Tissue-Like 3D Standard and Protocols for Microscope Quality Management.

This article outlines a global study conducted by the Association of Biomedical Resource Facilities (ABRF) Light Microscopy Research Group (LMRG). The results present a novel 3D tissue-like biologically relevant standard sample that is affordable and straightforward to prepare. Detailed sample preparation, instrument-specific image acquisition protocols and image analysis methods are presented and made available to the community. The standard consists of sub-resolution and large well characterized relative intensity fluorescence microspheres embedded in a 120 µm thick 3D gel with a refractive index of 1.365. The standard allows the evaluation of several properties as a function of depth. These include the following: 1) microscope resolution with automated analysis of the point-spread function (PSF), 2) automated signal-to-noise ratio analysis, 3) calibration and correction of fluorescence intensity loss, and 4) quantitative relative intensity. Results demonstrate expected refractive index mismatch dependent losses in intensity and resolution with depth, but the relative intensities of different objects at similar depths are maintained. This is a robust standard showing reproducible results across laboratories, microscope manufacturers and objective lens types (e.g., magnification, immersion medium). Thus, these tools will be valuable for the global community to benchmark fluorescence microscopes and will contribute to improved scientific rigor and reproducibility.

Detection of SARS-CoV-2 B.1.351 (Beta) Variant through Wastewater Surveillance before Case Detection in a Community, Oregon, USA.

Genomic surveillance has emerged as a critical monitoring tool during the SARS-CoV-2 pandemic. Wastewater surveillance has the potential to identify and track SARS-CoV-2 variants in the community, including emerging variants. We demonstrate the novel use of multilocus sequence typing to identify SARS-CoV-2 variants in wastewater. Using this technique, we observed the emergence of the B.1.351 (Beta) variant in Linn County, Oregon, USA, in wastewater 12 days before this variant was identified in individual clinical specimens. During the study period, we identified 42 B.1.351 clinical specimens that clustered into 3 phylogenetic clades. Eighteen of the 19 clinical specimens and all wastewater B.1.351 specimens from Linn County clustered into clade 1. Our results provide further evidence of the reliability of wastewater surveillance to report localized SARS-CoV-2 sequence information.

The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models.

Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.

In vitro antibacterial activity of the ceftazidime-avibactam combination against enterobacteriaceae, including strains with well-characterized ß-lactamases.

The novel ß-lactamase inhibitor avibactam is a potent inhibitor of class A, class C, and some class D enzymes. The in vitro antibacterial activity of the ceftazidime-avibactam combination was determined for a collection of Enterobacteriaceae clinical isolates; this collection was enriched for resistant strains, including strains with characterized serine ß-lactamases. The inhibitor was added either at fixed weight ratios to ceftazidime or at fixed concentrations, with the latter type of combination consistently resulting in greater potentiation of antibacterial activity. In the presence of 4 µg/ml of avibactam, the ceftazidime MIC50 and MIC90 (0.25 and 2 µg/ml, respectively) were both below the CLSI breakpoint for ceftazidime. Further comparisons with reference antimicrobial agents were performed using this fixed inhibitor concentration. Against most ceftazidime-susceptible and -nonsusceptible isolates, the addition of avibactam resulted in a significant increase in ceftazidime activity, with MICs generally reduced 256-fold for extended-spectrum ß-lactamase (ESBL) producers, 8- to 32-fold for CTX-M producers, and >128-fold for KPC producers. Overall, MICs of a ceftazidime-avibactam combination were significantly lower than those of the comparators piperacillin-tazobactam, cefotaxime, ceftriaxone, and cefepime and similar or superior to those of imipenem.

Efficacy of a Ceftazidime-Avibactam combination in a murine model of Septicemia caused by Enterobacteriaceae species producing ampc or extended-spectrum ß-lactamases.

