Stress-induced hematopoietic failure in the absence of immediate early response gene X-1 (IEX-1, IER3)

Expression of the immediate early response gene X-1 (IEX-1, IER3) is diminished significantly in hematopoietic stem cells in a subgroup of patients with early stage myelodysplastic syndromes, but it is not clear whether the deregulation contributes to the disease. The current study demonstrates increased apoptosis and a concomitant decrease in the number of hematopoietic stem cells lacking this early response gene. Null mutation of the gene also impeded platelet differentiation and shortened a lifespan of red blood cells. When bone marrow cells deficient in the gene were transplanted into wild-type mice, the deficient stem cells produced significantly fewer circulating platelets and red blood cells, despite their enhanced repopulation capability. Moreover, after exposure to a non-myeloablative dose of radiation, absence of the gene predisposed to thrombocytopenia, a significant decline in red blood cells, and dysplastic bone marrow morphology, typical characteristics of myelodysplastic syndromes. These findings highlight a previously unappreciated role for this early response gene in multiple differentiation steps within hematopoiesis, including thrombopoiesis, erythropoiesis and in the regulation of hematopoietic stem cell quiescence. The deficient mice offer a novel model for studying the initiation and progression of myelodysplastic syndromes as well as strategies to prevent this disorder.     

Laboratory misdiagnosis of von Willebrand disease in post-menarchal females: A multi-center study

Increased awareness of von Willebrand Disease (VWD) has led to more frequent diagnostic laboratory testing, which insurers often dictate be performed at a facility with off-site laboratory processing, instead of a coagulation facility with onsite processing. Off-site processing is more prone to preanalytical variables causing falsely low levels of von Willebrand Factor (VWF) due to the additional transport required. Our aim was to determine the percentage of discordance between off-site and onsite specimen processing for VWD in this multicenter, retrospective study. We enrolled females aged 12 to 50 years who had off-site specimen processing for VWF assays, and repeat testing performed at a consulting institution with onsite coagulation phlebotomy and processing. A total of 263 females from 17 institutions were included in the analysis. There were 251 subjects with both off-site and onsite VWF antigen (VWF:Ag) processing with 96 (38%) being low off-site and 56 (22%) low onsite; 223 subjects had VWF ristocetin co-factor (VWF:RCo), 122 (55%) were low off-site and 71 (32%) were low onsite. Similarly, 229 subjects had a Factor VIII (FVIII) assay, and 67 (29%) were low off-site with less than half, 29 (13%) confirmed low with onsite processing. Higher proportions of patients demonstrated low VWF:Ag, VWF:RCo, and/or FVIII with off-site processing compared to onsite (McNemarʼs test P-value <.0005, for all assays). These results emphasize the need to decrease delays from sample procurement to processing for VWF assays. The VWF assays should ideally be collected and processed at the same site under the guidance of a hematologist.     

Micromolded gelatin hydrogels for extended culture of engineered cardiac tissues

Defining the chronic cardiotoxic effects of drugs during preclinical screening is hindered by the relatively short lifetime of functional cardiac tissues in vitro, which are traditionally cultured on synthetic materials that do not recapitulate the cardiac microenvironment. Because collagen is the primary extracellular matrix protein in the heart, we hypothesized that micromolded gelatin hydrogel substrates tuned to mimic the elastic modulus of the heart would extend the lifetime of engineered cardiac tissues by better matching the native chemical and mechanical microenvironment. To measure tissue stress, we used tape casting, micromolding, and laser engraving to fabricate gelatin hydrogel muscular thin film cantilevers. Neonatal rat cardiac myocytes adhered to gelatin hydrogels and formed aligned tissues as defined by the microgrooves. Cardiac tissues could be cultured for over three weeks without declines in contractile stress. Myocytes on gelatin had higher spare respiratory capacity compared to those on fibronectin-coated PDMS, suggesting that improved metabolic function could be contributing to extended culture lifetime. Lastly, human induced pluripotent stem cell-derived cardiac myocytes adhered to micromolded gelatin surfaces and formed aligned tissues that remained functional for four weeks, highlighting their potential for human-relevant chronic studies.     

Antepartum Screening for Group B Streptococcus by Three FDA-Cleared Molecular Tests and Effect of Shortened Enrichment Culture on Molecular Detection Rates

Neonatal Streptococcus agalactiae infections cause significant morbidity and mortality, and antenatal screening is recommended. We compared three U.S. Food and Drug Administration (FDA)-cleared nucleic acid amplification tests (NAATs) to culture using 314 vaginal/rectal swabs after 18 to 24 h (recommended period) and 4 to 8 h (shortened period) of broth enrichment. Agreement of the NAATs with each other was high (97.1% to 98.4%), but culture was less sensitive than all NAATs (67% to 73%). A shortened period of broth culture enrichment resulted in 1 false-negative result in 68 (1.5%). The NAATs performed comparably and were more sensitive than culture.     

