Effects of parental age at mating on the fecundity and progeny sex ratio of <Emphasis Type="Italic">Campoletis chlorideae</Emphasis>, an endolarval parasitoid of the pod borer, <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), commonly called gram pod borer, is a serious pest of pulse crops in India and damages chickpea (Cicer arietinum L). On average a 30% crop loss is reported. One of the potential natural enemies reported for its biological control is Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae), an idiobiont parasitic wasp that attacks second instar larvae of the pod borer. Male-biased sex ratios hinder efforts to mass release parasitic Hymenoptera for biological control by making the production of females costly. Parental age at time of mating is known to affect the progeny sex ratio in some Braconidae. In this view, we examined the reproduction and survival of the parasitoid C. chlorideae in the laboratory [22 ± 4°C, 70 ± 10% R.H. and 10:14 h light:dark photoperiod]. All the nine (i.e. 3 × 3) combinations of possible mating were made for males and females, being each of different age groups (0–12, 48–60 and 96–108 h old). Second instar larvae of the pod borer were reared on chickpea and exposed to mated females of different mating age group combinations. Results revealed that (a) newly emerged females (0–12 h old) mated to aged males yielded more progeny contrasted to the older females inseminated by newly emerged males (0–12 h old). This implies that increased maternal age at mating caused greater reduction in the progeny yield than that of paternal age; (b) the progeny sex ratio was lowest (0.344 ± 0.057) (mean ± SD) when parents mated just after emergence (0–12 h old). It became more and more male biased as parents were deprived of mate and was highest (0.666 ± 0.701) for 96–108 h old parents. The analysis of variance of the data shows that mate deprived males caused greater contribution in the production of sons, resulting a male-biased progeny sex ratio. Our conclusion indicate that newly emerged parasitoids (0–12 h old) are most fecund and should be used in rearing practices in order to enhance the progeny yield, particularly with respect to daughters. Handling Editor: Torsten Meiners     

Pot1b deletion and telomerase haploinsufficiency in mice initiate an ATR-dependent DNA damage response and elicit phenotypes resembling dyskeratosis congenita

The Protection of telomeres 1 (POT1) protein is a single-stranded telomere binding protein that is essential for proper maintenance of telomere length. Disruption of POT1 function leads to chromosome instability and loss of cellular viability. Here, we show that targeted deletion of the mouse Pot1b gene results in increased apoptosis in highly proliferative tissues. In the setting of telomerase haploinsufficiency, loss of Pot1b results in depletion of germ cells and complete bone marrow failure due to increased apoptosis, culminating in premature death. Pot1b^−/− mTR^+/− hematopoietic progenitor and stem cells display markedly reduced survival potential in vitro. Accelerated telomere shortening, increased G overhang and elevated number of chromosome end-to-end fusions that initiate an ATR-dependent DNA damage response were also observed. These results indicate an essential role for Pot1b in the maintenance of genome integrity and the long-term viability of proliferative tissues in the setting of telomerase deficiency. Interestingly, these phenotypes closely resemble those found in the human disease dyskeratosis congenita (DC), an inherited syndrome characterized by bone marrow failure, hyperpigmentation, and nail dystrophy. We anticipate that this mouse will serve as a useful model to further understand the pathophysiology of DC.     

Reproducibility,sensitivity and compatibility of the ProteoExtract® subcellular fractionation kit with saturation labeling of laser microdissected tissues

Laser microdissection (LMD), a method of isolating specific microscopic regions of interest from a tissue that has been sectioned, is increasingly being applied to study proteomics. LMD generally requires tissues to be fixed and histologically stained, which can interfere with protein recovery and subsequent analysis. We evaluated the compatibility and reproducibility of protein extractions from laser microdissected human colon mucosa using a subcellular fractionation kit (ProteoExtract^®, Calbiochem). Four protein fractions corresponding to cytosol (fraction 1), membrane/organelle (fraction 2), nucleus (fraction 3) and cytoskeleton (fraction 4) were extracted, saturation labeled with Cy5 and 5 μg separated by both acidic (pH 4–7) and basic (pH 6–11) 2‐DE. The histological stains and fixation required for LMD did not interfere with the accurate subcellular fractionation of proteins into their predicted fraction. The combination of subcellular fractionation and saturation CyDye labeling produced very well resolved, distinct protein spot maps by 2‐DE for each of the subcellular fractions, and the total number of protein spots consistently resolved between three independent extractions for each fraction was 893, 1128, 1245 and 1577 for fractions 1, 2, 3 and 4, respectively. Although significant carryover of protein did occur between fractions, this carryover was consistent between experiments, and very low inter‐experimental variation was observed. In summary, subcellular fractionation kits are very compatible with saturation labeling DIGE of LMD tissues and provide greater coverage of proteins from very small amounts of microdissected material.     