Avibactam is a novel non-ß-lactam ß-lactamase inhibitor that has been shown in vitro to inhibit class A, class C, and some class D ß-lactamases. It is currently in phase 3 of clinical development in combination with ceftazidime. In this study, the efficacy of ceftazidime-avibactam was evaluated in a murine septicemia model against five ceftazidime-susceptible (MICs of 0.06 to 0.25 µg/ml) and 15 ceftazidime-resistant (MICs of 64 to >128 µg/ml) species of Enterobacteriaceae, bearing either TEM, SHV, CTX-M extended-spectrum, or AmpC ß-lactamases. In the first part of the study, ceftazidime-avibactam was administered at ratios of 4:1 and 8:1 (wt/wt) to evaluate the optimal ratio for efficacy. Against ceftazidime-susceptible isolates of Klebsiella pneumoniae and Escherichia coli, ceftazidime and ceftazidime-avibactam demonstrated similar efficacies (50% effective doses [ED50] of <1.5 to 9 mg/kg of body weight), whereas against ceftazidime-resistant ß-lactamase-producing strains (ceftazidime ED50 of >90 mg/kg), the addition of avibactam restored efficacy to ceftazidime (ED50 dropped to <5 to 65 mg/kg). In a subsequent study, eight isolates (two AmpC and six CTX-M producers) were studied in the septicemia model. Ceftazidime-avibactam was administered at a 4:1 (wt/wt) ratio, and the efficacy was compared to that of the 4:1 (wt/wt) ratio of either piperacillin-tazobactam or cefotaxime-avibactam. Against the eight isolates, ceftazidime-avibactam was the more effective combination, with ED50 values ranging from 2 to 27 mg/kg compared to >90 mg/kg and 14 to >90 mg/kg for piperacillin-tazobactam and cefotaxime-avibactam, respectively. This study demonstrates that the potent in vitro activity observed with the ceftazidime-avibactam combination against ceftazidime-resistant Enterobacteriaceae species bearing class A and class C ß-lactamases translated into good efficacy in the mouse septicemia model.

Activities of ceftazidime and avibactam against ß-lactamase-producing Enterobacteriaceae in a hollow-fiber pharmacodynamic model.

Avibactam is a novel non-ß-lactam ß-lactamase inhibitor that is currently undergoing phase 3 clinical trials in combination with ceftazidime. Ceftazidime is hydrolyzed by a broad range of ß-lactamases, but avibactam is able to inhibit the majority of these enzymes. The studies described here attempt to provide insight into the amount of avibactam required to suppress bacterial growth in an environment where the concentrations of both agents are varying as they would when administered to humans. Following the simulation of a single intravenous dose of the drug, ceftazidime alone had no effect on any test organism, but a ceftazidime-avibactam combination resulted in rapid killing of all of the strains, with growth suppressed for the 8 h of the study. For seven of eight strains, this was achieved with a 1-g-250-mg profile, but a 2-g-500-mg profile was necessary to completely suppress a high-level-AmpC-producing isolate. When ceftazidime was infused continuously for 24 h with a single bolus dose of avibactam, rapid killing of all of the strains was again observed, with growth suppressed for 10 to >24 h. Regrowth appeared to commence once the avibactam concentration dropped below a critical concentration of approximately 0.3 µg/ml. In a third series of studies, ceftazidime was administered every 8 h for 24 h with avibactam administered at fixed concentrations for short periods during each ceftazidime dose profile. Simulating a 1-g dose of ceftazidime, an avibactam pulse of >0.25 and <0.5 µg/ml was required to suppress growth for 24 h.

Structure-Guided Discovery of Selective USP7 Inhibitors with In Vivo Activity.

Inhibition of ubiquitin-specific protease 7, USP7, has been proposed as a mechanism to affect many disease processes, primarily those implicated in oncology. The bound crystal structure of a published high-throughput screening hit with low-micromolar affinity for USP7 identified three regions of the compound for structure-guided optimization. Replacing one side of the compound with different aromatic moieties gave little improvement in affinity, and the central piperidine could not be improved. However, the binding site for the other side of the compound was poorly defined in the crystal structure, which suggested a wide variety of synthetically accessible options for optimization. These were assessed by screening reaction mixtures that introduced different substituents to this other side. Subsequent optimization led to a compound with low-nanomolar affinity for USP7, which showed target engagement in tumors, was tolerated in mice, and showed efficacy in xenograft models.

International test results for objective lens quality, resolution, spectral accuracy and spectral separation for confocal laser scanning microscopes.

As part of an ongoing effort to increase image reproducibility and fidelity in addition to improving cross-instrument consistency, we have proposed using four separate instrument quality tests to augment the ones we have previously reported. These four tests assessed the following areas: (1) objective lens quality, (2) resolution, (3) accuracy of the wavelength information from spectral detectors, and (4) the accuracy and quality of spectral separation algorithms. Data were received from 55 laboratories located in 18 countries. The largest source of errors across all tests was user error which could be subdivided between failure to follow provided protocols and improper use of the microscope. This truly emphasizes the importance of proper rigorous training and diligence in performing confocal microscopy experiments and equipment evaluations. It should be noted that there was no discernible difference in quality between confocal microscope manufactures. These tests, as well as others previously reported, will help assess the quality of confocal microscopy equipment and will provide a means to track equipment performance over time. From 62 to 97% of the data sets sent in passed the various tests demonstrating the usefulness and appropriateness of these tests as part of a larger performance testing regiment.

