Comparative study of the cellular replication kinetics of rat mammary epithelial cells in vivo and in vitro

Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.     

Apolipoprotein H (beta-2-glycoprotein I) polymorphism in Asians.

Apolipoprotein H (APOH) (beta-2-glycoprotein I) polymorphism has been studied in 1159 Asians. The sample included 872 Chinese, 179 Asiatic Indians (Dravidian), 91 Filipinos, and 17 Malays. APOH polymorphism was determined by isoelectric focusing of sera in thin-layer polyacrylamide gels containing 3 M urea followed by immunoblotting. The frequencies of the three alleles--APOH*1, APOH*2, and APOH*3--were found to be 0.031, 0.900, and 0.069 in the Chinese; 0.061, 0.866, and 0.073 in the Dravidian Indians; 0.055, 0.923, and 0.022 in the Filipinos; and 0.088, 0.882, and 0.029 in the Malays. The phenotypic distribution was at Hardy-Weinberg equilibrium in all the populations.     

Identification and cloning of a novel heterogeneous nuclear ribonucleoprotein C-like protein that functions as a transcriptional activator of the hepatitis B virus enhancer II.

Liver specificity of hepatitis B virus (HBV) replication has been attributed to the action of its second enhancer (EII). We report here the characterization of EII and the subsequent isolation of a novel liver-specific DNA-binding protein which binds to and activates EII. The cDNA clone of the protein, designated E2BP, was isolated from a lambda gt11 expression library constructed from the hepatoma cell line HuH-6 which was screened with a binding site probe derived from EII. Sequence analysis of E2BP revealed 86.6% homology with a rat heterogeneous nuclear ribonucleoprotein C protein sequence, while conformational studies suggest a helix-loop-helix motif as a DNA-binding site. Cloned E2BP expressed in human fibroblasts by transient transfection displayed EII binding and activating characteristics similar to those of native E2BP in hepatocytes.     

Fine needle aspiration biopsy of hepatocellular carcinoma. Some unusual features.

The cytologic features of fine needle aspiration smears from 28 hepatocellular carcinomas (HCC) were reviewed. All aspirations except one were guided. There were 14 well-, 11 moderately and 3 poorly differentiated HCC. The better-differentiated HCC were characterized by similarity of the tumor cells to hepatocytes (83%), cohesive cell clusters with a trabecular arrangement (72%) and presence of sinusoidal endothelial cells (66%). Other features included bile production (38%), atypical hepatocytic naked nuclei (52%), acinar formation (31%), intracytoplasmic vacuoles (14%) and abnormal vascular patterns (14%). Poorly differentiated HCC showed dyshesive pleomorphic cells. Unusual cytologic features from a well-differentiated HCC with fatty change and an HCC with a prominent acinar component are described. The identification of fatty change in dissociated well-differentiated hepatocytes or cytologic features suggestive of an adenocarcinoma do not preclude the diagnosis of HCC. The usefulness of cell blocks is emphasized.     

Cell division cycles and circadian clocks : phase-response curves for light perturbations in synchronous cultures of euglena

The cell division rhythm in Euglena gracilis Klebs (Z strain) freeruns with a circadian period (30.2 ± 1.8 hours for 156 monitored oscillations) in aerated, magnetically stirred, 8-liter, axenic batch cultures grown photoautotrophically at 25°C in LD: 3,3, (7,500 lux, cool-white fluorescent) 6-hour light cycles from the moment of inoculation. Cell number was measured at 2-hour intervals with an automatic fraction collector and Coulter Electronic Particle Counter. At different circadian times throughout the 30-hour division cycle, 3-hour light perturbations were imposed on free-running cell populations by giving light during one of the intervals when dark would have fallen in the LD: 3,3 regimen. Using the onset of division as the phase reference point, the net steady-state phase advance or delay (±Δ) of the rhythm was determined after transients, if any, had subsided (usually in one or two days) relative to an unperturbed control culture. Both +Δ and −Δ were found, with maximum values of approximately ±11 to 12 hours being obtained at circadian time (CT) 20 to 22 (the `breakpoint'); little, if any phase shift occurred if the light signal was given between CT 6 and CT 12. The phase-resetting curve obtained by plotting new phase (′) versus old phase () was of the type 0 (`strong') variety. Light perturbations, no matter when imposed, engendered new phases which mapped to a relatively restricted portion (CT 6 to CT 13) of the circadian cycle.

