Regulation of Cell Division in Arabidopsis

The study of cell cycle control in plants is expected to contribute to the understanding of plants' unique developmental features. The principal regulators of the eukaryotic cell cycle, namely, cyclin-dependent kinases (CDKs) and cyclins, are also conserved in plants. This review is concerned with our present knowledge on cell cycle regulation in Arabidopsis thaliana, which is widely accepted as a model plant for the study of a broad range of biological questions. Up to the present, 2 CDKs and 11 cyclins have been identified in Arabidopsis. While the expression of one of these CDKs has been found to be positively correlated with the competence of cells to divide, cyc1A1 expression of the cyclin has been almost exclusively confined to dividing cells. Although much remains to be studied concerning upstream regulators of these genes, the successful introduction of mutant CDKs into plants demonstrates the potential of using such an approach to intentionally modulate the plant cell cycle and development.     

Persistence to anti-cancer treatments in the stationary to proliferating transition

The heterogeneous responses of clonal cancer cells to treatment is understood to be caused by several factors, including stochasticity, cell-cycle dynamics, and different micro-environments. In a tumor, cancer cells may encounter fluctuating conditions and transit from a stationary culture to a proliferating state, for example this may occur following treatment. Here, we undertake a quantitative evaluation of the response of single cancerous lymphoblasts (L1210 cells) to various treatments administered during this transition. Additionally, we developed an experimental system, a “Mammalian Mother Machine,” that tracks the fate of thousands of mammalian cells over several generations under transient exposure to chemotherapeutic drugs. Using our developed system, we were able to follow the same cell under repeated treatments and continuously track many generations. We found that the dynamics of the transition between stationary and proliferative states are highly variable and affect the response to drug treatment. Using cell-cycle markers, we were able to isolate a subpopulation of persister cells with distinctly higher than average survival probability. The higher survival rate encountered with cell-cycle phase specific drugs was associated with a significantly longer time-till-division, and was reduced by a non cell-cycle specific drug. Our results suggest that the variability of transition times from the stationary to the proliferating state may be an obstacle hampering the effectiveness of drugs and should be taken into account when designing treatment regimens.     

A Patient-Centered Understanding of the Referral System in Ethiopian Primary Health Care Units

Background

Primary healthcare systems in sub-Saharan Africa have undergone substantial development in an effort to expand access to appropriate facilities through a well-functioning referral system. The objective of this study was to evaluate the current patterns of seeking prior care before arriving at a health center or a hospital as a key aspect of the referral system of the primary health care unit (PHCU) in three regions in Ethiopia. We examined what percentage of patients had either sought prior care or had been referred to the present facility and identified demographic and clinical factors associated with having sought prior care or having been referred.

Methods and Findings

We conducted a cross-sectional study using face-to-face interviews in the local language with 796 people (99% response rate) seeking outpatient care in three primary health care units serving approximately 100,000 people each and reflecting regional and ethnic diversity; 53% (N = 418) of the sample was seeking care at hospital outpatient departments, and 47% of the sample was seeking care at health centers (N = 378). We used unadjusted and adjusted logistic regression to identify factors associated with having been referred or sought prior care. Our findings indicated that only 10% of all patients interviewed had been referred to their current place of care. Among those in the hospital population, 14% had been referred; among those in the health center population, only 6% had been referred. Of those who had been referred to the hospital, most (74%) had been referred by a health center. Among those who were referred to the health center, the plurality portion (32%) came from a nearby hospital (most commonly for continued HIV treatment or early childhood vaccinations); only 18% had come from a health post. Among patients who had not been formally referred, an additional 25% in the hospital sample and 10% in the health center sample had accessed some prior source of care for their present health concern. In the adjusted analysis, living a longer distance from the source of care and needing more specialized care were correlated with having sought prior care in the hospital sample. We found no factors significantly associated with having sought prior care in the health center sample.

Conclusions

The referral system among health facilities in Ethiopia is used by a minority of patients, suggesting that intended connections between health posts, health centers, and hospitals may need strengthening to increase the efficiency of primary care nationally.     

