A cryptic cycle in haematopoietic niches promotes initiation of malaria transmission and evasion of chemotherapy

Blood stage human malaria parasites may exploit erythropoietic tissue niches and colonise erythroid progenitors; however, the precise influence of the erythropoietic environment on fundamental parasite biology remains unknown. Here we use quantitative approaches to enumerate Plasmodium infected erythropoietic precursor cells using an in vivo rodent model of Plasmodium berghei. We show that parasitised early reticulocytes (ER) in the major sites of haematopoiesis establish a cryptic asexual cycle. Moreover, this cycle is characterised by early preferential commitment to gametocytogenesis, which occurs in sufficient numbers to generate almost all of the initial population of circulating, mature gametocytes. In addition, we show that P. berghei is less sensitive to artemisinin in splenic ER than in blood, which suggests that haematopoietic tissues may enable origins of recrudescent infection and emerging resistance to antimalarials. Continuous propagation in these sites may also provide a mechanism for continuous transmission and infection in malaria endemic regions

Origin of the transient unpulsed radio emission from the PSR B1259-63 binary system

We discuss the interpretation of transient, unpulsed radio emission detected from the unique pulsar/Be-star binary system PSR B1259-63. Extensive monitoring of the 1994 and 1997 periastron passages has shown that the source flares over a 100-day interval around periastron, varying on time-scales as short as a day and peaking at 60 mJy (~100 times the apastron flux density) at 1.4 GHz. Interpreting the emission as synchrotron radiation, we show that (i) the observed variations in flux density are too large to be caused by the shock interaction between the pulsar wind and an isotropic, radiatively driven, Be-star wind, and (ii) the radio emitting electrons do not originate from the pulsar wind. We argue instead that the radio electrons originate from the circumstellar disk of the Be star and are accelerated at two epochs, one before and one after periastron, when the pulsar passes through the disk. A simple model incorporating two epochs of impulsive acceleration followed by synchrotron cooling reproduces the essential features of the radio light curve and spectrum and is consistent with the system geometry inferred from pulsed radio data.Comment: To be published in Astrophysical Journal Letters 7 pages, 1 postscript figur

Single Synonymous Mutations in KRAS Cause Transformed Phenotypes in NIH3T3 Cells

Synonymous mutations in the KRAS gene are clustered at G12, G13, and G60 in human cancers. We constructed 9 stable NIH3T3 cell lines expressing KRAS, each with one of these synonymous mutations. Compared to the negative control cell line expressing the wild type human KRAS gene, all the synonymous mutant lines expressed more KRAS protein, grew more rapidly and to higher densities, and were more invasive in multiple assays. Three of the cell lines showed dramatic loss of contact inhibition, were more refractile under phase contrast, and their refractility was greatly reduced by treatment with trametinib. Codon usage at these glycines is highly conserved in KRAS compared to HRAS, indicating selective pressure. These transformed phenotypes suggest that synonymous mutations found in driver genes such as KRAS may play a role in human cancers

Phenylcyanamidocopper(I) and Silver(I) Complexes: Synthetic and Structural Studies