In vitro antibacterial activity of the ceftazidime-avibactam (NXL104) combination against Pseudomonas aeruginosa clinical isolates.

The ß-lactamase inhibitor avibactam (NXL104) displays potent inhibition of both class A and C enzymes. The in vitro antibacterial activity of the combination ceftazidime-avibactam was evaluated against a clinical panel of Pseudomonas aeruginosa isolates. Avibactam offered efficient protection from hydrolysis since 94% of isolates were susceptible to ceftazidime when combined with 4 µg/ml avibactam, compared with 65% susceptible to ceftazidime alone. Ceftazidime-avibactam also demonstrated better antipseudomonal activity than imipenem (82% susceptibility), a common reference treatment.

Evaluation of a Wastewater-Based Epidemiological Approach to Estimate the Prevalence of SARS-CoV-2 Infections and the Detection of Viral Variants in Disparate Oregon Communities at City and Neighborhood Scales.

BACKGROUND: Positive correlations have been reported between wastewater SARS-CoV-2 concentrations and a community's burden of infection, disease or both. However, previous studies mostly compared wastewater to clinical case counts or nonrepresentative convenience samples, limiting their quantitative potential. OBJECTIVES: This study examined whether wastewater SARS-CoV-2 concentrations could provide better estimations for SARS-CoV-2 community prevalence than reported cases of COVID-19. In addition, this study tested whether wastewater-based epidemiology methods could identify neighborhood-level COVID-19 hotspots and SARS-CoV-2 variants. METHODS: Community SARS-CoV-2 prevalence was estimated from eight randomized door-to-door nasal swab sampling events in six Oregon communities of disparate size, location, and demography over a 10-month period. Simultaneously, wastewater SARS-CoV-2 concentrations were quantified at each community's wastewater treatment plant and from 22 Newport, Oregon, neighborhoods. SARS-CoV-2 RNA was sequenced from all positive wastewater and nasal swab samples. Clinically reported case counts were obtained from the Oregon Health Authority. RESULTS: Estimated community SARS-CoV-2 prevalence ranged from 8 to 1,687/10,000 persons. Community wastewater SARS-CoV-2 concentrations ranged from 2.9 to 5.1 log10 gene copies per liter. Wastewater SARS-CoV-2 concentrations were more highly correlated (Pearson's r=0.96; R2=0.91) with community prevalence than were clinically reported cases of COVID-19 (Pearson's r=0.85; R2=0.73). Monte Carlo simulations indicated that wastewater SARS-CoV-2 concentrations were significantly better than clinically reported cases at estimating prevalence (p<0.05). In addition, wastewater analyses determined neighborhood-level COVID-19 hot spots and identified SARS-CoV-2 variants (B.1 and B.1.399) at the neighborhood and city scales. DISCUSSION: The greater reliability of wastewater SARS-CoV-2 concentrations over clinically reported case counts was likely due to systematic biases that affect reported case counts, including variations in access to testing and underreporting of asymptomatic cases. With these advantages, combined with scalability and low costs, wastewater-based epidemiology can be a key component in public health surveillance of COVID-19 and other communicable infections. https://doi.org/10.1289/EHP10289.

Quality assurance testing for modern optical imaging systems.

The days of being able to ascertain instrument performance by simply peering through the eye pieces at a specimen are gone. However, users and granting agencies need to be confident that data collected on these instruments is uniform and quantifiable both over time and between instruments. Ideally, a LASER should not fluctuate, illumination should be completely uniform, and colors should be perfectly aligned. To check the current performance of imaging equipment, we conducted a worldwide research study utilizing three image-based tests: long-/short-term illumination stability, co-registration of signals across various wavelengths, and field illumination uniformity. To differentiate between "acceptable" and "unacceptable" performance, the deviation in illumination power could not exceed 10% (long term) or 3% (short term), the difference in the center-of-mass of imaged multicolored beads could not exceed >1 pixel between different wavelengths, and field illumination values could not exceed 10% (horizontal) or 20% (diagonal) deviation. This study established the current state of microscope performance through simple, efficient, and robust tests, while defining relative standards to assist cores in maintaining their instruments in optimal operating conditions. We developed cross-platform performance standards that will improve the validity of quantitative measurements made using various light microscopes.

