Short-term culturing of teleost crystalline lenses combined with high-resolution optical measurements

Culturing whole lenses is a frequently used method for studying regulatory events on the lens in controlled environments. The evaluation methods used often fall under two categories, molecular or optical. The main benefit from optical measurements is that they directly detect changes in the lens’ main function, i.e. refracting light. However, these measurements often have rather low resolution or yield results open for subjective interpretation. Here we present a short-term crystalline lens culturing technique combined with a high-resolution optical measuring method. There are two main advantages of using teleost lenses compared to mammalian lenses. Teleost tissue generally has a higher tolerance than mammalian tissue with regard to temperature and nutrient fluctuations. Teleost lenses are structurally more robust and can be excised from the eye without disturbing form or function. The technique is developed for short-term culturing (3 h), however, the lenses appear viable for at least 24 h and longer culturing may be possible. The technique is resistant to small variations in osmolarity and yields quantitative datasets for further analyses and statistical treatment.     

The development of composite dispersal functions for estimating absolute pollen productivity in the Swiss Alps

Considering the complexity of real-world pollen dispersal, a single set of parameters may be inadequate to model pollen dispersal, especially as dispersal occurs on both local and regional scales. Here we combine more than one dispersal function into a composite dispersal function (CDF). The function incorporates multiple parameters and different modes of pollen transportation, and thus has the potential to better simulate the relationship between deposited pollen and the surrounding vegetation than would otherwise be possible. CDFs based on different dispersal functions and combinations of dispersal functions were evaluated using a pollen-trap dataset from the Swiss Alps. Absolute pollen productivity (APP) was estimated at 7,700 ± 2,000 grains cm⁻² year⁻¹ for Larix decidua, 13,500 ± 1,900 grains cm⁻² year⁻¹ for Picea abies and 95,600 ± 17,700 grains cm⁻² year⁻¹ for Pinus cembra (with 95% confidence level). The results are consistent with previous APP estimates made from the same dataset using different methods.     

Unfolding and refolding properties of S pili on extraintestinal pathogenic Escherichia coli

S pili are members of the chaperone-usher-pathway-assembled pili family that are predominantly associated with neonatal meningitis (S_II) and believed to play a role in ascending urinary tract infections (S_I). We used force-measuring optical tweezers to characterize the intrinsic biomechanical properties and kinetics of S_II and S_I pili. Under steady-state conditions, a sequential unfolding of the layers in the helix-like rod occurred at somewhat different forces, 26 pN for S_II pili and 21 pN for S_I pili, and there was an apparent difference in the kinetics, 1.3 and 8.8 Hz. Tests with bacteria defective in a newly recognized sfa gene (sfaX _II) indicated that absence of the sfaX _II gene weakens the interactions of the fimbrium slightly and decreases the kinetics. Data of S_I are compared with those of previously assessed pili primary associated with urinary tract infections, the P and type 1 pili. S pili have weaker layer-to-layer bonds than both P and type 1 pili, 21, 28 and 30 pN, respectively. In addition, the S pili kinetics are ~10 times faster than the kinetics of P pili and ~550 times faster than the kinetics of type 1 pili. Our results also show that the biomechanical properties of pili expressed ectopically from a plasmid in a laboratory strain (HB101) and pili expressed from the chromosome of a clinical isolate (IHE3034) are identical. Moreover, we demonstrate that it is possible to distinguish, by analyzing force-extension data, the different types of pili expressed by an individual cell of a clinical bacterial isolate.     

Activation of ERα is necessary for estradiol's anorexigenic effect in female rats

While there is considerable evidence that the ovarian hormone estradiol reduces food intake in female rats, it is unclear which estrogen receptor (ER) subtype, ERα or ERβ, mediates this effect. While several studies have demonstrated that activation of ERα, but not ERβ, is sufficient to reduce food intake in ovariectomized (OVX) rats, there are limited data regarding which receptor subtype is necessary. Here we used the selective ERα and ERß antagonists, MPrP and PHTPP, respectively, to investigate this question. We found that antagonism of ERα, but not ERβ, prevented the decrease in food intake following acute administration of estradiol in OVX rats. In addition, antagonism of ERα prevented the estrous-related, phasic reduction in food intake that occurs in response to the rise in circulating levels of estradiol in cycling rats. We conclude that activation of ERα is necessary for the anorexigenic effects of exogenous and endogenous estradiol in female rats.     

Genome-wide association study of blood pressure extremes identifies variant near UMOD associated with hypertension

Hypertension is a heritable and major contributor to the global burden of disease. The sum of rare and common genetic variants robustly identified so far explain only 1%–2% of the population variation in BP and hypertension. This suggests the existence of more undiscovered common variants. We conducted a genome-wide association study in 1,621 hypertensive cases and 1,699 controls and follow-up validation analyses in 19,845 cases and 16,541 controls using an extreme case-control design. We identified a locus on chromosome 16 in the 5′ region of Uromodulin (UMOD; rs13333226, combined P value of 3.6×10⁻¹¹). The minor G allele is associated with a lower risk of hypertension (OR [95%CI]: 0.87 [0.84–0.91]), reduced urinary uromodulin excretion, better renal function; and each copy of the G allele is associated with a 7.7% reduction in risk of CVD events after adjusting for age, sex, BMI, and smoking status (H.R. = 0.923, 95% CI 0.860–0.991; p = 0.027). In a subset of 13,446 individuals with estimated glomerular filtration rate (eGFR) measurements, we show that rs13333226 is independently associated with hypertension (unadjusted for eGFR: 0.89 [0.83–0.96], p = 0.004; after eGFR adjustment: 0.89 [0.83–0.96], p = 0.003). In clinical functional studies, we also consistently show the minor G allele is associated with lower urinary uromodulin excretion. The exclusive expression of uromodulin in the thick portion of the ascending limb of Henle suggests a putative role of this variant in hypertension through an effect on sodium homeostasis. The newly discovered UMOD locus for hypertension has the potential to give new insights into the role of uromodulin in BP regulation and to identify novel drugable targets for reducing cardiovascular risk.     