Phenylcyanamidocopper(I) and silver(I) complexes of the type, [{M(PPh3)2L}2] (M = Cu, L = 4-NO2pcyd or 4-Me2Npcyd; M = Ag, L = 4-Me2Npcyd), [Cu(PPh3)3L] (L = pcyd or 4-NO2-pcyd), [Ag-(PPh3)3L] (L = pcyd, 2-Clpcyd, 4-Clpcyd, 4-Brpcyd, 4-MeOpcyd, 4-NO2pcyd or 4-Me2Npcyd), [Ag(Me2phen)(2-Clpcyd)] (Me2phen = 2,9-dimethyl-1,10-phenanthroline) and [Ag(dppm)(4-Brpcyd)] (dppm = bis(diphenylphosphino)methane) have been synthesised and characterised and the crystal structures of four of the complexes determined. For both [{Cu(PPh3)2(4-Me2Npcyd)}2] ⋅ CH2Cl2 and [{Ag(PPh3)2(4-Me2Npcyd)}2], the cyanamide ligands bridge the metal atoms in a μ-1,3-fashion through the cyano and amido nitrogens. Each metal atom has a distorted tetrahedral geometry, being bound to two triphenylphosphine phosphorus atoms and two nitro-gen atoms from 4-Me2Npcyd ligands to give a \u27P2N2\u27 coordination sphere. In the case of the Cu complex the dimer is centrosymmetric but for the Ag complex the metal atoms are not equivalent. The complexes, [Ag(PPh3)3(4-Brpcyd)] and [Ag(PPh3)3(4-Me-Opcyd)], are discrete monomers, in which each of the Ag atoms adopts a distorted tetrahedral geometry, being bound to three triphenylphosphine phosphorus atoms and one phenylcyanamide ligand binding in a terminal fashion through the cyano nitrogen

Development and application of a positive–negative selectable marker system for use in reverse genetics in Plasmodium

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as ‘Hit and Run’ and ‘vector recycling’ which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems

4U2206+54 - an Unusual High Mass X-ray Binary with a 9.6 Day Orbital Period but No Strong Pulsations

Rossi X-ray Timing Explorer All-Sky Monitor observations of the X-ray source 4U2206+54, previously proposed to be a Be star system, show the X-ray flux to be modulated with a period of approximately 9.6 days. If the modulation is due to orbital variability then this would be one of the shortest orbital periods known for a Be star X-ray source. However, the X-ray luminosity is relatively modest whereas a high luminosity would be predicted if the system contains a neutron star accreting from the denser inner regions of a Be star envelope. Although a 392s pulse period was previously reported from EXOSAT observations, a reexamination of the EXOSAT light curves does not show this or any other periodicity. An analysis of archival RXTE Proportional Counter Array observations also fails to show any X-ray pulsations. We consider possible models that may explain the properties of this source including a neutron star with accretion halted at the magnetosphere and an accreting white dwarf.Comment: Accepted for publication in the Astrophysical Journa

Evidence for a very slow X-ray pulsar in 2S0114+650 from RXTE All-Sky Monitor Observations

Rossi X-ray Timing Explorer (RXTE) All-Sky Monitor (ASM) observations of the X-ray binary 2S0114+650 show modulations at periods close to both the optically derived orbital period (11.591 days) and proposed pulse period (~ 2.7 hr). The pulse period shows frequency and intensity variability during the more than 2 years of ASM observations analyzed. The pulse properties are consistent with this arising from accretion onto a rotating neutron star and this would be the slowest such period known. The shape of the orbital light curve shows modulation over the course of the entire orbit and a comparison is made with the orbital light curve of Vela X-1. However, the expected phase of eclipse, based on an extrapolation of the optical ephemeris, does not correspond with the observed orbital minimum. The orbital period derived from the ASM light curve is also slightly longer than the optical period.Comment: To be published in the Astrophysical Journal, 1999, volume 511. 9 figure

Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host

Background: The Plasmodium Cysteine Repeat Modular Proteins (PCRMP) are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse. Methods: In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants,.pcrmp3 and.pcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Delta pcrmp3 and Delta pcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine. Results: Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Delta crmp3 and Delta crmp4 sporozoites does not confer protective immunity upon subsequent challenge. Conclusions: PCRMP3 and 4 play multiple roles during the Plasmodium life cycle; they are essential for the establishment of sporozoite infection in the mosquito salivary gland, and subsequently for development in hepatocytes. However, although Delta pcrmp3 and Delta pcrmp4 parasites are completely growth-impaired in the liver, immunization with live sporozoites does not induce the protective immune responses that have been shown for other genetically-attenuated parasites.Host-parasite interactio