These data provide the first detailed phase-response curve for a circadian mitotic clock. The findings, therefore, not only further support the hypothesis that a circadian oscillator (perhaps exhibiting limit cycle behavior) can modulate cell division in eukaryotic cells, but also provide a useful basis for the dissection of the nature and extent of the coupling between cell division and circadian cycles.

     

Structural investigations of DNA-histone complexes. A spin label study.

We have prepared two acridine spin labels, 6-chloro-9-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)amino]-2-methoxyacridine (I) and 9-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)amino]-acridine (II) and have used them to study the binding of lysine-rich histone (H1) to DNA using electron spin resonance (ESR). ESR spectra of I in the presence of DNA, polydA-polydT and polydG-polydC were characteristic of highly immobilized radicals with maximum hyperfine splitting (2T11) of 59G, 62.5G and 59G respectively. However, the 2T11 values for II in the same systems were 55.5G, 55.5G and 62.5G respectively. Addition of H1 at a low P/D released ionically bound I and II from DNA. In the presence of 0.1 M NaCl, which prevents ionic binding, H1 still caused a significant release of bound II but not I from DNA. At a high P/D (with or without NaCl) H1 caused no displacement of either I or II. Our findings suggest that H1 does not affect the intercalating sites and probably binds to one of the grooves of DNA, most probably the major groove, and specifically in the A-T-rich regions.     

Whole-genome sequencing to detect mutations associated with resistance to insecticides and Bt proteins in Spodoptera frugiperda

The fall armyworm (FAW), Spodoptera frugiperda, is a major pest native to the Americas that has recently invaded the Old World. Point mutations in the target-site proteins acetylcholinesterase-1 (ace-1), voltage-gated sodium channel (VGSC) and ryanodine receptor (RyR) have been identified in S. frugiperda as major resistance mechanisms to organophosphate, pyrethroid and diamide insecticides respectively. Mutations in the adenosine triphosphate-binding cassette transporter C2 gene (ABCC2) have also been identified to confer resistance to Cry1F protein. In this study, we applied a whole-genome sequencing (WGS) approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China, Malawi, Uganda and Brazil. This approach revealed three amino acid substitutions (A201S, G227A and F290V) of S. frugiperda ace-1, which are known to be associated with organophosphate resistance. The Brazilian population had all three ace-1 point mutations and the 227A allele (mean frequency = 0.54) was the most common. Populations from China, Malawi and Uganda harbored two of the three ace-1 point mutations (A201S and F290V) with the 290V allele (0.47–0.58) as the dominant allele. Point mutations in VGSC (T929I, L932F and L1014F) and RyR (I4790M and G4946E) were not detected in any of the 150 individuals. A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations. Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations, but also provide insights for improvement of resistance management tactics in S. frugiperda.     

Integration of a multicomponent intervention for hypertension into primary healthcare services in Singapore—A cluster randomized controlled trial