Deuterium equilibrium isotope effects in a supramolecular receptor for the hydrochalcogenide and halide anions

We highlight a convenient synthesis to selectively deuterate an aryl C–H hydrogen bond donor in an arylethynyl bisurea supramolecular anion receptor and use the Perrin method of competitive titrations to study the deuterium equilibrium isotope effects (DEIE) of anion binding for HS⁻, Cl⁻, and Br⁻. This work highlights the utility and also challenges in using this method to determine EIE with highly reactive and/or weakly binding anions.

We highlight a convenient synthesis to selectively deuterate an aryl C–H hydrogen bond donor in a supramolecular anion receptor and use competitive titrations to study the deuterium equilibrium isotope effects (DEIE) in binding HS⁻, Cl⁻, and Br⁻.

Molecular recognition and host–guest binding in both biological and synthetic systems are often driven by a mixture of competitive and additive primarily non-covalent interactions. Understanding the role of each of these forces in a host–guest system can reveal insights into the driving forces behind binding and help inform on the molecular design of future hosts.^1–3 Equilibrium isotope effects (EIE), also referred to as binding isotope effects (BIE) in structural molecular biology,⁴ measure the effect of isotopic substitution on supramolecular interactions through changes in the vibrational energy of the substituted bond. These studies can be used to elucidate the complex non-covalent forces involved in host conformational changes and host–guest binding.^5–8Examples from structural molecular biology have demonstrated that EIEs can reveal mechanistic information in enzyme–ligand binding events.^4,9 Isotopic substitution in synthetic supramolecular systems has been used both for labelling purposes and for studying individual non-covalent interactions. For example, Bergman, Raymond, and coworkers used deuterium equilibrium isotope effects (DEIE) to study benzylphosphonium cation guest binding in a self-assembled supramolecular complex in aqueous solution.¹⁰ From these DEIE studies, the authors found that attractive cation⋯π interactions in the interior of the host were important for promoting guest binding, and that C–H⋯π and π⋯π interactions were relatively small contributors. In another example, Shimizu and coworkers studied the DEIE on the strength of C–H⋯π interactions in their molecular balances.¹¹ Both computational and experimental results showed that the strength of C–H⋯π and C–D⋯π interactions were about equal, settling the debate on which interaction is stronger and easing concerns about using deuteration for spectroscopic and labelling applications.Previously, we used DEIE to study Cl⁻ binding with the arylethynyl bisurea anion receptor 1^H/D (Fig. 1) in DMSO-d₆.¹² We found an experimental DEIE of 1.019 ± 0.010, which matched the computationally-predicted DEIE of 1.020. Further computational analysis determined that the DEIE was due to a distorted N–H⋯Cl⁻ hydrogen bond geometry, which resulted in changes in the C–H/D bond vibrational energy in the host–guest complex. In addition, Paneth and coworkers performed a computational study with 1^H and other hydrogen bonding supramolecular Cl⁻ receptors to determine the EIE of ^35/37Cl⁻ binding in these hosts.¹³ Because isotope effects, both equilibrium and kinetic, originate solely from changes in the vibrational energy of the isotopically labelled bond, the EIE arising from this study came from changes in the vibrational energies of the bonds in the supramolecular hosts when participating in hydrogen bonding with Cl⁻ isotopes. Indeed, a linear relationship was observed between the hydrogen bond donor (D) D–H bond lengths in the host–guest complex and the computed ^35/37Cl EIE.Open in a separate windowFig. 1Arylethynyl bisurea receptors 1^H and 1^D used in our previous DEIE study of Cl⁻ binding. Related receptors 2^H and 2^D are used in this study to avoid reaction of the nitro group with HS⁻.Previous EIE studies with receptor 1^H/D have focused on Cl⁻ binding; however, to the best of our knowledge, no work has yet investigated the EIE of hydrosulfide (HS⁻) binding in this or other systems. HS⁻ is a highly reactive anion that plays crucial roles in biology. At physiological pH, HS⁻ is favored in solution by a 3 : 1 ratio over its conjugate acid, hydrogen sulfide (H₂S). H₂S has been identified as the third physiological gasotransmitter alongside CO and NO and plays essential roles in physiological systems.¹⁵ Despite its high nucleophilicity and redox activity, HS⁻ has been observed to be bound through non-covalent interactions in the protein crystal structure of a bacterial ion channel¹⁶ and in the turn-over state of vanadium-containing nitrogenase.¹⁷ The supramolecular chemistry of HS⁻ is under-studied in synthetic supramolecular receptors, likely due to the inherent high reactivity of HS⁻. Indeed, we are aware of only three families of receptors that have been shown to reversibly bind HS⁻.^18–21Recently, we used a series of arylethynyl bisurea anion receptors to investigate and demonstrate a linear free energy relationship between the polarity of a non-traditional C–H hydrogen bond donor and the solution binding energy of HS⁻, HSe⁻, Cl⁻ and Br⁻.