Chromatographic single-step purification of tagless proteins using gp41-1 split inteins.

The current trend in biopharmaceutical drug manufacturing is towards increasing potency and complexity of products such as peptide scaffolds, oligonucleotides and many more. Therefore, a universal affinity purification step is important in order to meet the requirements for cost and time efficient drug production. By using a self-splicing intein affinity tag, a purification template is generated that allows for a universal chromatographic affinity capture step to generate a tagless target protein without the use of proteases for further tag removal. This study describes the successful implementation of gp41-1-based split inteins in a chromatographic purification process for, e.g., E. coli-derived targets. The tagless target is generated in a single-step purification run. The on-column cleavage is induced by triggering a simple pH change in the buffer conditions without the need for additives such as Zn2+ or thiols. This system has proven to be reusable for at least ten purification cycles that use 150 mM H3PO4 as the cleaning agent.

Comparison of longitudinal diary and actigraphy-assessed sleep in pregnant women.

STUDY OBJECTIVES: Various methods are employed to assess sleep in pregnant women, including self-report, sleep diary, and actigraphy. Unfortunately, the data are often contradictory, and interpretations are often inconsistent. The current aims are to compare subjective and objective sleep data in pregnant women collected longitudinally in early pregnancy. METHODS: In this secondary analysis of 104 pregnant women, sleep was collected via diary and actigraphy for 14 days during three separate occasions (10-12 weeks; 14-16 weeks; and 18-20 weeks). Sleep variables included wake after sleep onset (WASO), sleep efficiency (SE), bedtime/lights out, sleep onset latency (SL), and total sleep duration (TST). Repeated measures ANOVAs compared each sleep variable across Time and by Method of data collection, while controlling for parity and daytime naps. RESULTS: Significant differences were noted for only the method of data collection for sleep component studied for WASO, F (1, 98) = 147.20, p < 0.001; SE, F (1, 98) = 129.41, p < 0.001); bedtime/lights out, F (1, 103) = 5.33, p < 0.05); and sleep duration, F (1, 104) = 182.75, p < 0.001). Significant variation was not seen in any variable across time-period. CONCLUSIONS: There are substantial discrepancies between diary- and actigraphy-assessed sleep measures in pregnant women which is in alignment with previous literature. These data highlight that these methodologies assess different constructs. We contend that these data may be useful as a reference to compare high-risk women or those with sleep disorders. Using a multi-modal approach to identify sleep disturbance in pregnancy is likely a more clinically useful option.

Proteins Marking the Sequence of Genotoxic Signaling from Irradiated Mesenchymal Stromal Cells to CD34+ Cells.

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Genotoxic Bystander Signals from Irradiated Human Mesenchymal Stromal Cells Mainly Localize in the 10-100 kDa Fraction of Conditioned Medium.

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10-100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10-100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10-100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

DNA Damage and DNA Damage Response in Chronic Myeloid Leukemia.

DNA damage and alterations in the DNA damage response (DDR) are critical sources of genetic instability that might be involved in BCR-ABL1 kinase-mediated blastic transformation of chronic myeloid leukemia (CML). Here, increased DNA damage is detected by γH2AX foci analysis in peripheral blood mononuclear cells (PBMCs) of de novo untreated chronic phase (CP)-CML patients (n = 5; 2.5 γH2AX foci per PBMC ± 0.5) and blast phase (BP)-CML patients (n = 3; 4.4 γH2AX foci per PBMC ± 0.7) as well as CP-CML patients with loss of major molecular response (MMR) (n = 5; 1.8 γH2AX foci per PBMC ± 0.4) when compared to DNA damage in PBMC of healthy donors (n = 8; 1.0 γH2AX foci per PBMC ± 0.1) and CP-CML patients in deep molecular response or MMR (n = 26; 1.0 γH2AX foci per PBMC ± 0.1). Progressive activation of erroneous non-homologous end joining (NHEJ) repair mechanisms during blastic transformation in CML is indicated by abundant co-localization of γH2AX/53BP1 foci, while a decline of the DDR is suggested by defective expression of (p-)ATM and (p-)CHK2. In summary, our data provide evidence for the accumulation of DNA damage in the course of CML and suggest ongoing DNA damage, erroneous NHEJ repair mechanisms, and alterations in the DDR as critical mediators of blastic transformation in CML.

Antileukemic Efficacy in Vitro of Talazoparib and APE1 Inhibitor III Combined with Decitabine in Myeloid Malignancies.

Malignant hematopoietic cells of myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemias (CMML) and acute myeloid leukemias (AML) may be vulnerable to inhibition of poly(ADP ribose) polymerase 1/2 (PARP1/2) and apurinic/apyrimidinic endonuclease 1 (APE1). PARP1/2 and APE1 are critical enzymes involved in single-strand break repair and base excision repair, respectively. Here, we investigated the cytotoxic efficacy of talazoparib and APE1 inhibitor III, inhibitors of PARP1/2 and APE1, in primary CD34+ MDS/CMML cell samples (n = 8; 4 MDS and 4 CMML) and in primary CD34+ or CD34- AML cell samples (n = 18) in comparison to healthy CD34+ donor cell samples (n = 8). Strikingly, talazoparib and APE1 inhibitor III demonstrated critical antileukemic efficacy in selected MDS/CMML and AML cell samples. Low doses of talazoparib and APE1 inhibitor III further increased the cytotoxic efficacy of decitabine in MDS/CMML and AML cells. Moreover, low doses of APE1 inhibitor III increased the cytotoxic efficacy of talazoparib in MDS/CMML and AML cells. In summary, talazoparib and APE1 inhibitor III demonstrated substantial antileukemic efficacy as single agents, in combination with decitabine, and combined with each other. Hence, our findings support further investigation of these agents in sophisticated clinical trials.

