Brief Report: Quantitative detection of hydrogen peroxide in rain,air, exhaled breath,and biological fluids by NMR spectroscopy

Hydrogen peroxide (H₂O₂) plays a key role in environmental chemistry, biology, and medicine. H₂O₂ concentrations typically are 6 to 10 orders of magnitude lower than that of water, making its quantitative detection challenging. We demonstrate that optimized NMR spectroscopy allows direct, interference-free, quantitative measurements of H₂O₂ down to submicromolar levels in a wide range of fluids, ranging from exhaled breath and air condensate to rain, blood, urine, and saliva. NMR measurements confirm the previously reported spontaneous generation of H₂O₂ in microdroplets that form when condensing water vapor on a hydrophobic surface, which can interfere with atmospheric H₂O₂ measurements. Its antimicrobial activity and strong seasonal variation speculatively could be linked to the seasonality of respiratory viral diseases.     

Serum Vitamin D Level among Multiple Sclerosis Patients in the Tropics: Experience from a Private Clinic in Addis Ababa,Ethiopia

BackgroundMultiple sclerosis (MS) is an immune mediated disabling neurological disorder. Very little is known about MS in Ethiopia. The objective of this study was to determine the prevalence of hypovitaminosis D and associated factors in cohort of MS patients in Ethiopia.MethodA cross-sectional observational study was conducted among 25 multiple sclerosis patients at Yehuleshet Specialty Clinic, Addis Ababa, Ethiopia.ResultsThe prevalence of vitamin D deficiency was 96% (n=24). The average serum vitamin D was 14.8 (±10.4) ng/mL. The mean age was 35.8 (±10) years. Females accounted for 80% (n=20). Relapsing and remitting MS was the commonest variant. Motor, sensory, and mixed symptoms accounted for 40% (n=10), 20% (n=5), and 24% (n=6), respectively. Cold or hot weather and stress were reported as worsening factors in 24% (n=6). Relapse rate was 44% (n=11). Fatigue and seizure disorder were reported by 80% (n=20) and 16% (n=4) respectively. Steroid is the commonest prescribed medication for the patients. A negative correlation was found between serum vitamin D and age (r = -0.062, p = 0.7). Similarly, a negative association was observed between vitamin D and duration of illness (r = -0.311, p = 0.1). Fatigue was reported by those with moderate hypovitaminosis compared to those having severe hypovitaminosis (p=0.002). Seizure was reported more by those with vitamin D below 10ng/mL compared to those having above 10 ng/mL (p=0.004).ConclusionOur study demonstrates a high prevalence of hypovitaminosis D in Ethiopian MS patients. Hypovitaminosis D was associated with increment in age and duration of illness.     

The role of self-help voluntary associations for women empowerment and social capital: the experience of women's iddirs (burial societies) in Ethiopia

Mutual help is essential to the human race to progress to a full humanity. Participating in voluntary associations has multiple benefits and makes a bigger contribution to resiliency in socio-economically deprived neighborhoods. Therefore, the objective of the study is to assess the role of women’s iddirs (burial societies) in promoting their lives, particularly in terms of their social and economic well-being. We employed a quantitative cross-sectional survey. The study population from which the survey data were collected was members of five women’s iddirs’. Majority of them (52.5%) reported that their iddirs helped them to a great extent to become more connected with the people in their local community and 36.4% reported that their confidence has increased to have their own say. Nearly three-fourth (70.3%) of the participants indicated that members were willing to help each other. About 71.6% totally agreed that participation in iddirs alleviates stress and difficulty when a relative or family member dies. Majority of the participants (93.3%) stated that their iddirs didn’t help them to have access to money. All participants (100%) reported that their iddirs don’t have explicitly stated credit mechanisms and nearly all (94.8%) didn’t have any experience of borrowing money from banks. Most study participants appeared to appreciate the social benefits of iddirs much higher than that of their economic benefits. This suggests that iddirs need to be well-organized and co-operate with each other and ensuring flexible lending and interest payment criteria need to be established logistically to serve the needs of the poor members.     

In vitro cultivation of an African strain of Babesia bigemina,its characterisation and infectivity in cattle