¹⁴ A major and unexpected finding of this study was that HS⁻ demonstrated a significant increase in sensitivity towards the polarity of the C–H hydrogen donor over HSe⁻, Cl⁻ and Br⁻. Although increasing the polarity of the C–H hydrogen bond donor did not lead to changes in selectivity between Cl⁻, Br⁻, and HSe⁻, we observed a 9-fold increase in selectivity for HS⁻ over Cl⁻, suggesting a fresh approach to selective HS⁻ recognition using non-covalent interactions. In this current study, we label the C–H hydrogen bond donor in an arylethynyl bisurea receptor with a deuterium atom (2^H/D, Fig. 1) to further investigate this apparent preference of polar C–H H-bond donors for HS⁻ over Cl⁻ and Br⁻ through DEIE.Receptor 2^H is a previously reported anion receptor for HS⁻, Cl⁻, and Br⁻ and was prepared by established methods.¹⁴ Deuterium labelling of the isotopologue 2^D was achieved by selective monodeuteration of intermediates through methods similar to those reported in the literature (Scheme 1).²² The diazonium salt 3 was synthesized in a 71% yield from 2,6-diiodo-4-trifluoromethylaniline.²³ Dediazonation in DMF-d₇ is catalyzed by FeSO₄ and allows for selective synthesis of monodeuterated intermediate 4. The deuteration step proceeds through a radical pathway that uses DMF-d₇ as the deuterium source. This deuteration reaction provides efficient deuterium incorporation even with up to 50% by volume H₂O in the reaction solution due to the differential bond strengths in DMF and H₂O.²² Sonogashira cross-coupling reaction of 4 and 4-t-butyl-2-ethynylaniline²⁴ afforded 5 in 45% yield. Subsequent addition with 4-methoxyphenyl isocyanate gave 2^D in 34% yield. Compound 2^D and intermediates were characterized through ¹H, ²H, ¹³C{¹H}, and ¹⁹F NMR spectroscopy and high-resolution mass spectrometry (see ESI^†).Open in a separate windowScheme 1Synthetic route for the selective deuteration of anion receptor 2^D.Previous work on the DEIE of Cl⁻ binding with 1^H/D in DMSO revealed an experimental isotope effect of 1.019 ± 0.010. Therefore, we expected similar small DEIEs for HS⁻, Cl⁻, and Br⁻ binding with 2^H/D. Typical methods to determine binding constants (K_a) in supramolecular systems use non-linear regression fitting of titration data. Results from this method can be affected by small errors in the known initial host and guest concentration, quality of the titration isotherm, and subsequent data fitting, which when taken together often results in 2–15% errors in K_a. To increase the precision in K^H_a/K^D_a data for this study, we used the Perrin method of competitive titrations,²⁵ which has been shown previously to reduce errors in EIE values significantly with errors as small as 0.0004.²⁶ In this method, a linearized plot of the chemical shifts of 2^H (δ_H) and 2^D (δ_D) in fast exchange with an anionic guest is fit by linear regression to eqn (1):(δ⁰_H − δ_H)(δ_D − δ^f_D) = DEIE(δ⁰_D − δ_D)(δ_H − δ^f_H)1The slope of the linear regression is equal to the DEIE of the system. Because the linear regression only relies on chemical shift values and is independent of host and guest concentration, the precision of the method is limited to the precision of the NMR instrument and quality of data fitting.In addition, ¹³C NMR spectroscopy is sensitive to isotopic labelling and can show changes in chemical shifts between isotopomers. We were able to differentiate between the ¹³C NMR signals for C^ab, C¹ and C² for free and bound 2^H and 2^D (Fig. 2a) in 10% DMSO-d₆/CD₃CN, which were similar to those reported for 1^H/D in DMSO-d₆.¹² Competitive ¹³C NMR spectroscopy titrations were performed in anaerobic and anhydrous 10% DMSO-d₆/CD₃CN at 25 °C with mixtures of 2^H and 2^D in combined concentrations between 5.71 and 13.46 mM. Aliquots of the tetrabutylammonium (TBA) salts of HS⁻, Cl⁻, and Br⁻ were added until the system had reached saturation (Titration method A in ESI^†). In an effort to decrease reactivity of HS⁻ with 2^H/D and DMSO over long periods of time and decrease oxygen and water contaminations, some titrations with HS⁻ were performed by splitting the host solution of 2^H/D between four J-young NMR tubes. For each point in the competitive titration, TBASH was added to a new solution of 2^H/D inside an N₂-filled glovebox shortly before obtaining a ¹³C NMR spectra (Titration method B in ESI^†). The C^ab, C¹ and C^{2 13}C NMR signals were tracked for 2^H and 2^D in each titration for each anion. A representative competitive titration and linearized plots for Cl⁻ binding is shown in Fig. 2.Open in a separate windowFig. 2(a) Representation of the host–guest equilibrium between 2^H/D and Cl⁻. (b) Differences in the chemical shifts between the 2^H and 2^D isotopologues are observed in the ¹³C NMR signals for the C^ab, C¹, and C² carbons. ¹³C NMR signals for the C^ab, C¹, and C² carbons in 2^H and 2^D are tracked throughout a titration. (c–e) Linearized plots from fitting the chemical shifts of the C^ab, C¹, and C² throughout a titration to eqn (1).The DEIE data calculated from tracking the chemical shifts of the C^ab, C¹ and C^{2 13}C NMR signals from Cl⁻ and Br⁻ binding are summarized in eqn (1) through linear regression is included in parentheses

