Genotypic differences in thermotolerance are dependent upon prestress capacity for antioxidant protection of the photosynthetic apparatus in Gossypium hirsutum

Numerous studies have illustrated the need for antioxidant enzymes in acquired photosynthetic thermotolerance, but information on their possible role in promoting innate thermotolerance is lacking. We investigated the hypothesis that genotypic differences in source leaf photosynthetic thermostability would be dependent upon prestress capacity for antioxidant protection of the photosynthetic apparatus in Gossypium hirsutum. To test this hypothesis, thermosensitive (cv. ST4554) and reportedly thermotolerant (cv. VH260) G. hirsutum plants were exposed to control (30/20°C) or high‐day temperature (38/20°C) conditions during flowering and source leaf gas exchange, chlorophyll content and maximum photochemical efficiency (F_v/F_m) were measured for each treatment. The relationship between source leaf thermostability and prestress antioxidant capacity was quantified by monitoring the actual quantum yield response of photosystem II (PSII) (Φ_PSII) to a range of temperatures for both cultivars grown under the control temperature regime and measuring antioxidant enzyme activity for those same leaves. VH260 was more thermotolerant than ST4554 as evidenced by photosynthesis and F_v/F_m being significantly lower under high temperature for ST4554 but not VH260. Under identical growth conditions, VH260 had significantly higher optimal and threshold temperatures for Φ_PSII and glutathione reductase (GR; EC 1.8.1.7) activity than ST4554, and innate threshold temperature was dependent upon endogenous GR and superoxide dismutase (SOD; EC 1.15.1.1) activity. We conclude that maintaining a sufficient antioxidant enzyme pool prior to heat stress is an innate mechanism for coping with rapid leaf temperature increases that commonly occur under field conditions.     

The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis,cell division and virulence in the mammalian host

Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.     

Noggin suppression enhances in vitro osteogenesis and accelerates in vivo bone formation

Several investigations have demonstrated a precise balance to exist between bone morphogenetic protein (BMP) agonists and antagonists, dictating BMP signaling and osteogenesis. We report a novel approach to manipulate BMP activity through a down-regulation of the potent BMP antagonist Noggin, and examined the effects on the bone forming capacity of osteoblasts. Reduction of noggin enhanced BMP signaling and in vitro osteoblast bone formation, as demonstrated by both gene expression profiles and histological staining. The effects of noggin suppression on in vivo bone formation were also investigated using critical-sized calvarial defects in mice repaired with noggin-suppressed osteoblasts. Radiographic and histological analyses revealed significantly more bone regeneration at 2 and 4 weeks post-injury. These findings strongly support the concept of enhanced osteogenesis through a down-regulation in Noggin and suggest a novel approach to clinically accelerate bone formation, potentially allowing for earlier mobilization of patients following skeletal injury or surgical resection.     

Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH

Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant (foz/foz) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a) vehicle control, b) ezetimibe (5 mg/kg/day), c) atorvastatin (20 mg/kg/day), or d) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα-positive (activated) KCs forming CLSs (≥3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation.     

Competitive binding of Fe<Superscript>3+</Superscript>, Cr<Superscript>3+</Superscript>, and Ni<Superscript>2+</Superscript> to transferrin

Competitive binding of Fe³⁺, Cr³⁺, and Ni²⁺ to transferrin (Tf) was investigated at various physiological iron to Tf concentration ratios. Loading percentages for these metal ions are based on a two M ⁿ⁺ to one Tf (i.e., 100% loading) stoichiometry and were determined using a particle beam/hollow cathode–optical emission spectroscopy (PB/HC-OES) method. Serum iron concentrations typically found in normal, iron-deficient, iron-deficient from chronic disease, iron-deficient from inflammation, and iron-overload conditions were used to determine the effects of iron concentration on iron loading into Tf. The PB/HC-OES method allows the monitoring of metal ions in competition with Fe³⁺ for Tf binding. Iron-overload concentrations impeded the ability of chromium (15.0 μM) or nickel (10.3 μM) to load completely into Tf. Low Fe³⁺ uptake by Tf under iron-deficient or chronic disease iron concentrations limited Ni²⁺ loading into Tf. Competitive binding kinetic studies were performed with Fe³⁺, Cr³⁺, and Ni²⁺ to determine percentages of metal ion uptake into Tf as a function of time. The initial rates of Fe³⁺ loading increased in the presence of nickel or chromium, with maximal Fe³⁺ loading into Tf in all cases reaching approximately 24%. Addition of Cr³⁺ to 50% preloaded Fe³⁺–Tf showed that excess chromium (15.0 μM) displaced roughly 13% of Fe³⁺ from Tf, resulting in 7.6 ± 1.3% Cr³⁺ loading of Tf. The PB/HC-OES method provides the ability to monitor multiple metal ions competing for Tf binding and will help to understand metal competition for Tf binding.     

Efficient suspension bioreactor expansion of murine embryonic stem cells on microcarriers in serum-free medium

Large numbers of cells will be required for successful embryonic stem cell (ESC)-based cellular therapies or drug discovery, thus raising the need to develop scaled-up bioprocesses for production of ESCs and their derived progeny. Traditionally, ESCs have been propagated in adherent cultures in static flasks on fibroblasts layers in serum-containing medium. Direct translation of two-dimensional flatbed cultures to large-scale production of the quantities of cells required for therapy simply by increasing the number of dishes or flasks is not practical or economical. Here, we describe successful scaled-up production of ESCs on microcarriers in a stirred culture system in a serum-free medium. Cells expanded on CultiSpher S, Cytodex 3, and Collagen microcarriers showed superior cell-fold expansions of 439, 193, and 68, respectively, without excessive agglomeration, compared with 27 in static culture. In addition, the ESCs maintained their pluripotency after long-term culture (28 days) in serum-free medium. This is the first time mESCs have been cultured on microcarriers without prior exposure to serum and/or fibroblasts, while also eliminating the excessive agglomeration plaguing earlier studies. These protocols provide an economical, practical, serum-free means for expanding ESCs in a stirred suspension bioprocess.     

Factors influencing the seasonal life history of the pitcher-plant mosquito, Wyeomyia smithii

Abstract.  1. The effects of resource levels, thermal microclimate, and seasonal oviposition patterns on fecundity and survivorship in the pitcher-plant mosquito, Wyeomyia smithii (Coq.), were examined at a northern Wisconsin bog over the course of 2 years. Wyeomyia smithii are bivoltine at this locality, thereby enabling the study of summer and overwintering generations separately.
2. Nutrient resources of W. smithii were not limiting and there was no indication of density-dependent survivorship or fecundity.
3. Oviposition rates were highest in young, large pitchers and individual mosquitoes appeared to allocate only a few eggs to any one leaf.
4. Winter was the harsh season, and the principal manifestation of seasonal harshness was reduced survivorship.
5. Overwintering W. smithii that had been oviposited later in the summer had a higher odds of survival than those oviposited earlier in the summer.
6. It was concluded that dispersal of eggs among many pitchers serves to spread the risk of encountering lethal winter temperatures among spatially unpredictable patches.