BackgroundDespite availability of clinical practice guidelines for hypertension management, blood pressure (BP) control remains sub-optimal (<30%) even in high-income countries. This study aims to assess the effectiveness of a potentially scalable multicomponent intervention integrated into primary care system compared to usual care on BP control.Methods and findingsA cluster-randomized controlled trial was conducted in 8 government clinics in Singapore. The trial enrolled 916 patients aged ≥40 years with uncontrolled hypertension (systolic BP (SBP) ≥140 mmHg or diastolic BP (DBP) ≥90 mmHg).Multicomponent intervention consisted of physician training in risk-based treatment of hypertension, subsidized losartan-HCTZ single-pill combination (SPC) medications, nurse training in motivational conversations (MCs), and telephone follow-ups. Usual care (controls) comprised of routine care in the clinics, no MC or telephone follow-ups, and no subsidy on SPCs. The primary outcome was mean SBP at 24 months’ post-baseline. Four clinics (447 patients) were randomized to intervention and 4 (469) to usual care. Patient enrolment commenced in January 2017, and follow-up was during December 2018 to September 2020. Analysis used intention-to-treat principles. The primary outcome was SBP at 24 months. BP at baseline, 12 and 24 months was modeled at the patient level in a likelihood-based, linear mixed model repeated measures analysis with treatment group, follow-up, treatment group × follow-up interaction as fixed effects, and random cluster (clinic) effects.A total of 766 (83.6%) patients completed 2-year follow-up. A total of 63 (14.1%) and 87 (18.6%) patients in intervention and in usual care, respectively, were lost to follow-up. At 24 months, the adjusted mean SBP was significantly lower in the intervention group compared to usual care (−3.3 mmHg; 95% CI: −6.34, −0.32; p = 0.03). The intervention led to higher BP control (odds ratio 1.51; 95% CI: 1.10, 2.09; p = 0.01), lower odds of high (>20%) 10-year cardiovascular risk score (OR 0.67; 95% CI: 0.47, 0.97; p = 0.03), and lower mean log albuminuria (−0.22; 95% CI: −0.41, −0.02; p = 0.03). Mean DBP, mortality rates, and serious adverse events including hospitalizations were not different between groups. The main limitation was no masking in the trial.ConclusionsA multicomponent intervention consisting of physicians trained in risk-based treatment, subsidized SPC medications, nurse-delivered motivational conversation, and telephone follow-ups improved BP control and lowered cardiovascular risk. Wide-scale implementation of a multicomponent intervention such as the one in our trial is likely to reduce hypertension-related morbidity and mortality globally.Trial registrationTrial Registration: Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02972619","term_id":"NCT02972619"}}NCT02972619.

Tazeen H Jafar and colleagues present findings from a cluster-randomized controlled trial conducted to evaluate the effectiveness of an intervention designed to manage hypertension.     

A hybrid anaerobic solid-liquid bioreactor for food waste digestion

A hybrid anaerobic solid-liquid (HASL) bioreactor is an enhanced two-phase anaerobic system, that consists of a solid waste reactor as the acidification reactor and a wastewater reactor, i.e. an upflow anaerobic sludge blanket (UASB) reactor as the methanogenic reactor. Food waste digestion in HASL bioreactors with pre-acidification and HASL operation stages was investigated in two separate runs. After 8 days of pre-acidification in Run A and 4 days in Run B, total volatile fatty acid (TVFA) and chemical oxygen demand (COD) concentrations in the leachates of both acidification reactors were similar. During HASL operation stage, TVFA and COD removal in the methanogenic phase were 77–100% and 75–95%, respectively. Some 99% of the total methane generated was from the methanogenic phase with a content of 68–70% methane. At the end of operation, about 59–60% of the added volatile solids (VS) were removed with a methane yield of 0.25 l g^–1 VS.     

Dissecting the telomere region of barley chromosome 5HL using rice genomic sequences as references: new markers for tracking a complex region in breeding

The terminal region of barley chromosome 5HL controls malt extract, diastatic power, free amino acid nitrogen, alpha-amylase activity, seed dormancy and pre-harvest sprouting. Comparative analysis of the barley and rice maps has established that the terminal region of barley chromosome 5HL is syntenic to rice chromosome 3L near the telomere end. The rice BAC (Bacterial Artificial Chromosome) sequences covering the region of chromosome 3L were used to search barley expressed sequenced tags database. Thirty-three genes were amplified by PCR (polymerase chain reaction) with the primers designed from barley ESTs (expressed sequence tag). Comparison of the sequences of the PCR generated DNA fragments revealed polymorphisms including single nucleotide polymorphism (SNP), insertions or deletions between the barley varieties. Seven new PCR based molecular markers were developed and mapped within 10 cM in three doubled haploid barley populations (Stirling × Harrington, Baudin × AC Metcalfe and Chebec × Harrington). The mapped genes maintain the micro-syntenic relationship between barley and rice. These gene specific markers provide simple and efficient tools for germplasm characterization and marker-assisted selection for barley malting quality, and ultimately lead to isolation and identification of the major gene(s) controlling multiple quality traits on barley chromosome 5HL.