Pevonedistat and azacitidine upregulate NOXA (PMAIP1) to increase sensitivity to venetoclax in preclinical models of acute myeloid leukemia

Dysregulation of apoptotic machinery is one mechanism by which acute myeloid leukemia (AML) acquires a clonal survival advantage. B-cell lymphoma protein-2 (BCL2) overexpression is a common feature in hematologic malignancies. The selective BCL2 inhibitor, venetoclax (VEN) is used in combination with azacitidine (AZA), a DNAmethyltransferase inhibitor (DNMTi), to treat patients with AML. Despite promising response rates to VEN/AZA, resistance to the agent is common. One identified mechanism of resistance is the upregulation of myeloid cell leukemia-1 protein (MCL1). Pevonedistat (PEV), a novel agent that inhibits NEDD8-activating enzyme, and AZA both upregulate NOXA (PMAIP1), a BCL2 family protein that competes with effector molecules at the BH3 binding site of MCL1. We demonstrate that PEV/AZA combination induces NOXA to a greater degree than either PEV or AZA alone, which enhances VEN-mediated apoptosis. Herein, using AML cell lines and primary AML patient samples ex vivo, including in cells with genetic alterations linked to treatment resistance, we demonstrate robust activity of the PEV/VEN/AZA triplet. These findings were corroborated in preclinical systemic engrafted models of AML. Collectively, these results provide rational for combining PEV/VEN/AZA as a novel therapeutic approach in overcoming AML resistance in current therapies.     

Ongoing rabies outbreak in dogs of unprecedented scale and human cases in Nelson Mandela Bay Municipality,South Africa,up to 13 February 2022

More than 430 cases of rabies have been confirmed in dogs in the Nelson Mandela Bay Metropolitan Municipality of South Africa since July 2021. We describe the ongoing outbreak, its geographical spread and six related human deaths that have occurred. Further investigation of the outbreak and vaccination of the dog population is required. Raising awareness among healthcare providers, the public, and among international travellers planning to visit the region, is key for action to protect human and animal health.     

CDK13-related disorder: Report of a series of 18 previously unpublished individuals and description of an epigenetic signature

PurposeRare genetic variants in CDK13 are responsible for CDK13-related disorder (CDK13-RD), with main clinical features being developmental delay or intellectual disability, facial features, behavioral problems, congenital heart defect, and seizures. In this paper, we report 18 novel individuals with CDK13-RD and provide characterization of genome-wide DNA methylation.MethodsWe obtained clinical phenotype and neuropsychological data for 18 and 10 individuals, respectively, and compared this series with the literature. We also compared peripheral blood DNA methylation profiles in individuals with CDK13-RD, controls, and other neurodevelopmental disorders episignatures. Finally, we developed a support vector machine–based classifier distinguishing CDK13-RD and non–CDK13-RD samples.ResultsWe reported health and developmental parameters, clinical data, and neuropsychological profile of individuals with CDK13-RD. Genome-wide differential methylation analysis revealed a global hypomethylated profile in individuals with CDK13-RD in a highly sensitive and specific model that could aid in reclassifying variants of uncertain significance.ConclusionWe describe the novel features such as anxiety disorder, cryptorchidism, and disrupted sleep in CDK13-RD. We define a CDK13-RD DNA methylation episignature as a diagnostic tool and a defining functional feature of the evolving clinical presentation of this disorder. We also show overlap of the CDK13 DNA methylation profile in an individual with a functionally and clinically related CCNK-related disorder.