Long-term use of thiazolidinediones and fractures in type 2 diabetes: a meta-analysis

Background

Rosiglitazone and pioglitazone may increase the incidence of fractures. We aimed to determine systematically the risk of fractures associated with thiazolidinedione therapy and to evaluate the effect of the therapy on bone density.

Methods

We searched MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials (CENTRAL), other trial registries and product information sheets through June 2008. We selected long-term (≥ 1 year) randomized controlled trials involving patients with type 2 diabetes and controlled observational studies that described the risk of fractures or changes in bone density with thiazolidinediones. We calculated pooled odds ratios (ORs) for fractures and the weighted mean difference in bone density.

Results

We analyzed data from 10 randomized controlled trials involving 13 715 participants and from 2 observational studies involving 31 679 participants. Rosiglitazone and pioglitazone were associated with a significantly increased risk of fractures overall in the 10 randomized controlled trials (OR 1.45, 95% confidence interval [CI] 1.18–1.79; p < 0.001). Five randomized controlled trials showed a significantly increased risk of fractures among women (OR 2.23, 95% CI 1.65–3.01; p < 0.001) but not among men (OR 1.00, 95% CI 0.73–1.39; p = 0.98). The 2 observational studies demonstrated an increased risk of fractures associated with rosiglitazone and pioglitazone. Bone mineral density in women exposed to thiazolidinediones was significantly reduced at the lumbar spine (weighted mean difference –1.11%, 95% CI –2.08% to –0.14%; p = 0.02) and hip (weighted mean difference –1.24%, 95%CI –2.34% to –0.67%; p < 0.001) in 2 randomized controlled trials.

Interpretation

Long-term thiazolidinedione use doubles the risk of fractures among women with type 2 diabetes, without a significant increase in risk of fractures among men with type 2 diabetes.Recent systematic reviews have focused on the adverse cardiovascular effects of the thiazolidinediones rosiglitazone and pioglitazone.^1–5 In late 2006, the risk of fractures with the use of rosiglitazone was raised in a footnote of the report of the A Diabetes Outcome and Progression Trial (ADOPT).⁶ The manufacturers of rosiglitazone⁷ and pioglitazone followed this up by issuing warning letters about the risk of fractures.^7–9Women with type 2 diabetes are at an increased risk of nonvertebral fractures,¹⁰ with a near doubling in the risk of hip fractures.¹¹ Any additional risk from thiazolidinedione therapy could have a considerable impact. Our primary objective was to determine systematically the relative and absolute risks of fractures with long-term thiazolidinedione therapy for type 2 diabetes. We also reviewed the effect of thiazolidinedione therapy on bone mineral density to ascertain its biological plausibility.     

