Coupling DNA-binding and ATP hydrolysis in Escherichia coli RecQ: role of a highly conserved aromatic-rich sequence

RecQ enzymes are broadly conserved Superfamily-2 (SF-2) DNA helicases that play critical roles in DNA metabolism. RecQ proteins use the energy of ATP hydrolysis to drive DNA unwinding; however, the mechanisms by which RecQ links ATPase activity to DNA-binding/unwinding are unknown. In many Superfamily-1 (SF-1) DNA helicases, helicase sequence motif III links these activities by binding both single-stranded (ss) DNA and ATP. However, the ssDNA-binding aromatic-rich element in motif III present in these enzymes is missing from SF-2 helicases, raising the question of how these enzymes link ATP hydrolysis to DNA-binding/unwinding. We show that Escherichia coli RecQ contains a conserved aromatic-rich loop in its helicase domain between motifs II and III. Although placement of the RecQ aromatic-rich loop is topologically distinct relative to the SF-1 enzymes, both loops map to similar tertiary structural positions. We examined the functions of the E.coli RecQ aromatic-rich loop using RecQ variants with single amino acid substitutions within the segment. Our results indicate that the aromatic-rich loop in RecQ is critical for coupling ATPase and DNA-binding/unwinding activities. Our studies also suggest that RecQ's aromatic-rich loop might couple ATP hydrolysis to DNA-binding in a mechanistically distinct manner from SF-1 helicases

On Estimating the High-Energy Cutoff in the X-ray Spectra of Black Holes via Reflection Spectroscopy

The fundamental parameters describing the coronal spectrum of an accreting black hole are the slope $\Gamma$ of the power-law continuum and the energy $E_{cut}$ at which it rolls over. Remarkably, this parameter can be accurately measured for values as high as 1 MeV by modeling the spectrum of X-rays reflected from a black hole accretion disk at energies below 100 keV. This is possible because the details in the reflection spectrum, rich in fluorescent lines and other atomic features, are very sensitive to the spectral shape of the hardest coronal radiation illuminating the disk. We show that fitting simultaneous NuSTAR (3-79 keV) and low-energy (e.g., Suzaku) data with the most recent version of our reflection model RELXILL, one can obtain reasonable constraints on $E_{cut}$ at energies from tens of keV up to 1 MeV, for a source as faint as 1 mCrab in a 100 ks observation.Comment: Accepted for publication in ApJL, 6 pages, 5 figure

Molecular insights into the prototypical single-stranded DNA-binding protein from E. coli

The SSB protein o

Binding Mechanism of Metal⋅NTP Substrates and Stringent-Response Alarmones to Bacterial DnaG-Type Primases

SummaryPrimases are DNA-dependent RNA polymerases found in all cellular organisms. In bacteria, primer synthesis is carried out by DnaG, an essential enzyme that serves as a key component of DNA replication initiation, progression, and restart. How DnaG associates with nucleotide substrates and how certain naturally prevalent nucleotide analogs impair DnaG function are unknown. We have examined one of the earliest stages in primer synthesis and its control by solving crystal structures of the S. aureus DnaG catalytic core bound to metal ion cofactors and either individual nucleoside triphosphates or the nucleotidyl alarmones, pppGpp and ppGpp. These structures, together with both biochemical analyses and comparative studies of enzymes that use the same catalytic fold as DnaG, pinpoint the predominant nucleotide-binding site of DnaG and explain how the induction of the stringent response in bacteria interferes with primer synthesis

Assessing and Responding to Palliative Care Needs in Rural Sub-Saharan Africa: Results from a Model Intervention and Situation Analysis in Malawi

