation and beta tests, revealing average scores of more than 4 points (on a scale of 1-5) for both perceptibility and utility. CONCLUSIONS: The present findings suggest that PDAs are useful and supplement the shared decision-making practice, helping patients make decisions about preferences and consider the pros and cons of treatment regimens, along with insurance reimbursement options.

Evaluation of Time-of-Flight Secondary Ion Mass Spectrometry Spectra of Peptides by Random Forest with Amino Acid Labels: Results from a Versailles Project on Advanced Materials and Standards Interlaboratory Study.

We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the identification of peptide sample TOF-SIMS spectra by machine learning. More than 1000 time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra of six peptide model samples (one of them was a test sample) were collected using 27 TOF-SIMS instruments from 25 institutes of six countries, the U. S., the U. K., Germany, China, South Korea, and Japan. Because peptides have systematic and simple chemical structures, they were selected as model samples. The intensity of peaks in every TOF-SIMS spectrum was extracted using the same peak list and normalized to the total ion count. The spectra of the test peptide sample were predicted by Random Forest with 20 amino acid labels. The accuracy of the prediction for the test spectra was 0.88. Although the prediction of an unknown peptide was not perfect, it was shown that all of the amino acids in an unknown peptide can be determined by Random Forest prediction and the TOF-SIMS spectra. Moreover, the prediction of peptides, which are included in the training spectra, was almost perfect. Random Forest also suggests specific fragment ions from an amino acid residue Q, whose fragment ions detected by TOF-SIMS have not been reported, in the important features. This study indicated that the analysis using Random Forest, which enables translation of the mathematical relationships to chemical relationships, and the multi labels representing monomer chemical structures, is useful to predict the TOF-SIMS spectra of an unknown peptide.

In vitro activity of imipenem/relebactam, meropenem/vaborbactam, ceftazidime/avibactam, cefepime/zidebactam and other novel antibiotics against imipenem-non-susceptible Gram-negative bacilli from Taiwan.

OBJECTIVES: To investigate the susceptibility of imipenem-non-susceptible Escherichia coli (INS-EC), Klebsiella pneumoniae (INS-KP), Acinetobacter baumannii (INS-AB) and Pseudomonas aeruginosa (INS-PA) to novel antibiotics. METHODS: MICs were determined using the broth microdilution method. Carbapenemase and ESBL phenotypic testing and PCR for genes encoding ESBLs, AmpCs and carbapenemases were performed. RESULTS: Zidebactam, avibactam and relebactam increased the respective susceptibility rates to cefepime, ceftazidime and imipenem of 17 INS-EC by 58.8%, 58.8% and 70.6%, of 163 INS-KP by 77.9%, 88.3% and 76.1% and of 81 INS-PA by 45.7%, 38.3% and 85.2%, respectively. Vaborbactam increased the meropenem susceptibility of INS-EC by 41.2% and of INS-KP by 54%. Combinations of ß-lactams and novel ß-lactamase inhibitors or ß-lactam enhancers (BLI-BLE) were inactive against 136 INS-AB. In 58 INS-EC and INS-KP with exclusively blaKPC-like genes, zidebactam, avibactam, relebactam and vaborbactam increased the susceptibility of the partner ß-lactams by 100%, 96.6%, 84.5% and 75.9%, respectively. In the presence of avibactam, ceftazidime was active in an additional 85% of 20 INS-EC and INS-KP with exclusively blaOXA-48-like genes while with zidebactam, cefepime was active in an additional 75%. INS-EC and INS-KP with MBL genes were susceptible only to cefepime/zidebactam. The ß-lactam/BLI-BLE combinations were active against INS-EC and INS-KP without detectable carbapenemases. For INS-EC, INS-KP and INS-AB, tigecycline was more active than omadacycline and eravacycline but eravacycline had a lower MIC distribution. Lascufloxacin and delafloxacin were active in <35% of these INS isolates. CONCLUSIONS: ß-Lactam/BLI-BLE combinations were active in a higher proportion of INS-EC, INS-KP and INS-PA. The susceptibility of novel fluoroquinolones and tetracyclines was not superior to that of old ones.