Localization of a Subset of Yeast mRNAs Depends on Inheritance of Endoplasmic Reticulum

Localization of messenger RNA (mRNAs) contributes to generation and maintenance of cellular asymmetry, embryonic development and neuronal function. The She1‐3 protein machinery in Saccharomyces cerevisiae localizes >30 mRNAs to the bud tip, including 13 mRNAs encoding membrane or secreted proteins. Ribonucleoprotein (RNP) particles can co‐localize with tubular endoplasmic reticulum (ER) structures that form the initial elements for segregation of cortical ER (cER), suggesting a coordination of mRNA localization and cER distribution. By investigating localization of MS2‐tagged mRNAs in yeast defective at various stages of cER segregation, we demonstrate that proper cER segregation is required for localization of only a subset of mRNAs. These mRNAs include WSC2, IST2, EAR1 and SRL1 that encode membrane or ER associated proteins and are expressed during S and G2 phases of the cell cycle when tubular ER movement into the bud occurs. Translation of WSC2 is not required for localization, ruling out co‐translational targeting of this mRNA. Localization of ASH1 mRNA is independent of cER segregation, which is consistent with the expression pattern of ASH1 at late mitosis. Our findings indicate the presence of two different pathways to localize mRNAs to the yeast bud.     

First evidence of maternal transmission of algal endosymbionts at an oocyte stage in a triploblastic host, with observations on reproduction in Waminoa brickneri (Acoelomorpha)

Abstract. Examination of sexual reproduction in a symbiotic acoelomorph worm, Waminoa brickneri from Eilat (Red Sea), presents the first definitive evidence for maternal transmission of dinoflagellate algal symbionts in a triploblastic organism. Sexually mature worms were removed from the stony coral Plesiastrea laxa and raised in the laboratory. Eggs were detected 18 d after the collection of the worms and hatched 4 d later. Histological sections performed on sexually mature worms showed an ovary with oocytes containing two distinct types of algal endosymbionts within their ooplasm. Transmission electron microscopy corroborated the presence of algal symbionts within the developing embryos. Our findings regarding maternal transmission of symbionts shed new light on the diversity of modes of algal symbiont acquisition known in triploblastic organisms.     

3-18F-fluoropropane-1-thiol and 18F-PEG4-1-thiol: Versatile prosthetic groups for radiolabeling maleimide functionalized peptides

The efficient radiosynthesis of biomolecules utilizing minute quantities of maleimide substrate is important for availability of novel peptide molecular imaging agents. We evaluated both 3-¹⁸F-fluoropropane-1-thiol and 2-(2-(2-(2-¹⁸F-fluoroethoxy)ethoxy)ethoxy)ethane-1-thiol (¹⁸F-fluoro-PEG₄ thiol) as prosthetic groups for radiolabeling under physiological conditions. The precursor employed a benzoate for protection of the thiol and an arylsulfonate leaving group. The radiofluorination was fully automated on an Eckert & Ziegler synthesis system using standard Kryptofix₂₂₂/K₂CO₃ conditions. In order to minimize the amount of biological molecule required for subsequent conjugation, the intermediates, S-(3-¹⁸F-fluoropropyl) benzothioate and ¹⁸F-fluoro-PEG₄ benzothioate, were purified by HPLC. The intermediates were isolated from the HPLC in yields of 37–47% and 28–35%, respectively, and retrieved from eluate using solid phase extraction. Treatment of the benzothioates with sodium methoxide followed by acetic acid provided the free thiols. The desired maleimide substrate in acetonitrile or phosphate buffer was then added and incubated at room temperature for 15 min. The final radiolabeled bioconjugate was purified on a separate HPLC or NAP-5 column. Maleimides utilized for the coupling reaction included phenyl maleimide, an Evans Blue maleimide derivative, a dimeric RGDfK maleimide (E[c(RGDfK)]₂), two aptamer maleimides, and PSMA maleimide derivative. Isolated radiochemical yields (non-decay corrected) of maleimide addition products based on starting ¹⁸F-fluoride ranged from 6 to 22% in a synthesis time of about 90 min.¹⁸F-thiol prosthetic groups were further tested in vivo by conjugation to E[c(RGDfK)]₂ maleimide in a U87MG xenograft model. PET studies demonstrated similar tumor accumulation of both prosthetic groups. ¹⁸F-fluoro-PEG₄-S-E[c(RGDfK)]₂ displayed a somewhat favorable pharmacokinetics compared to ¹⁸F-fluoropropyl-S-E[c(RGDfK)]₂. Bone uptake was low for both indicating in vivo stability.