Dopamine Induces Optical Changes in the Cichlid Fish Lens

The crystalline lens in the cichlid fish Aequidens pulcher undergoes a transformation of its optical properties every dawn and dusk as the eye adapts to changes in light conditions. During dusk the transformation result in an increase of the refractive power in the lens cortex, the outermost 40 percent. The change is thought to match the optical properties of the lens to the requirements of the retina. Using a short term in vitro lens culturing system together with optical measurements we here present data that confirm that the optical properties of the lens can change within hours and that dopamine influences the optical properties of the lens. Dopamine yields dose-dependent decrease of the refractive power in the lens cortex. The D1-agonist SKF-38393 induces a similar decrease of the refractive power in the cortex, while the D2-agonist quinpirole has no effect. The effect of dopamine can be blocked by using the D1-antagonist SCH 23390. Our results suggest that dopamine alone could be responsible for the light/dark adaptive optical changes in the lens, but the involvement of other signaling substances cannot be ruled out.     

Interactions between M proteins of Streptococcus pyogenes and glycosaminoglycans promote bacterial adhesion to host cells.

Several microbial pathogens have been reported to interact with glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix. Here we demonstrate that M protein, a major surface-expressed virulence factor of the human bacterial pathogen, Streptococcus pyogenes, mediates binding to various forms of GAGs. Hence, S. pyogenes strains expressing a large number of different types of M proteins bound to dermatan sulfate (DS), highly sulfated fractions of heparan sulfate (HS) and heparin, whereas strains deficient in M protein surface expression failed to interact with these GAGs. Soluble M protein bound DS directly and could also inhibit the interaction between DS and S. pyogenes. Experiments with M protein fragments and with streptococci expressing deletion constructs of M protein, showed that determinants located in the NH2-terminal part as well as in the C-repeat region of the streptococcal proteins are required for full binding to GAGs. Treatment with ABC-chondroitinase and HS lyase that specifically remove DS and HS chains from cell surfaces, resulted in significantly reduced adhesion of S. pyogenes bacteria to human epithelial cells and skin fibroblasts. Together with the finding that exogenous DS and HS could inhibit streptococcal adhesion, these data suggest that GAGs function as receptors in M protein-mediated adhesion of S. pyogenes.     

Intramuscular pressure and EMG relate during static contractions but dissociate with movement and fatigue.

Intramuscular pressure (IMP) and electromyography (EMG) mirror muscle force in the nonfatigued muscle during static contractions. The present study explores whether the constant IMP-EMG relationship with increased force may be extended to dynamic contractions and to fatigued muscle. IMP and EMG were recorded from shoulder muscles in three sessions: 1). brief static arm abductions at angles from 0 to 90 degrees, with and without 1 kg in the hands; 2). dynamic arm abductions at angular velocities from 9 to 90 degrees /s, with and without 1 kg in the hands; and 3). prolonged static arm abduction at 30 degrees for 30 min followed by recovery. IMP and EMG increased in parallel with increasing shoulder torque during brief static tasks. During dynamic contractions, peak IMP and EMG increased to values higher than those during static contractions, and EMG, but not IMP, increased significantly with speed of abduction. In the nonfatigued supraspinatus muscle, a linear relationship was found between IMP and EMG; in contrast, during fatigue and recovery, significant timewise changes of the IMP-to-EMG ratio occurred. The results indicate that IMP should be included along with EMG when mechanical load sharing between muscles is evaluated during dynamic and fatiguing contractions.     

Liver asialoglycoprotein receptor levels correlate with severity of alcoholic liver damage in rats.

It has been demonstrated that the oral administration of ethanol (Lieber-DeCarli liquid diet) to rats results in a decreased expression and content of the asialoglycoprotein receptor (ASGP-R) in the resultant fatty liver. In the present study, we wanted to determine whether the extent of impaired receptor content was correlated with the severity of liver pathology by using the intragastric feeding model. When ASGP-R protein and mRNA levels were measured in animals infused with ethanol or dextrose in the presence of fish oil (FO) or medium-chain triglyceride as the source of fat, more significant impairments to the ASGP-R were observed in the FO-ethanol group compared with the medium-chain triglyceride-ethanol group. Furthermore, only the FO-ethanol group showed pathological liver changes. These results demonstrate that a correlation exists between the progression of alcohol-associated liver injury, as defined by the severity of liver pathology, and an ethanol-induced decline in ASGP-R content.     

A novel plant ferredoxin-like protein and the regulator Hor are quorum-sensing targets in the plant pathogen Erwinia carotovora

Quorum sensing (QS), a population-density-sensing mechanism, controls the production of the main virulence determinants, the plant cell-wall-degrading enzymes (PCWDEs) of the soft-rot phytopathogen Erwinia carotovora subsp. carotovora. In this study, we used random transposon mutagenesis with a gusA reporter construct to identify two new QS-controlled genes encoding the regulator Hor and a plant ferredoxin-like protein, FerE. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and mediated by the global repressor RsmA. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production. Our results showed that FerE contributes to oxidative stress tolerance and in planta fitness of the bacteria and suggest that QS could be central to control of oxidative stress tolerance. The presence of the FerE protein appears to be rather unique in heterotrophic bacteria and suggests an acquisition of the corresponding gene from plant host by horizontal gene transfer.