The short-chain fatty acid pentanoate suppresses autoimmunity by modulating the metabolic-epigenetic crosstalk in lymphocytes.

Short-chain fatty acids (SCFAs) have immunomodulatory effects, but the underlying mechanisms are not well understood. Here we show that pentanoate, a physiologically abundant SCFA, is a potent regulator of immunometabolism. Pentanoate induces IL-10 production in lymphocytes by reprogramming their metabolic activity towards elevated glucose oxidation. Mechanistically, this reprogramming is mediated by supplying additional pentanoate-originated acetyl-CoA for histone acetyltransferases, and by pentanoate-triggered enhancement of mTOR activity. In experimental mouse models of colitis and multiple sclerosis, pentanoate-induced regulatory B cells mediate protection from autoimmune pathology. Additionally, pentanoate shows a potent histone deacetylase-inhibitory activity in CD4+ T cells, thereby reducing their IL-17A production. In germ-free mice mono-colonized with segmented filamentous bacteria (SFB), pentanoate inhibits the generation of small-intestinal Th17 cells and ameliorates SFB-promoted inflammation in the central nervous system. Taken together, by enhancing IL-10 production and suppressing Th17 cells, the SCFA pentanoate might be of therapeutic relevance for inflammatory and autoimmune diseases.

Virtual Screening Identifies Irreversible FMS-like Tyrosine Kinase 3 Inhibitors with Activity toward Resistance-Conferring Mutations.

The use of covalent irreversible binding inhibitors is an established concept for drug development. Usually, the discovery of new irreversible kinase inhibitors occurs serendipitously, showing that efficient rational approaches for the rapid discovery of new drugs are needed. Herein, we report a virtual screening strategy that led to the discovery of irreversible inhibitors of FMS-like tyrosine kinase 3 (FLT3) involved in the pathogenesis of acute myeloid leukemia. A virtual screening library was designed to target the highly conserved Cys828 residue preceding the DFG motif by modification of reported reversible inhibitors with chemically reactive groups. Prospective covalent docking allowed the identification of two lead series, resulting in a massive increase in inhibition of kinase activity and cell viability by irreversible inhibitors compared to the corresponding reversible scaffolds. Lead compound 4b (BSc5371) displays superior cytotoxicity in FLT3-dependent cell lines to compounds in recent clinical trials and overcomes drug-resistant mutations.

The global burden of road injury: its relevance to the emergency physician.

Background. Road traffic crash fatalities in the United States are at the lowest level since 1950. The reduction in crash injury burden is attributed to several factors: public education and prevention programs, traffic safety policies and enforcement, improvements in vehicle design, and prehospital services coupled with emergency and acute trauma care. Globally, the disease burden of road traffic injuries is rising. In 1990, road traffic injuries ranked ninth in the ten leading causes of the global burden of disease. By 2030, estimates show that road traffic injuries will be the fifth leading causes of death in the world. Historically, emergency medicine has played a pivotal role in contributing to the success of the local, regional, and national traffic safety activities focused on crash and injury prevention. Objective. We report on the projected trend of the global burden of road traffic injuries and fatalities and describe ongoing global initiatives to reduce road traffic morbidity and mortality. Discussion. We present key domains where emergency medicine can contribute through international collaboration to address global road traffic-related morbidity and mortality. Conclusion. International collaborative programs and research offer important opportunities for emergency medicine physicians to make a meaningful impact on the global burden of disease.

Crash injury prediction and vehicle damage reporting by paramedics.

OBJECTIVE: The accuracy of pre-hospital crash scene details and crash victim assessment has important implications for initial trauma care assessment and management. Similarly, it is known to influence physician perception of crash victim injury severity. The goal of this feasibility study was to examine paramedic accuracy in predicting crash victim injury profile, disability outcome at hospital discharge, and reporting vehicle damage with other crash variables. METHODS: This prospective case series study was undertaken at a Southern California, Level I trauma center certified by the American College of Surgeons. Paramedics transporting crash injured motor vehicle occupants to our emergency department (ED)/trauma center were surveyed. We abstracted ED and in-patient records of injured vehicle occupants. Vehicle and crash scene data were obtained from a professional crash reconstruction, which included the assessment of deformation, crash forces, change in velocity, and the source of each injury. RESULTS: We used survey, injury, and crash reconstruction data from 22 collision cases in the final analysis. The median Injury Severity Score (ISS) was five (range 1-24). No enrolled patients died, and none were severely disabled at the time of discharge from the hospital. The paramedic crash injury severity predictions were sensitive for an Abbreviated Injury Scale (AIS) of 2-4. Paramedics often agreed with the crash reconstruction on restraint use, ejection, and other fatalities at the scene, and had lower levels of agreement for front airbag deployment, steering wheel damage, and window/windshield impact. Paramedics had 80% accuracy in predicting any disability at the time of hospital discharge. CONCLUSION: Paramedic prediction of injury profile was sensitive, and prediction of disability outcome at discharge was accurate when compared to discharge diagnosis. Their reporting of vehicle specific crash variables was less accurate. Further study should be undertaken to assess the benefits of crash biomechanics education for paramedics and other pre-hospital care providers.