Introduction: Palliative care is rarely accessible in rural sub-Saharan Africa. Partners In Health and the Malawi government established the Neno Palliative Care Program (NPCP) to provide palliative care in rural Neno district. We conducted a situation analysis to evaluate early NPCP outcomes and better understand palliative care needs, knowledge, and preferences. Methods: Employing rapid evaluation methodology, we collected data from 3 sources: 1) chart review of all adult patients from the NPCP’s first 9 months; 2) structured interviews with patients and caregivers; 3) semi-structured interviews with key stakeholders. Results: The NPCP enrolled 63 patients in its first 9 months. Frequent diagnoses were cancer (n = 50, 79%) and HIV/AIDS (n = 37 of 61, 61%). Nearly all (n = 31, 84%) patients with HIV/AIDS were on antiretroviral therapy. Providers registered 112 patient encounters, including 22 (20%) home visits. Most (n = 43, 68%) patients had documented pain at baseline, of whom 23 (53%) were treated with morphine. A majority (n = 35, 56%) had ≥1 follow-up encounter. Mean African Palliative Outcome Scale pain score decreased non-significantly between baseline and follow-up (3.0 vs. 2.7, p = 0.5) for patients with baseline pain and complete pain assessment documentation. Providers referred 48 (76%) patients for psychosocial services, including community health worker support, socioeconomic assistance, or both. We interviewed 36 patients referred to the NPCP after the chart review period. Most had cancer (n = 19, 53%) or HIV/AIDS (n = 10, 28%). Patients frequently reported needing income (n = 24, 67%) or food (n = 22, 61%). Stakeholders cited a need to make integrated palliative care widely available. Conclusions: We identified a high prevalence of pain and psychosocial needs among patients with serious chronic illnesses in rural Malawi. Early NPCP results suggest that comprehensive palliative care can be provided in rural Africa by integrating disease-modifying treatment and palliative care, linking hospital, clinic, and home-based services, and providing psychosocial support that includes socioeconomic assistance

Simultaneous evaluation of treatment efficacy and toxicity for bispecific T-cell engager therapeutics in a humanized mouse model.

Immuno-oncology (IO)-based therapies such as checkpoint inhibitors, bi-specific antibodies, and CAR-T-cell therapies have shown significant success in the treat- ment of several cancer indications. However, these therapies can result in the de- velopment of severe adverse events, including cytokine release syndrome (CRS). Currently, there is a paucity of in vivo models that can evaluate dose-response relationships for both tumor control and CRS-related safety issues. We tested an in vivo PBMC humanized mouse model to assess both treatment efficacy against specific tumors and the concurrent cytokine release profiles for individual human donors after treatment with a CD19xCD3 bispecific T-cell engager (BiTE). Using this model, we evaluated tumor burden, T-cell activation, and cytokine release in response to bispecific T-cell-engaging antibody in humanized mice generated with different PBMC donors. The results show that PBMC engrafted NOD-scid Il2rgnull mice lacking expression of mouse MHC class I and II (NSG-MHC-DKO mice) and implanted with a tumor xenograft predict both efficacy for tumor control by CD19xCD3 BiTE and stimulated cytokine release. Moreover, our find- ings indicate that this PBMC-engrafted model captures variability among donors for tumor control and cytokine release following treatment. Tumor control and cytokine release were reproducible for the same PBMC donor in separate experi- ments. The PBMC humanized mouse model described here is a sensitive and re- producible platform that identifies specific patient/cancer/therapy combinations for treatment efficacy and development of complications

Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy.

Establishment of an in vivo small animal model of human tumor and human immune system interaction would enable preclinical investigations into the mechanisms underlying cancer immunotherapy. To this end, nonobese diabetic (NOD).Cg- PrkdcscidIL2rgtm1Wjl/Sz (null; NSG) mice were transplanted with human (h)CD34+ hematopoietic progenitor and stem cells, which leads to the development of human hematopoietic and immune systems [humanized NSG (HuNSG)]. HuNSG mice received human leukocyte antigen partially matched tumor implants from patient-derived xenografts [PDX; non-small cell lung cancer (NSCLC), sarcoma, bladder cancer, and triple-negative breast cancer (TNBC)] or from a TNBC cell line-derived xenograft (CDX). Tumor growth curves were similar in HuNSG compared with nonhuman immune-engrafted NSG mice. Treatment with pembrolizumab, which targets programmed cell death protein 1, produced significant growth inhibition in both CDX and PDX tumors in HuNSG but not in NSG mice. Finally, inhibition of tumor growth was dependent on hCD8+ T cells, as demonstrated by antibody-mediated depletion. Thus, tumor-bearing HuNSG mice may represent an important, new model for preclinical immunotherapy research. FASEB J 2018 Mar; 32(3):1537-1549