Menopausal hormone therapy and women’s health: An umbrella review

BackgroundThere remains uncertainty about the impact of menopausal hormone therapy (MHT) on women’s health. A systematic, comprehensive assessment of the effects on multiple outcomes is lacking. We conducted an umbrella review to comprehensively summarize evidence on the benefits and harms of MHT across diverse health outcomes.Methods and findingsWe searched MEDLINE, EMBASE, and 10 other databases from inception to November 26, 2017, updated on December 17, 2020, to identify systematic reviews or meta-analyses of randomized controlled trials (RCTs) and observational studies investigating effects of MHT, including estrogen-alone therapy (ET) and estrogen plus progestin therapy (EPT), in perimenopausal or postmenopausal women in all countries and settings. All health outcomes in previous systematic reviews were included, including menopausal symptoms, surrogate endpoints, biomarkers, various morbidity outcomes, and mortality. Two investigators independently extracted data and assessed methodological quality of systematic reviews using the updated 16-item AMSTAR 2 instrument. Random-effects robust variance estimation was used to combine effect estimates, and 95% prediction intervals (PIs) were calculated whenever possible. We used the term MHT to encompass ET and EPT, and results are presented for MHT for each outcome, unless otherwise indicated. Sixty systematic reviews were included, involving 102 meta-analyses of RCTs and 38 of observational studies, with 102 unique outcomes. The overall quality of included systematic reviews was moderate to poor. In meta-analyses of RCTs, MHT was beneficial for vasomotor symptoms (frequency: 9 trials, 1,104 women, risk ratio [RR] 0.43, 95% CI 0.33 to 0.57, p < 0.001; severity: 7 trials, 503 women, RR 0.29, 95% CI 0.17 to 0.50, p = 0.002) and all fracture (30 trials, 43,188 women, RR 0.72, 95% CI 0.62 to 0.84, p = 0.002, 95% PI 0.58 to 0.87), as well as vaginal atrophy (intravaginal ET), sexual function, vertebral and nonvertebral fracture, diabetes mellitus, cardiovascular mortality (ET), and colorectal cancer (EPT), but harmful for stroke (17 trials, 37,272 women, RR 1.17, 95% CI 1.05 to 1.29, p = 0.027) and venous thromboembolism (23 trials, 42,292 women, RR 1.60, 95% CI 0.99 to 2.58, p = 0.052, 95% PI 1.03 to 2.99), as well as cardiovascular disease incidence and recurrence, cerebrovascular disease, nonfatal stroke, deep vein thrombosis, gallbladder disease requiring surgery, and lung cancer mortality (EPT). In meta-analyses of observational studies, MHT was associated with decreased risks of cataract, glioma, and esophageal, gastric, and colorectal cancer, but increased risks of pulmonary embolism, cholelithiasis, asthma, meningioma, and thyroid, breast, and ovarian cancer. ET and EPT had opposite effects for endometrial cancer, endometrial hyperplasia, and Alzheimer disease. The major limitations include the inability to address the varying effects of MHT by type, dose, formulation, duration of use, route of administration, and age of initiation and to take into account the quality of individual studies included in the systematic reviews. The study protocol is publicly available on PROSPERO (CRD42017083412).ConclusionsMHT has a complex balance of benefits and harms on multiple health outcomes. Some effects differ qualitatively between ET and EPT. The quality of available evidence is only moderate to poor.

In an umbrella review, Guo-Qiang Zhang and colleagues comprehensively summarize evidence on the benefits and harms of menopausal hormone therapy across diverse health outcomes.     

Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5′-terminal phosphorylation both in vitro and in vivo

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2′, 3′ seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5′-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5′-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.     

Raman Tweezers Spectroscopy of Live,Single Red and White Blood Cells

An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC) and white blood cells (WBC) under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm) is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW). Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip.     

Metagenomes from High-Temperature Chemotrophic Systems Reveal Geochemical Controls on Microbial Community Structure and Function

The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14–15,000 Sanger reads per site) was obtained for five high-temperature (>65°C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O₂ influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H₂-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, Fe, O₂) control microbial community structure and function in YNP geothermal springs.     

Cell growth: how to grow and where to grow

Root hairs provide a model system for studying tip growth in plants. The recent cloning of genes required for tip growth has shed new light on the link between ionic regulation, cell wall assembly and the cytoskeleton in cell growth.     

Hydroxylamine potentiates the effect of low dose hydrogen peroxide in glioma cells independent of p53

We had earlier shown that higher concentration of hydrogen peroxide (H₂O₂) induced p53-dependent apoptosis in glioma cell line with wild type p53 but had minimal effect on cells with mutated p53. Here we show a potentiating effect of hydroxylamine (HA), an inhibitor of catalase, on a nontoxic dose of H₂O₂ in glioma cells. HA sensitized both p53 wild type and mutated glioma cells to 0.25 mM H₂O₂. Potentiating effect of HA was independent of p53. Higher levels of reactive oxygen species (ROS) generation were observed in cells treated with HA+H₂O₂ as compared to cells treated with each component alone in both the cell lines. Dimethyl sulfoxide (DMSO) protected cells. Cytosolic cytochrome c and activated caspase 3 were detected at 4 h. The results suggest that higher levels of intracellular ROS, generated by HA+H₂O₂ act as a molecular switch in activating a rapidly acting p53-independent mitochondrial apoptotic pathway.