An examination of health care utilization during the COVID-19 pandemic among women with early-stage hormone receptor-positive breast cancer

Background: Women undergoing treatment for breast cancer require frequent clinic visits for maintenance of therapy. With COVID-19 causing health care disruptions, it is important to learn about how this population’s access to health care has changed. This study compares self-reported health care utilization and changes in factors related to health care access among women treated at a cancer center in the mid-South US before and during the pandemic. Methods: Participants (N = 306) part of a longitudinal study to improve adjuvant endocrine therapy (AET) adherence completed pre-intervention baseline surveys about their health care utilization prior to AET initiation. Questions about the impact of COVID-19 were added after the pandemic started assessing financial loss and factors related to care. Participants were categorized into three time periods based on the survey completion date: (1) pre-COVID (December 2018 to March 2020), (2) early COVID (April 2020 – December 2020), and later COVID (January 2021 to June 2021). Negative binomial regression analyses used to compare health care utilization at different phases of the pandemic controlling for patient characteristics. Results: Adjusted analyses indicated office visits declined from pre-COVID, with an adjusted average of 17.7 visits, to 12.1 visits during the early COVID period (p = 0.01) and 9.9 visits during the later COVID period (p < 0.01). Hospitalizations declined from an adjusted average 0.45 admissions during early COVID to 0.21 during later COVID, after vaccines became available (p = 0.05). Among COVID period participants, the proportion reporting changes/gaps in health insurance coverage increased from 9.5% participants during early-COVID to 14.8% in the later-COVID period (p = 0.05). The proportion reporting financial loss due to the pandemic was similar during both COVID periods (34.3% early- and 37.7% later-COVID, p = 0.72). The proportion of participants reporting delaying care or refilling prescriptions decreased from 15.2% in early-COVID to 4.9% in the later-COVID period (p = 0.04). Conclusion: COVID-19 caused disruptions to routine health care for women with breast cancer. Patients reported having fewer office visits at the start of the pandemic that continued to decrease even after vaccines were available. Fewer patients reported delaying in-person care as the pandemic progressed.National Cancer Institute ; Division of Cancer Prevention, National Cancer Institute ; Center for Strategic Scientific Initiatives, National Cancer Institute ; Division of Cancer Epidemiology and Genetics, National Cancer Institut

Polycyclic aromatic hydrocarbons as skin carcinogens:Comparison of benzo [a]pyrene, dibenzo[def,p]chrysene and three environmental mixtures in the FVB/N mouse

The polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), was compared to dibenzo[def,p]chrysene (DBC) and combinations of three environmental PAH mixtures (coal tar, diesel particulate and cigarette smoke condensate) using a two stage, FVB/N mouse skin tumor model. DBC (4 nmol) was most potent, reaching 100% tumor incidence with a shorter latency to tumor formation, less than 20 weeks of 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion compared to all other treatments. Multiplicity was 4 times greater than BaP (400 nmol). Both PAHs produced primarily papillomas followed by squamous cell carcinoma and carcinoma in situ. Diesel particulate extract (1 mg SRM 1650b; mix 1) did not differ from toluene controls and failed to elicit a carcinogenic response. Addition of coal tar extract (1 mg SRM 1597a; mix 2) produced a response similar to BaP. Further addition of 2 mg of cigarette smoke condensate (mix 3) did not alter the response with mix 2. PAH-DNA adducts measured in epidermis 12 h post initiation and analyzed by (32)P post- labeling, did not correlate with tumor incidence. PAH- dependent alteration in transcriptome of skin 12 h post initiation was assessed by microarray. Principal component analysis (sum of all treatments) of the 922 significantly altered genes (p<0.05), showed DBC and BaP to cluster distinct from PAH mixtures and each other. BaP and mixtures up-regulated phase 1 and 2 metabolizing enzymes while DBC did not. The carcinogenicity with DBC and two of the mixtures was much greater than would be predicted based on published Relative Potency Factors (RPFs)