Structure and function of the regulatory C-terminal HRDC domain from Deinococcus radiodurans RecQ

RecQ helicases are critical for maintaining genome integrity in organisms ranging from bacteria to humans by participating in a complex network of DNA metabolic pathways. Their diverse cellular functions require specialization and coordination of multiple protein domains that integrate catalytic functions with DNA–protein and protein–protein interactions. The RecQ helicase from Deinococcus radiodurans (DrRecQ) is unusual among RecQ family members in that it has evolved to utilize three ‘Helicase and RNaseD C-terminal’ (HRDC) domains to regulate its activity. In this report, we describe the high-resolution structure of the C-terminal-most HRDC domain of DrRecQ. The structure reveals unusual electrostatic surface features that distinguish it from other HRDC domains. Mutation of individual residues in these regions affects the DNA binding affinity of DrRecQ and its ability to unwind a partial duplex DNA substrate. Taken together, the results suggest the unusual electrostatic surface features of the DrRecQ HRDC domain may be important for inter-domain interactions that regulate structure-specific DNA binding and help direct DrRecQ to specific recombination/repair sites

Phase 1-2a multicenter dose-escalation study of ezatiostat hydrochloride liposomes for injection (Telintra®, TLK199), a novel glutathione analog prodrug in patients with myelodysplastic syndrome

<p>Abstract</p> <p>Background</p> <p>Ezatiostat hydrochloride liposomes for injection, a glutathione S-transferase P1-1 inhibitor, was evaluated in myelodysplastic syndrome (MDS). The objectives were to determine the safety, pharmacokinetics, and hematologic improvement (HI) rate. Phase 1-2a testing of ezatiostat for the treatment of MDS was conducted in a multidose-escalation, multicenter study. Phase 1 patients received ezatiostat at 5 dose levels (50, 100, 200, 400 and 600 mg/m²) intravenously (IV) on days 1 to 5 of a 14-day cycle until MDS progression or unacceptable toxicity. In phase 2, ezatiostat was administered on 2 dose schedules: 600 mg/m²IV on days 1 to 5 or days 1 to 3 of a 21-day treatment cycle.</p> <p>Results</p> <p>54 patients with histologically confirmed MDS were enrolled. The most common adverse events were grade 1 or 2, respectively, chills (11%, 9%), back pain (15%, 2%), flushing (19%, 0%), nausea (15%, 0%), bone pain (6%, 6%), fatigue (0%, 13%), extremity pain (7%, 4%), dyspnea (9%, 4%), and diarrhea (7%, 4%) related to acute infusional hypersensitivity reactions. The concentration of the primary active metabolites increased proportionate to ezatiostat dosage. Trilineage responses were observed in 4 of 16 patients (25%) with trilineage cytopenia. Hematologic Improvement-Erythroid (HI-E) was observed in 9 of 38 patients (24%), HI-Neutrophil in 11 of 26 patients (42%) and HI-Platelet in 12 of 24 patients (50%). These responses were accompanied by improvement in clinical symptoms and reductions in transfusion requirements. Improvement in bone marrow maturation and cellularity was also observed.</p> <p>Conclusion</p> <p>Phase 2 studies of ezatiostat hydrochloride liposomes for injection in MDS are supported by the tolerability and HI responses observed. An oral formulation of ezatiostat hydrochloride tablets is also in phase 2 clinical development.</p> <p>Trial Registration</p> <p>Clinicaltrials.gov: NCT00035867</p