Discovery of S64315, a Potent and Selective Mcl-1 Inhibitor.

Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1. Starting from a fragment derived lead compound, we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compounds against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has drug-like properties that have enabled its development and entry into clinical trials.

In vitro activity of the {beta}-lactamase inhibitor NXL104 against KPC-2 carbapenemase and Enterobacteriaceae expressing KPC carbapenemases.

BACKGROUND: NXL104 is a novel-structure beta-lactamase inhibitor with potent activity against both class A and class C enzymes. Among the class A carbapenemases, KPC-type enzymes are now spreading rapidly and KPC-related carbapenemase resistance is an emerging phenomenon of great clinical importance. The activity of NXL104 against KPC beta-lactamases was examined. METHODS: Enzymatic activity of purified recombinant KPC-2 was measured with nitrocefin as reporter substrate and inhibition by NXL104 was measured by determination of IC(50) values. Antimicrobial susceptibility testing of various beta-lactams combined with a fixed concentration of NXL104 at 4 mg/L against strains producing KPC enzymes was performed by the broth microdilution method. RESULTS: NXL104 was a potent inhibitor of KPC-2 with an IC(50) of 38 nM. NXL104 restored the antimicrobial activity of ceftazidime, ceftriaxone, imipenem and piperacillin against Enterobacteriaceae strains producing KPC-2 or KPC-3. MIC values of ceftazidime against KPC producers were reduced by up to 1000-fold by combination with NXL104. CONCLUSIONS: NXL104 inhibitory activity is unique in terms of spectrum, encompassing class A extended-spectrum beta-lactamases, class C enzymes and class A carbapenemases. Given the limited therapeutic options available for infections caused by multiresistant Enterobacteriaceae isolates, NXL104 beta-lactamase inhibitor is a promising agent to be used in combination with a beta-lactam to protect its antibacterial activity.

Mechanism of action of the antibiotic NXL101, a novel nonfluoroquinolone inhibitor of bacterial type II topoisomerases.

NXL101 is one of a new class of quinoline antibacterial DNA gyrase and topoisomerase IV inhibitors showing potent activity against gram-positive bacteria, including methicillin- and fluoroquinolone-resistant strains. NXL101 inhibited topoisomerase IV more effectively than gyrase from Escherichia coli, whereas the converse is true of enzymes from Staphylococcus aureus. This apparent target preference is opposite to that which is associated with most fluoroquinolone antibiotics. In vitro isolation of S. aureus mutants resistant to NXL101 followed by cloning and sequencing of the genes encoding gyrase and topoisomerase IV led to the identification of several different point mutations within, or close to, the quinolone resistance-determining region (QRDR) of GyrA. However, the mutations were not those that are most frequently associated with decreased sensitivity to quinolones. A fluoroquinolone-resistant mutant variant of gyrase generated in vitro was highly resistant to inhibition by the fluoroquinolones ciprofloxacin and moxifloxacin but remained fully susceptible to inhibition by NXL101. Two mutant gyrases constructed in vitro, with mutations in gyrA engineered according to those most frequently found in S. aureus strains resistant to NXL101, were insensitive to inhibition by NXL101 and had a diminished sensitivity to ciprofloxacin and moxifloxacin. Certain combinations of mutations giving rise to NXL101 resistance and those giving rise to fluoroquinolone resistance may be mutually exclusive.

A human T-cell lymphotropic virus type 1 enhancer of Myc transforming potential stabilizes Myc-TIP60 transcriptional interactions.

The human T-cell lymphotropic virus type 1 (HTLV-1) infects and transforms CD4+ lymphocytes and causes adult T-cell leukemia/lymphoma (ATLL), an aggressive lymphoproliferative disease that is often fatal. Here, we demonstrate that the HTLV-1 pX splice-variant p30II markedly enhances the transforming potential of Myc and transcriptionally activates the human cyclin D2 promoter, dependent upon its conserved Myc-responsive E-box enhancer elements, which are associated with increased S-phase entry and multinucleation. Enhancement of c-Myc transforming activity by HTLV-1 p30II is dependent upon the transcriptional coactivators, transforming transcriptional activator protein/p434 and TIP60, and it requires TIP60 histone acetyltransferase (HAT) activity and correlates with the stabilization of HTLV-1 p30II/Myc-TIP60 chromatin-remodeling complexes. The p30II oncoprotein colocalizes and coimmunoprecipitates with Myc-TIP60 complexes in cultured HTLV-1-infected ATLL patient lymphocytes. Amino acid residues 99 to 154 within HTLV-1 p30II interact with the TIP60 HAT, and p30II transcriptionally activates numerous cellular genes in a TIP60-dependent or TIP60-independent manner, as determined by microarray gene expression analyses. Importantly, these results suggest that p30II functions as a novel retroviral modulator of Myc-TIP60-transforming interactions that may contribute to adult T-cell leukemogenesis.