An African (Kenyan) strain of Babesia bigemina, Muguga (B_2-1), was inoculated into a calf from a stabilate and blood from the calf was used to establish the parasite in vitro. The strain has been cultured continuously for 20 months, initially in bovine erythrocytes with 60% adult bovine serum, later, with 50%. Cultures were incubated at 37 °C in RPMI 1640 medium with a gas mixture of 1% O₂, 5% CO₂, 94% N₂. Adaptation in vitro was demonstrated when serum from a calf which had recovered from infection with B_2-1 bound to proteins of M_r 46 kDa, 49 kDa, 52 kDa, 61 kDa and 72 kDa on Western blots of B_2-1 antigens from cattle blood but did not recognise the 49 kDa or 52 kDa antigens from in-vitro-derived parasites. These proteins were considered specific for B_2-1, as they were not recognised by the same serum on profiles of a Mexican isolate of B. bigemina or an African isolate of B. bovis (Kwanyange). After 9 months of in vitro culture, a stabilate of the cultured parasite was injected into two splenectomised calves and one intact calf. The calves experienced a drop in packed cell volume and low parasitaemias but recovered spontaneously. Two of these animals, one splenectomised and one intact, were challenged with virulent B_2-1 and experienced only mild babesiosis, in contrast to a previously uninfected calf also challenged with B_2-1, which had to be euthanised after 5 days with severe babesiosis. Received: 24 February 1997 / Accepted: 5 August 1997     

Phenotypic screening of the ReFRAME drug repurposing library to discover new drugs for treating sickle cell disease

Stem cell transplantation and genetic therapies offer potential cures for patients with sickle cell disease (SCD), but these options require advanced medical facilities and are expensive. Consequently, these treatments will not be available for many years to the majority of patients suffering from this disease. What is urgently needed now is an inexpensive oral drug in addition to hydroxyurea, the only drug approved by the FDA that inhibits sickle-hemoglobin polymerization. Here, we report the results of the first phase of our phenotypic screen of the 12,657 compounds of the Scripps ReFRAME drug repurposing library using a recently developed high-throughput assay to measure sickling times following deoxygenation to 0% oxygen of red cells from sickle trait individuals. The ReFRAME library is a very important collection because the compounds are either FDA-approved drugs or have been tested in clinical trials. From dose-response measurements, 106 of the 12,657 compounds exhibit statistically significant antisickling at concentrations ranging from 31 nM to 10 μM. Compounds that inhibit sickling of trait cells are also effective with SCD cells. As many as 21 of the 106 antisickling compounds emerge as potential drugs. This estimate is based on a comparison of inhibitory concentrations with free concentrations of oral drugs in human serum. Moreover, the expected therapeutic potential for each level of inhibition can be predicted from measurements of sickling times for cells from individuals with sickle syndromes of varying severity. Our results should motivate others to develop one or more of these 106 compounds into drugs for treating SCD.