S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth.

Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

Inhibitors of Civ1 kinase belonging to 6-aminoaromatic-2-cyclohexyldiamino purine series as potent anti-fungal compounds.

There is today a blatant need for new antifungal agents, because of the recent increase in life-threatening infections involving an ever-greater number of fungal strains. Fungi make extensive use of kinases in the regulation of essential processes, in particular the cell cycle. Most fungal kinases, however, are shared with higher eukaryotes. Only the kinases which have no human homologs, such as the histidine kinases, can be used as targets for antifungal drugs design. This review describes efforts directed towards the discovery of drugs active against a novel target, the atypical cell cycle kinase, Civ1.

[Backward flow of blood in the extracorporal circuit pressure transducers of the generator-monitors of hemodialysis]. / Inondation des capteurs de pression sanguine des générateurs-moniteurs d'hémodialyse.

Transmission of hepatitis C virus between hemodialysis patients occurs mainly between the patients treated simultaneously in the same sector or in the same room. The other mode of viral transmission occurs between two patients treated successively with the same hemodialysis generator-monitor and sharing the same environment. One of the vectors of this last mode of transmission could be the contamination of the system of measurement of pressure of the extracorporal blood circuit. In July 2004, the French health products safety agency (Agence française de sécurité sanitaire des produits de santé, Afssaps) published a recommendation on these systems of measurement of pressure. i) The recommendation points out the optional preventive measures that users and manufacturers can carry out: (a) the manufacturers can propose tight systems of measurement of pressure, based on the deformation of a device; (b) the users can add a second protective filter on the external tubing connecting the blood circuit to the machine; (c) the manufacturers can add additional protective filters on the internal tubings of the machine. ii)The recommendation indicates the actions to be carried out obligatorily in the event of backward flow of blood in the last external protective filter: the soiled filter must be replaced and the causes of the backward flow must be analyzed and corrected; after the session, the monitor must be taken out of the unit and checked by a technician. The technician must replace all the elements located downstream from a soiled protective filter: tubings, drip chamber level control system, and pressure transducer. When these elements are protected by internal filters, only tubings and the first internal filter not soiled must be replaced. Precautions with regard to the systems of measurement of pressure must supplement standard precautions of hygiene essential in hemodialysis units.

The Werner syndrome helicase is a cofactor for HIV-1 long terminal repeat transactivation and retroviral replication.

The Werner syndrome helicase (WRN) participates in DNA replication, double strand break repair, telomere maintenance, and p53 activation. Mutations of wrn cause Werner syndrome (WS), an autosomal recessive premature aging disorder associated with cancer predisposition, atherosclerosis, and other aging related symptoms. Here, we report that WRN is a novel cofactor for HIV-1 replication. Immortalized human WRN(-/-) WS fibroblasts, lacking a functional wrn gene, are impaired for basal and Tat-activated HIV-1 transcription. Overexpression of wild-type WRN transactivates the HIV-1 long terminal repeat (LTR) in the absence of Tat, and WRN cooperates with Tat to promote high-level LTR transactivation. Ectopic WRN induces HIV-1 p24(Gag) production and retroviral replication in HIV-1-infected H9(HIV-1IIIB) lymphocytes. A dominant-negative helicase-minus mutant, WRN(K577M), inhibits LTR transactivation and HIV-1 replication. Inhibition of endogenous WRN, through co-expression of WRN(K577M), diminishes recruitment of p300/CREB-binding protein-associated factor (PCAF) and positive transcription elongation factor b (P-TEFb) to Tat/transactivation response-RNA complexes, and immortalized WRN(-/-) WS fibroblasts exhibit comparable defects in recruitment of PCAF and P-TEFb to the HIV-1 LTR. Our results demonstrate that WRN is a novel cellular cofactor for HIV-1 replication and suggest that the WRN helicase participates in the recruitment of PCAF/P-TEFb-containing transcription complexes. WRN may be a plausible target for antiretroviral therapy.