An abnormal hemoglobin (HbS) causes sickle cell disease (SCD), the first example of a “molecular disease” reported by the legendary chemist, Linus Pauling, in 1949 (1, 2). Pauling’s discovery gave birth to the field of molecular medicine. This monogenic disease results from a single nucleotide change from A to T in the codon (GAG) of the gene for the Hb beta chain. The mutation results in the replacement of a negatively charged glutamate with a neutral, hydrophobic valine on the molecular surface, which creates a sticky patch that promotes polymerization of HbS upon deoxygenation to form fibers (3, 4). HbS polymerization in the tissues is the root cause of SCD pathology because the fibers stiffen and distort (“sickle”) red cells. Sickled cells can cause vaso-occlusion in the smallest vessels of almost every tissue (5, 6), resulting in intermittent episodes of acute clinical events, the most prominent of which is pain that is so severe that it is commonly referred to as a sickle cell crisis (5, 6). In addition, the irreversible structural changes in the red cell cytoskeleton associated with sickling in the tissues and unsickling in the lungs cause red cell fragility and consequent hemolysis that underlies the chronic hemolytic anemia. The two hallmarks of recurrent vaso-occlusion and chronic hemolysis trigger and sustain a continuing background of inflammatory response leading to vasculopathy and acute and chronic damage to the brain, lungs, kidney, liver, and others. Inhibition of HbS polymerization is therefore the most rational drug treatment for the disease (7).Hydroxyurea (HU), approved by the Food and Drug Administration (FDA) in 1998, is currently the only successful antisickling drug (8)^*, but its use is limited by its safety profile, which includes myelosuppression and thrombocytopenia. The efficacy of HU is due to its effect on various aspects of sickle pathophysiology; induction of fetal Hb (HbF), which inhibits HbS polymerization, is considered the most important. However, individual responses to HbF are variable based on clinical response and changes in hematological parameters (12, 13), and the increase in HbF is not evenly distributed in all cells (7, 14). Consequently, it is only partially effective (7, 14, 15). Thus, additional inexpensive oral drugs that inhibit sickling in all cells are urgently needed because the majority of patients in the world do not have, and for many years will not have, the necessary advanced medical facilities and resources required for curative therapies such as hematopoietic stem cell transplantation or gene therapy (16, 17). Now that large compound libraries are available, basic research scientists can engage in drug development using screening assays that are pathophysiologically relevant. We have recently developed a relevant high-throughput assay (18) to screen the Scripps California Institute of Biomedical Research (Calibr) ReFRAME drug repurposing library. This library is a unique and very important collection because the compounds are either FDA-approved drugs for other indications or have been tested in clinical trials with extensive drug annotation in an open-access format (reframedb.org) (19). Consequently, compounds that inhibit sickling at serum concentrations found in humans, without known side effects that would be deleterious to SCD patients, can be rapidly brought to clinical trial without extensive preclinical studies that markedly slow drug development. Since there is no specific molecular target, this approach is referred to as phenotypic drug discovery (20). In this work, we present the results so far from screening the 12,657 compounds of the Calibr library using our sickling assay (18).Our previous screening assay used a 96-well plate format and laser photodissociation of carbon monoxide (CO) saturated sickle trait cells to rapidly create deoxyHbS in <1 s (21). The assay is extremely sensitive because it determines the delay time before sickle fiber formation for each cell, but it is low throughput and requires the use of sodium dithionite, a very strong reducing agent that could react with the test compound (9, 21). We were therefore motivated to develop a high-throughput assay under more physiological conditions without dithionite. Our current assay uses a 384-well plate format and is based on deoxygenation of sickle trait cells with nitrogen to 0% oxygen and acquisition of images of 100 to 300 cells in each well every 15 min using an Agilent “Lionheart” automated microscope system (18). With one instrument, up to ∼1,300 compounds at a single concentration can be tested in 1 week, making our screen relatively high throughput; we now have four such instruments. As in the previous CO photolysis assay, the time at which individual cells change shape (sickle) is determined using automated image algorithms for characterizing red cell morphology, refined by machine learning (18).There are many advantages to using cells from donors with sickle trait, the heterozygous condition (21). The delay time for polymerization is ∼1,000-fold longer for trait cells than for SCD cells because of the much lower fraction of HbS in trait cells (35 to 40% HbS in trait cells compared to 85 to 95% in SCD cells) (22, 23). With longer delay times, there are many fewer primary nucleation events, resulting in only a few domains of fibers (24, 25). Consequently, cellular distortion is much greater (26), making the determination of sickling times more accurate. In addition, trait cells are less damaged and therefore are a much more homogeneous cell population than SCD cells (6, 27). There are many more trait blood donors, who very often have relatives with SCD and are therefore willing to donate as frequently as requested. While polymerization in trait cells is much slower, there is no evidence that the mechanism of sickle fiber formation is different (25). Nevertheless, it is important that we demonstrate that inhibitors of trait cell sickling also inhibit the sickling of SCD cells.There is every reason to expect that new drugs will be discovered from our screen because it tests for all but one of the five independent approaches that have been proposed to inhibit sickling as therapy (7). These four classes of inhibitory mechanisms, discussed in detail in reference 7, are (1) blocking intermolecular contacts in the sickle fiber; (2) destabilizing the fiber by decreasing 2,3-diphosphoglycerate (28, 29), with added destabilization resulting from the concomitant increase in intracellular pH (30–32); (3) decreasing the intracellular hemoglobin concentration, for example, by swelling red cells (21, 33); and (iv) preferentially shifting the R-T quaternary equilibrium toward the nonpolymerizing R conformation with a drug that both binds and dissociates very rapidly from the R quaternary conformation (9, 25, 34, 35). Since the assay measures the inhibition of mature cells in peripheral blood, it does not test for compounds that induce HbF synthesis in stem cells (14, 36–40). To assess this fifth independent approach, we make considerable use of the assay to measure the sickling of blood from individuals with SCD and sickle trait on various protocols and from patients who have been treated by using a virus to add globin genes to their stem cells for the nonpolymerizing HbF mimicking mutant, HbA(T87Q) (41).     

Synthesis of natural product-inspired inhibitors of Mycobacterium tuberculosis mycothiol-associated enzymes: the first inhibitors of GlcNAc-Ins deacetylase

Synthesis and evaluation of a chemical library of inhibitors of the mycothiol biosynthesis enzyme GlcNAc-Ins deacetylase (MshB) and the mycothiol-dependent detoxification enzyme mycothiol- S-conjugate amidase (MCA) from Mycobacterium tuberculosis are reported. The library was biased to include structural features of a group of natural products previously shown to competitively inhibit MCA. Molecular docking studies that reproducibly placed the inhibitors in the active site of the enzyme MshB reveal the mode of binding and are consistent with observed biological activity.     

Evaluation of the tubulin-bound paclitaxel conformation: synthesis, biology, and SAR studies of C-4 to C-3' bridged paclitaxel analogues

The important anticancer drug paclitaxel binds to the beta-subunit of the alphabeta-tubulin dimer in the microtubule in a stoichiometric ratio, promoting microtubule polymerization and stability. The conformation of microtubule-bound drug has been the subject of intense study, and various suggestions have been proposed. In previous work we presented experimental and theoretical evidence that paclitaxel adopts a T-shaped conformation when it is bound to tubulin. In this study we report additional experimental data and calculations that delineate the allowable parameters for effective paclitaxel-tubulin interactions.